滞育诱导温度和光照节律对家蚕滞育相关基因表达的影响
|
摘要
家蚕(Bombyx mori)是卵滞育性鳞翅目模式昆虫,受亲代卵孵化期(催青期)温度、光节律等环境因子诱导,并受产下后温度诱导决定表现。本文以经典的家蚕滞育卵诱导条件催青期蚕卵及胚后幼虫与蚕蛹,调查家蚕滞育激素基因dh、滞育特征能量代谢限速酶山梨醇脱氢酶基因sdh、家蚕滞育生物钟蛋白基因ea4以及家蚕抗氧化酶基因sod和过氧化氢酶基因cat的表达谱,从滞育诱导与活化调控相关基因表达与环境互作的角度研究了家蚕活化的分子机制。主要获得了以下结果:
1家蚕5个滞育相关基因的表达存在显著的组织和性别差异性
家蚕大造品种5龄幼虫期基因芯片表达谱显示,dh基因、sdh基因和cat表达水平存在性别差异与组织差异,雌蚕的多个组织dh基因表达水平都显著低于雄性;雄蚕的sdh基因表达丰度显著高于雌蚕,而卵巢的sdh基因表达丰度高于精巢;cat基因除在卵巢的表达水平高于精巢外,其它组织的表达量都低于雄性。ea4基因、sod基因和cat基因在不同组织中的表达有显著差异性,ea4基因和sod基因在后部丝腺都只有痕迹量表达,而表达丰度最高的组织分别为头部和体壁,cat则在不同组织中的表达有极显著的差异性。
10个家蚕品种的ESTs表达谱分析显示,ea4基因表达具有组织(器官)和发育时期的差异性,前胸腺表达最高,而生殖腺没有表达;成虫期表达量显著高于其它发育时期,其中刚羽化成虫的前胸腺与被微生物感染5龄幼虫的脂肪体中ea4基因表达丰度显著上调。
2诱导子代滞育卵发生的高温与光照条件上调家蚕dh基因表达
催青期在诱导子代滞育卵发生的高温25℃与持续光照条件下,蚕卵有稳定的、高水平dh基因mRNA存在,而诱导子代非滞育卵发生的低温15℃与持续黑暗条件下,蚕卵中几乎检测不到dh基因的mRNA存在。在25LL或者20LD催青环境中,胚胎发育至单眼形成后(积温高于2160℃·h),蚕卵中的dh基因表达水平开始显著上调,这与在此阶段的蚕卵胚胎对滞育诱导环境温度和光节律更加敏感相一致,但dh基因表达水平没有出现随不同滞育诱导温度或光节律变化的振荡节律,表明蚕卵胚胎的dh基因表达受到滞育诱导温度和光照这两个近日节律授时因子的影响,但不是节律授时因子直接调控的基因。
从催青期开始的整个世代保护温度和光照,对家蚕胚后时期dh基因的表达水平影响,呈现高温25℃上调dh基因的表达。亲代卵孵化期的催青温度与光照,影响了子代卵的滞育性,也影响了子代卵内的dh基因mRNA水平,但这种影响与蚕卵的滞育性不完全一致。
3亲代卵高温环境催青能够下调家蚕整个世代sdh基因表达
催青期的25℃高温和持续光照,显著下调蚕卵sdh基因表达;在20LD催青条件下,蚕卵sdh基因灵敏地受到滞育诱导温度和光照这两个近日节律授时因子的影响,但没有出现随不同滞育诱导温度变化的振荡节律;整个世代维持在诱导子代卵发生滞育的25LL环境下,幼虫至蚕蛾时期各阶段体内(生殖腺)sdh基因的表达水平总体低于20LD环境;亲代不同温度孵化处理结果表明,25℃条件下子代卵在孵化后期进入滞育,sdh基因的转录水平也随之降低。
4光照对该环境中的蚕卵和子代卵的sdh基因表达影响不同
亲代卵催青期低温条件下的持续光照,有显著下调蚕卵sdh基因表达的作用;亲代催青期低温条件下的持续光照,有上调子代卵sdh基因表达的作用。催青期光照对处于该光照环境中的亲代蚕卵和子代卵的sdh基因表达影响呈现相反的结果。
5家蚕TIME-EA4的滞育生物钟活性不依赖基因表达水平的上调
虽然催青期在15DD环境中,蚕卵ea4基因转录水平显著低于25LL和20LD环境,但温度和光照节律的振荡改变,没有出现相应的蚕卵胚胎的ea4基因转录水平变化。胚后时期家蚕幼虫与蚕蛹的ea4基因具有发育阶段与组织(器官)的显著表达差异,在不同滞育诱导环境之间也有差异,但与滞育诱导发生条件无明显关联,而是与自由基代谢相关。
子代卵滞育发生和活化过程中ea4基因的表达结果显示,授精前(处女蛾卵)都具有高水平ea4 mRNA存在,而授精后0.5 h-12 h迅速被降解;合子形成后,转录本水平逐渐升高,滞育性卵在卵龄12 h、无滞育性卵在卵龄24 h后达到授精前水平,并在24 h-72 h的滞育发生期,以及72 h以上的滞育完成期,一直维持此水平,也无滞育性差异。盐酸活化处理卵龄24 h的滞育性卵,卵内EA4的ATPase活性在处理后1.5 h左右出现;5℃低温冷藏卵龄48 h的滞育性卵,活性则在冷藏13d左右出现,但这2个过程都没有检测到ea4基因转录水平升高。提示家蚕滞育性卵活化启动时的EA4生物钟活性出现不依赖ea4基因表达。
6棉铃虫ea4同源基因的克隆、分子进化及组织表达
Haea4基因全长cDNA 799bp,编码199个氨基酸。蛋白序列分析表明,Haea4分子量18768.88,等电点5.45。Haea4基因有明显的信号肽序列,属于分泌蛋白。含有Cu/Zn SOD核心结构域、N-糖基化位点、N-酰基化位点、酪蛋白激酶Ⅱ磷酸化位点和蛋白激酶C磷酸化位点。蛋白同源性分析结果表明,HaEA4在不同物种中的保守性较高。分子进化分析表明,棉铃虫和家蚕、野桑蚕的TIME-EA4同源蛋白质都与Cu-Zn/SOD蛋白的进化距离较近,蛋白质二级结构分析也显示,棉铃虫与家蚕的TIME-EA4基本特性有很大相似性。该基因在棉铃虫不同组织中的表达有一定差异,中肠、脂肪体、体壁表达量较高,头部、生殖腺、丝腺表达量相对较低,提示Haea4基因的分布与酯酶发挥代谢解毒的功能是一致的。氨基酸遗传距离和同源性分析表明,棉铃虫的TIME-EA4与家蚕和野蚕间的遗传距离分别为0.536,0.548,同源性均为44%。
7滞育诱导温度和光节律对家蚕抗氧化酶基因sod和过氧化氢酶基因cat表达谱的影响
催青期25LL环境,蚕卵sod基因表达除孵化时显著下调外,其它时期有高水平的sod mRNA存在;20LD催青环境,蚕卵sod基因表达量高于15DD,在孵化后期胚胎神经系统发育完全后更加显著;cat基因在25LL、20LD和15DD环境之间的表达量没有显著差异,孵化后期(积温2160-3600℃·h),cat基因表达水平显著上调,这与在此阶段的蚕卵胚胎对滞育诱导环境温度和光节律更加敏感相一致。
sod基因在人工活化卵中的表达水平显著高于非浸酸的滞育性卵;卵龄24-192h,25LL条件下子代卵逐渐进入滞育,sod基因表达量下调;温度和光照对家蚕子代卵期sod基因的表达水平影响,呈现低温黑暗上调sod基因的表达。cat基因在人工活化卵初期mRNA水平下降,低于滞育性卵,卵龄72h转录水平开始上调,96h时接近滞育性卵,提示盐酸活化抑制了初期家蚕CAT活性。亲代15℃恒温环境,黑暗条件下的子代卵cat基因表达水平一直高于光照条件,光照与温度协同处理时,20LD处理,子代卵中cat基因表达水平总体低于25LL和15DD环境。
Bombyx mori is the model insect of Lepioloptera with diapausing during its egg stage. Diapause inducements are due to temperature and photoperiod in the incubation process of parental eggs. However, whether or not eggs at progeny enter into diapause depends on temperature after oviposition. This paper investigated the expression of diapause-related genes in Bombyx mori using the classic diapause-inducing incubation conditions, which contained diapause hormone gene, sorbitol dehydrogenase gene, time interval measuring enzyme-esterase A4 gene, superoxide dismutase and catalase genes. The major results are as follows:
1 Diapause-related genes had sex specific and tissue specific expression characteristics in Bombyx mori
Gene microarray analysis displayed that five genes, containing dh, sdh, ea4, sod and cat, had sex specific and tissue specific expression characteristics during postembryonic stage. dh gene expression level in multiple tissues of female is significantly lower than male. sdh gene expression abundance in male is higher than female, but the abundance in testis is obviously lower than ovary. Expression analysis of ea4 showed that it had higher transcription level in head than in posterior silk gland. sod and cat genes also had the tissue specificity, moreover, cat gene significantly had sex specific expression, its expression abundances in male are higher than female except gonads.
Express Sequence Tag (EST) profile showed that ea4 shows stage-specific and tissue-specific expression during postembryonic stages, with high ea4 mRNA in pheromone gland, but with low levels in gonads. Expression was relatively high in the adult stage, and the expression was up-regulated in the microbe-infected fat body of fifth-instar larvae.
2 The temperature and photoperiod inducing diapause of offspring eggs up-regulated the expression level of dh gene
Eggs at progeny are completely diapause when parental eggs are under constant illumination at 25℃(25LL) incubation, and they are completely non-diapause under constant darkness at 15℃(15DD). The dh mRNA of eggs in 25LL stayed at a high level, but it was very low in 15LL. Under 25LL or 20LD, the dh level of parental eggs up-regulated in the late incubation stage, which indicated that B. mori would be sensitive to the external illumination and temperature after embryonic nervous developed. However, the mRNA level of dh was not oscillated with the periodic oscillation of incubation photoperiod and temperature. The dh expression level rose at 25℃during postembryonic developmental stage. In conclusion, the incubation temperature and photoperiod of parental eggs affected the diapause of offspring eggs, but dh was not the direct gene regulated by the zeitgebers.
3 The expression level of sdh gene of the whole generation down-regulated when parental eggs were under high temperature
Eggs incubated under high temperature and constant illumination (25LL), the expression level of sdh gene would decrease. In 20LD, diapause-inducing temperature and photoperiod directly affected the expression of sdh gene in parental eggs but it did not have the obvious rhythmicity. The expression level of sdh gene in 25LL was lower than 20LD from larva to pupa. And sdh mRNA level was lower in the offspring eggs at 25℃than at 15℃, which indicated that as offspring eggs went into the diapausing stage, the transcription level of sdh subsequently fell.
4 The effect of photoperiod on the expression of sdh gene was different between parental eggs and offspring eggs
When parental eggs were incubated under the low temperature and constant illumination, the expression level of sdh gene in this generation was significantly down-regulated, however, its level of offspring eggs was up-regulated. This indicated that the constant illumination played a reverse role beween parental eggs and offspring eggs.
5 The TIME-ATPase activity did not depend on the up-regulated expression level of ea4 gene
The expression level of Bmea4 gene was significantly different among eggs incubated in 25LL, 15DD, and 20LD, respectively. However, they were not oscillated with the periodic oscillation of incubation photoperiod and temperature. The expression level of ea4 is different during postembryonic developmental stage, no matter development stages or tissues (organs), and the difference also existed among different diapause-inducing conditions. So it might be not related to diapause inducing conditions but related to free radical metabolism.
The expression of ea4 in the processes of diapause incidence and termination showed that eggs from virgin moths (0h eggs) exhibited a high level of ea4 mRNA, but 0.5h-12h after oviposition, the ea4 mRNA level decreased steeply, from 24 to 72 hours after oviposition, and after age-72h, the levels of ea4 mRNA were consistent among DDE, DTE, and NDE during the above two periods. The EA4-ATPase activity in eggs was transiently elevated at 1.5h after common acid-treatment, while the typical peak presented on day-13 at 5℃from age-48h, but ea4 mRNA level did not increase at the same time.
6 The research on Haea4 gene from Helicoverpa armigera
The ORF of Haea4 which codes 199 amino acids is 799 bp. Protein analysis show that the molecular weight of Haea4 is 18768.88(18.8 kD), and the isoelectric point is 5.45. HaEA4 shows the significant signal peptide sequence which indicates that it is a secretory protein. N-glycosylation site, Protein kinase C phosphorylation site, Tyrosine kinase II phosphorylation site, N-myristoylation site, Casein kinase phosphorylation site, Cu/Zn SOD core domain are identified in HaEA4. The protein homology analysis reveals that HaEA4 is of high conservative in different species. Analysis of molecular evolution indicates that the evolution distances between HaEA4/BmEA4/BmaEA4 and Cu/Zn SOD are far. Secondary structure prognosticates results reveal that the characteristics of HaEA4 are much similar with BmEA4. Semi-quantitative RT-PCR showed that Haea4 was highly expressed in midgut, integument and fat body, lowly expressed in head, gonad and silk gonad. It was postulated that this Haea4 gene might be involved in the detoxification of xenobiotics. Genetic distance and homology analysis showed that the TIME-EA4 genetic distances of Helicoverpa armigera and Bombyx mori, Helicoverpa armigera and Bombyx mandarina were 0.536 and 0.548, respectively, in addition, the sequence identities of both them are 44%.
7 The effect of diapause-inducing incubation temperature and photoperiod on the expression of sod and cat genes
When eggs were incubated in 25LL, their sod mRNA would keep at a high level, but the transcription level was rapidly down-regulated at EAT1800℃.h and 3600℃.h. The expression of sod gene in 20LD was significantly higher than in 15DD, this phenomenon was more obvious when the embryonic nervous had fully grown in the later incubation stage. The expression of cat gene had no obvious differences when eggs were incubated in 25LL, 20LD or 15DD but it was notable that the mRNA level of cat all increased in the late incubation stage, which was in accordance with the fact that the eggs were more sensitive to diapause-inducing incubation temperature and photoperiod of this time.
After artificial hatching treatment with hydrochloric acid at 24h after oviposition, the sod mRNA in diapause-terminated eggs (DTE) was much higher than diapause-destined eggs (DDE). From 24 to 192 hours after oviposition, eggs at progeny went into the diapause stage, the transcription level of sod subsequently decreased. After we examined the common effect of illumination and temperature on the sod gene of progeny eggs, it was observed that the sod mRNA increased under constant darkness at 15℃. The cat mRNA level of DTE was lower than DDE from 24 to 48 hours after oviposition, but after age-72h, the mRNA increased, which indicated that the expression of cat might be inhibited by hydrochloric acid at the early activation. We also investigated the common effect of illumination and temperature on the cat gene of progeny eggs and found that the expression level of cat in 20LD was lower than in 25LL and 15DD.
1家蚕5个滞育相关基因的表达存在显著的组织和性别差异性
家蚕大造品种5龄幼虫期基因芯片表达谱显示,dh基因、sdh基因和cat表达水平存在性别差异与组织差异,雌蚕的多个组织dh基因表达水平都显著低于雄性;雄蚕的sdh基因表达丰度显著高于雌蚕,而卵巢的sdh基因表达丰度高于精巢;cat基因除在卵巢的表达水平高于精巢外,其它组织的表达量都低于雄性。ea4基因、sod基因和cat基因在不同组织中的表达有显著差异性,ea4基因和sod基因在后部丝腺都只有痕迹量表达,而表达丰度最高的组织分别为头部和体壁,cat则在不同组织中的表达有极显著的差异性。
10个家蚕品种的ESTs表达谱分析显示,ea4基因表达具有组织(器官)和发育时期的差异性,前胸腺表达最高,而生殖腺没有表达;成虫期表达量显著高于其它发育时期,其中刚羽化成虫的前胸腺与被微生物感染5龄幼虫的脂肪体中ea4基因表达丰度显著上调。
2诱导子代滞育卵发生的高温与光照条件上调家蚕dh基因表达
催青期在诱导子代滞育卵发生的高温25℃与持续光照条件下,蚕卵有稳定的、高水平dh基因mRNA存在,而诱导子代非滞育卵发生的低温15℃与持续黑暗条件下,蚕卵中几乎检测不到dh基因的mRNA存在。在25LL或者20LD催青环境中,胚胎发育至单眼形成后(积温高于2160℃·h),蚕卵中的dh基因表达水平开始显著上调,这与在此阶段的蚕卵胚胎对滞育诱导环境温度和光节律更加敏感相一致,但dh基因表达水平没有出现随不同滞育诱导温度或光节律变化的振荡节律,表明蚕卵胚胎的dh基因表达受到滞育诱导温度和光照这两个近日节律授时因子的影响,但不是节律授时因子直接调控的基因。
从催青期开始的整个世代保护温度和光照,对家蚕胚后时期dh基因的表达水平影响,呈现高温25℃上调dh基因的表达。亲代卵孵化期的催青温度与光照,影响了子代卵的滞育性,也影响了子代卵内的dh基因mRNA水平,但这种影响与蚕卵的滞育性不完全一致。
3亲代卵高温环境催青能够下调家蚕整个世代sdh基因表达
催青期的25℃高温和持续光照,显著下调蚕卵sdh基因表达;在20LD催青条件下,蚕卵sdh基因灵敏地受到滞育诱导温度和光照这两个近日节律授时因子的影响,但没有出现随不同滞育诱导温度变化的振荡节律;整个世代维持在诱导子代卵发生滞育的25LL环境下,幼虫至蚕蛾时期各阶段体内(生殖腺)sdh基因的表达水平总体低于20LD环境;亲代不同温度孵化处理结果表明,25℃条件下子代卵在孵化后期进入滞育,sdh基因的转录水平也随之降低。
4光照对该环境中的蚕卵和子代卵的sdh基因表达影响不同
亲代卵催青期低温条件下的持续光照,有显著下调蚕卵sdh基因表达的作用;亲代催青期低温条件下的持续光照,有上调子代卵sdh基因表达的作用。催青期光照对处于该光照环境中的亲代蚕卵和子代卵的sdh基因表达影响呈现相反的结果。
5家蚕TIME-EA4的滞育生物钟活性不依赖基因表达水平的上调
虽然催青期在15DD环境中,蚕卵ea4基因转录水平显著低于25LL和20LD环境,但温度和光照节律的振荡改变,没有出现相应的蚕卵胚胎的ea4基因转录水平变化。胚后时期家蚕幼虫与蚕蛹的ea4基因具有发育阶段与组织(器官)的显著表达差异,在不同滞育诱导环境之间也有差异,但与滞育诱导发生条件无明显关联,而是与自由基代谢相关。
子代卵滞育发生和活化过程中ea4基因的表达结果显示,授精前(处女蛾卵)都具有高水平ea4 mRNA存在,而授精后0.5 h-12 h迅速被降解;合子形成后,转录本水平逐渐升高,滞育性卵在卵龄12 h、无滞育性卵在卵龄24 h后达到授精前水平,并在24 h-72 h的滞育发生期,以及72 h以上的滞育完成期,一直维持此水平,也无滞育性差异。盐酸活化处理卵龄24 h的滞育性卵,卵内EA4的ATPase活性在处理后1.5 h左右出现;5℃低温冷藏卵龄48 h的滞育性卵,活性则在冷藏13d左右出现,但这2个过程都没有检测到ea4基因转录水平升高。提示家蚕滞育性卵活化启动时的EA4生物钟活性出现不依赖ea4基因表达。
6棉铃虫ea4同源基因的克隆、分子进化及组织表达
Haea4基因全长cDNA 799bp,编码199个氨基酸。蛋白序列分析表明,Haea4分子量18768.88,等电点5.45。Haea4基因有明显的信号肽序列,属于分泌蛋白。含有Cu/Zn SOD核心结构域、N-糖基化位点、N-酰基化位点、酪蛋白激酶Ⅱ磷酸化位点和蛋白激酶C磷酸化位点。蛋白同源性分析结果表明,HaEA4在不同物种中的保守性较高。分子进化分析表明,棉铃虫和家蚕、野桑蚕的TIME-EA4同源蛋白质都与Cu-Zn/SOD蛋白的进化距离较近,蛋白质二级结构分析也显示,棉铃虫与家蚕的TIME-EA4基本特性有很大相似性。该基因在棉铃虫不同组织中的表达有一定差异,中肠、脂肪体、体壁表达量较高,头部、生殖腺、丝腺表达量相对较低,提示Haea4基因的分布与酯酶发挥代谢解毒的功能是一致的。氨基酸遗传距离和同源性分析表明,棉铃虫的TIME-EA4与家蚕和野蚕间的遗传距离分别为0.536,0.548,同源性均为44%。
7滞育诱导温度和光节律对家蚕抗氧化酶基因sod和过氧化氢酶基因cat表达谱的影响
催青期25LL环境,蚕卵sod基因表达除孵化时显著下调外,其它时期有高水平的sod mRNA存在;20LD催青环境,蚕卵sod基因表达量高于15DD,在孵化后期胚胎神经系统发育完全后更加显著;cat基因在25LL、20LD和15DD环境之间的表达量没有显著差异,孵化后期(积温2160-3600℃·h),cat基因表达水平显著上调,这与在此阶段的蚕卵胚胎对滞育诱导环境温度和光节律更加敏感相一致。
sod基因在人工活化卵中的表达水平显著高于非浸酸的滞育性卵;卵龄24-192h,25LL条件下子代卵逐渐进入滞育,sod基因表达量下调;温度和光照对家蚕子代卵期sod基因的表达水平影响,呈现低温黑暗上调sod基因的表达。cat基因在人工活化卵初期mRNA水平下降,低于滞育性卵,卵龄72h转录水平开始上调,96h时接近滞育性卵,提示盐酸活化抑制了初期家蚕CAT活性。亲代15℃恒温环境,黑暗条件下的子代卵cat基因表达水平一直高于光照条件,光照与温度协同处理时,20LD处理,子代卵中cat基因表达水平总体低于25LL和15DD环境。
Bombyx mori is the model insect of Lepioloptera with diapausing during its egg stage. Diapause inducements are due to temperature and photoperiod in the incubation process of parental eggs. However, whether or not eggs at progeny enter into diapause depends on temperature after oviposition. This paper investigated the expression of diapause-related genes in Bombyx mori using the classic diapause-inducing incubation conditions, which contained diapause hormone gene, sorbitol dehydrogenase gene, time interval measuring enzyme-esterase A4 gene, superoxide dismutase and catalase genes. The major results are as follows:
1 Diapause-related genes had sex specific and tissue specific expression characteristics in Bombyx mori
Gene microarray analysis displayed that five genes, containing dh, sdh, ea4, sod and cat, had sex specific and tissue specific expression characteristics during postembryonic stage. dh gene expression level in multiple tissues of female is significantly lower than male. sdh gene expression abundance in male is higher than female, but the abundance in testis is obviously lower than ovary. Expression analysis of ea4 showed that it had higher transcription level in head than in posterior silk gland. sod and cat genes also had the tissue specificity, moreover, cat gene significantly had sex specific expression, its expression abundances in male are higher than female except gonads.
Express Sequence Tag (EST) profile showed that ea4 shows stage-specific and tissue-specific expression during postembryonic stages, with high ea4 mRNA in pheromone gland, but with low levels in gonads. Expression was relatively high in the adult stage, and the expression was up-regulated in the microbe-infected fat body of fifth-instar larvae.
2 The temperature and photoperiod inducing diapause of offspring eggs up-regulated the expression level of dh gene
Eggs at progeny are completely diapause when parental eggs are under constant illumination at 25℃(25LL) incubation, and they are completely non-diapause under constant darkness at 15℃(15DD). The dh mRNA of eggs in 25LL stayed at a high level, but it was very low in 15LL. Under 25LL or 20LD, the dh level of parental eggs up-regulated in the late incubation stage, which indicated that B. mori would be sensitive to the external illumination and temperature after embryonic nervous developed. However, the mRNA level of dh was not oscillated with the periodic oscillation of incubation photoperiod and temperature. The dh expression level rose at 25℃during postembryonic developmental stage. In conclusion, the incubation temperature and photoperiod of parental eggs affected the diapause of offspring eggs, but dh was not the direct gene regulated by the zeitgebers.
3 The expression level of sdh gene of the whole generation down-regulated when parental eggs were under high temperature
Eggs incubated under high temperature and constant illumination (25LL), the expression level of sdh gene would decrease. In 20LD, diapause-inducing temperature and photoperiod directly affected the expression of sdh gene in parental eggs but it did not have the obvious rhythmicity. The expression level of sdh gene in 25LL was lower than 20LD from larva to pupa. And sdh mRNA level was lower in the offspring eggs at 25℃than at 15℃, which indicated that as offspring eggs went into the diapausing stage, the transcription level of sdh subsequently fell.
4 The effect of photoperiod on the expression of sdh gene was different between parental eggs and offspring eggs
When parental eggs were incubated under the low temperature and constant illumination, the expression level of sdh gene in this generation was significantly down-regulated, however, its level of offspring eggs was up-regulated. This indicated that the constant illumination played a reverse role beween parental eggs and offspring eggs.
5 The TIME-ATPase activity did not depend on the up-regulated expression level of ea4 gene
The expression level of Bmea4 gene was significantly different among eggs incubated in 25LL, 15DD, and 20LD, respectively. However, they were not oscillated with the periodic oscillation of incubation photoperiod and temperature. The expression level of ea4 is different during postembryonic developmental stage, no matter development stages or tissues (organs), and the difference also existed among different diapause-inducing conditions. So it might be not related to diapause inducing conditions but related to free radical metabolism.
The expression of ea4 in the processes of diapause incidence and termination showed that eggs from virgin moths (0h eggs) exhibited a high level of ea4 mRNA, but 0.5h-12h after oviposition, the ea4 mRNA level decreased steeply, from 24 to 72 hours after oviposition, and after age-72h, the levels of ea4 mRNA were consistent among DDE, DTE, and NDE during the above two periods. The EA4-ATPase activity in eggs was transiently elevated at 1.5h after common acid-treatment, while the typical peak presented on day-13 at 5℃from age-48h, but ea4 mRNA level did not increase at the same time.
6 The research on Haea4 gene from Helicoverpa armigera
The ORF of Haea4 which codes 199 amino acids is 799 bp. Protein analysis show that the molecular weight of Haea4 is 18768.88(18.8 kD), and the isoelectric point is 5.45. HaEA4 shows the significant signal peptide sequence which indicates that it is a secretory protein. N-glycosylation site, Protein kinase C phosphorylation site, Tyrosine kinase II phosphorylation site, N-myristoylation site, Casein kinase phosphorylation site, Cu/Zn SOD core domain are identified in HaEA4. The protein homology analysis reveals that HaEA4 is of high conservative in different species. Analysis of molecular evolution indicates that the evolution distances between HaEA4/BmEA4/BmaEA4 and Cu/Zn SOD are far. Secondary structure prognosticates results reveal that the characteristics of HaEA4 are much similar with BmEA4. Semi-quantitative RT-PCR showed that Haea4 was highly expressed in midgut, integument and fat body, lowly expressed in head, gonad and silk gonad. It was postulated that this Haea4 gene might be involved in the detoxification of xenobiotics. Genetic distance and homology analysis showed that the TIME-EA4 genetic distances of Helicoverpa armigera and Bombyx mori, Helicoverpa armigera and Bombyx mandarina were 0.536 and 0.548, respectively, in addition, the sequence identities of both them are 44%.
7 The effect of diapause-inducing incubation temperature and photoperiod on the expression of sod and cat genes
When eggs were incubated in 25LL, their sod mRNA would keep at a high level, but the transcription level was rapidly down-regulated at EAT1800℃.h and 3600℃.h. The expression of sod gene in 20LD was significantly higher than in 15DD, this phenomenon was more obvious when the embryonic nervous had fully grown in the later incubation stage. The expression of cat gene had no obvious differences when eggs were incubated in 25LL, 20LD or 15DD but it was notable that the mRNA level of cat all increased in the late incubation stage, which was in accordance with the fact that the eggs were more sensitive to diapause-inducing incubation temperature and photoperiod of this time.
After artificial hatching treatment with hydrochloric acid at 24h after oviposition, the sod mRNA in diapause-terminated eggs (DTE) was much higher than diapause-destined eggs (DDE). From 24 to 192 hours after oviposition, eggs at progeny went into the diapause stage, the transcription level of sod subsequently decreased. After we examined the common effect of illumination and temperature on the sod gene of progeny eggs, it was observed that the sod mRNA increased under constant darkness at 15℃. The cat mRNA level of DTE was lower than DDE from 24 to 48 hours after oviposition, but after age-72h, the mRNA increased, which indicated that the expression of cat might be inhibited by hydrochloric acid at the early activation. We also investigated the common effect of illumination and temperature on the cat gene of progeny eggs and found that the expression level of cat in 20LD was lower than in 25LL and 15DD.
引文
1. Adamek G, Fischer J. The oxygen consumption of nondormant and dormant larvae of Chironomus plumosus (Diptera) [J]. J Insect Physiol, 1985, 31: 757-772
2. Andrewartha HG, Miethke PM, Wells A. Induction of diapause in the pupa of Phalaenoides glycinae by a hormone from the suboesophageal ganglion. J Insect Physiol. 1974, 20: 679 - 702
3. Barynin VV, Grebenko AI. T-catalase is a nonheme catalase of extremely thermophilic bacterium Thermus thermophi lus [J]. DoklAkad Nauk SSSR, 1986 , 286 (2): 461-464
4. Beck SD, Hanec W. Diapause in the European corn borer (ECB), Ostrinia nubilalis [J]. J.Insect Physiol., 1960, 4: 304-318
5. Berry SJ, Hormones and metabolism in the pupal diapause of silkmoths (Lepidoptera: Saturniidae) [J]. Entomol Gen, 1981, 7: 233-243
6. Boothroyd CE, Wijnen H, Naef F, et al. Integration of light and temperature in the regulation of circadian gene expression in Drosophila [J]. PLoS Genet, 2007, 3(4): 0492-0507
7. Chino H. Carbohydrate metabolism in the diapause egg of the silkworm, Bombyx mori. II. Conversion of glycogen into sorbitol and glycerol during diapause [J]. J Insect Physiol, 1958, 2: 1-12
8. Chippendale GM & Yin CM. Endocrine interactions controlling the larval diapause of the southwestern Corn borer, Diatraea grandiosella [J]. J Insect Physiol, 1976, 22: 989-995
9. Chippendale GM, Yin CM. Reappraisal of proctodone involvement in the hormonal regulation of larval diapause[J] Biol Bull, 1976, 149:151-164
10. Chippendale GM. Hormonal regulation of larval diapause [J]. Annu. Rev. Entomol., 1977, 22: 121-138
11. De Bie I, Marcinkiewicz M, Malide D, et al. The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments[J] J Cell Bio, 1996,135:1261-1275
12. De Wilde J., Hormones and insect diapause. Mem.Soc. Endocrinol, 1970, 18, 487-514
13. Denlinger DL. Hormonal control of diapause in comprehensive insect physiology, biochemistry and pharmacology (Eds Kerkut GA & Gilbert LI) [M]. Oxford: Pergamon Press, 1985, 8: 353-412
14. Denlinger DL. Regulation of diapause. Annual Review of Entomology, 2002, 47: 93-122.
15. Dismukes GC. Manganese enzymes with binuclear active sites. Chem.Rev, 1996, 96 (7): 2909-2926
16. Fan JY, Muskus MJ, Price JL. Entrainment of the Drosophila circadian clock: more heat than light [J/OL]. Sci. STKE, 2007, (413): pe65. [2008-03-19]. http://www.stke.org/cgi/content/full/2007/413/pe65
17. Frank V, Eva V, James FD, et al. The role of active oxygen species in plant signal transduction. Plant Science.2001, 161: 405- 414
18. Fukuda S. The production of diapause eggs by transplanting the suboesophageal ganglion in the silkworm. Proc Jap. Acad. 1951, 27: 672– 676
19. Glaser FT, Stanewsky R. Temperature synchronization of the Drosophila circadian clock [J]. Current Biology, 2005, 15(15): 1352-1363
20. Glaser FT, Stanewsky R. Temperature synchronization of the Drosophila circadian clock [J]. Current Biology, 2005, 15(15): 1352-1363
21. Goldberg I, Hochrnan A. Three different types of catalase in Klebsiel lapneumoniae [J]. Arch Biochem Biophys, 1989, 268(1):124-128
22. Goncalves VM , Cezar de Cerqueira Leite L, Raw I, et al . Purification of catalase from human placenta [J]. Biotechnology and Applied Biochemistry, 1999, 29 (1):73-77
23. Levin G, Mendive F, Targovnik HM, et al. Genetically engineered horseradish peroxidase for facilitated purification from baculovirus cultures by cation-exchange chromatography [J]. J Biotechnol, 2005, 118 (4): 363-369
24. Hasegawa K. Studies in voltinism in the silkworm Bombyx mori I. with special reference to the organs concerning determination of voltinism. J Fac. Agric TottoriUniv 1952, 1: 83 - 124
25. Hasegawa K. The diapauses hormone of the silkworm, Bombyx mori [J]. Nature, 1957, 179: 1300-1301
26. Hiraki T, Shibayama N, Akashi S, et al. Crystal Structures of the Clock Protein EA4 from the Silkworm Bombyx mori [J]. J Mol Biol, 2008, 377: 630-635
27. Hong B, Zhang Z-F, Tang S-M, et al. Protein-DNA interaction in the promoter region of the gene encoding DH-PBAN of the cotton bollworm, Helicoverpa armigera [J]. Biochim.Biophys. Acta, 2006, 1759:177-185
28. Isobe K, Konno T, Nakazawa I, et al. Further characterization of the silkworm diapause hormone [ J ] A. J Insect Physiol, 1975, 21 :1221 1920
29. Isobe M, Kai H, Kurahashi T, et al. The molecular mechanism of the termination of insect diapause: Part 1. A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo-glycoprotein [J]. Chem Bio Chem, 2006, 7: 1590-1598
30. Isobe M, Suwan S, Kai H, et al. Amino acid sequence of PIN peptides conducting TIME(Time-Interval-Measuring-Esterase) activation for resumption of embryonic development in the silkworm, Bombyx mori. Bioorganic & Medicinal Chemistry Letters, 1995, 5: 2851-2854
31. Iwai S, Fukui Y, Fujiwara Y, et al. Structure and expressions of two circadian clock gene, period and timeless in the commercial silk moth, Bombyx mori [J]. J Insect Physiol, 2006, 52: 625–637
32. Imai K, Konno T, Nakazaw Y, et al. Isolation and structure of diapause hormone of the silkworm [J] . Bombyx mori. Proc. Jap. Acad. 1991, 67: 98– 101
33. Jacek S, Loewen PC. Diversity of properties among catalases. Archivers of biochemistry and biophysiscs.2002, 401: 145- 154
34. Kai H, Kawai T. Similar action of acid-treatment to long chilling for breaking silkworm diapause in respect esterase A activities in eggs [J]. Seric. Sci. Jpn., 1981, 50: 373-378
35. Kai H, Nishi K. Diapause development in Bombyx eggs in relation to ?esterase A‘activity [J]. Journal of Insect Physiology., 1976, 22: 1315-1320
36. Kai H, Arai T, Yasuda F. Accomplishment of time-interval activation of esterase A4 by simple removal of PIN fraction. Chronobiology International, 1999, 16: 51–58
37. Kai H, Kawai T, Kaneto A. Esterase A4 elevation mechanism in relation to Bombyx (Lepidoptera: Bombycidae) eggs diapause development [J]. Appl. Ent. Zool., 1984, 19(1): 8-14
38. Kai H, Kawai T, Kawai Y. A time-interval activation of esterase A4 by cold-relation to the termination of embryonic diapause in the silkworm, Bombyx mori [J]. Insect Biochem, 1987, 17: 367-372
39. Kai H, Kotani H, Oda Y. Presence of PIN factor(s) responsible for the Time-interval activation of esterase A4 in the diapause duration timer of Bombyx mori. Journal of Sericultural Science of Japan, 1996, 65: 31-38
40. Kai H, Kotani Y, Miao Y, et al. Time interval measuring enzyme for resumption of embryonic development in the silkworm, Bombyx mori. J Insect Physiol. 1995,41(10): 905-910
41. Kai H, Miao Y, Kawai T. Activation of esterase A4 by hydrogen peroxide. J Seric. Sci. Japan, 1988,57(6): 533-534
42. Kai H, Miao Y, Xu PX, et al. Effective acid-treatment in vitro to elevate the time measuring esterase A4 [J]. Seric Sci Jpn,1988, 57: 313-317
43. Kai H, Nishi K. Diapause development in Bombyx eggs in relation to "esterase A" activity. Journal of Insect Physiology, 1976, 22: 1315–1320
44. Kai H, Nishi K. Diapause development in Bombyx eggs in relation to ?esterase A‘activity. J. Insect Physical., 1976, 22, 1315-1320
45. Kai, H. Molecular biology of an alarm clock: a timer-protein involved in an active resumption of embryogenesis in the Bombyx silkworm. Biophysics, 2002, 42: 168-173(in Japanese)
46. Kai H., et al. Electrophoretic protein patterns and esterase zymograms in ovaries and mature eggs of Bombyx mori in relation to diapause. J. Insect Physical., 1972a, 18, 133-142
47. Kai H., et al. Studies on the mode of action of the diapause hormone with special reference to the protein metabolism in the silkworm, Bombyx mori L.Ⅲ. Effects ofacid-treatment and detergents on ?Esterase A‘in diapause eggs. J. Seric. sci. Jpn., 1972b, 41(4): 253-262
48. Kai H, Kawai T, Kawai Y. A time-interval activation of esterase A4 by cold. Insect Biochemistry, 1987, 17: 367-372
49. Kaushik R, Nawathean P, Busza A, et al. PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila [J]. PLoS Biol, 2007, 5(6): 1257-1266
50. Khansen TE, Viik MO. The content of free amino acids in wintering insects [J]. Zool. ZH., 1985, 63: 1634-1640
51. Kitagawa N, Shiomi K, Imai K, et al. Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori. Zoological Science, 2005, 22: 213-221
52. Kono Y, Fridovich I. Isolation and characterization of the pseudocatalase of L actobacillus plantarum A new manganese containing enzyme [J]. Biol Chem, 1983, 258 (10): 6015-6019
53. Kostal V, Hodek I. Photoperiodism and control of summer diapause in the Mediterranean tiger minth Cymbalophora pudica [J]. Insect Physiol, 1997, 43: 767 - 777
54. Krishnan B, Levine JD, Lynch MK, et al. A new role for cryptochrome in a Drosophila circadian oscillator [J]. Nature, 2001, 411: 313-317
55. McCord J M & Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein) [J]. Biol. Chem, 1969, 244: 6049-6055
56. McGuire JU, Schechter MS. Synthesis of macromolecules in diapausing and nondiapausing larvae of the European corn borer [J]. Econ. Entomol., 1971, 65: 676-679
57. Nakamura K, Watanabe M, Sasaki Y,et al. Purification and characterization of liver catalase in acatalasemic beagle dog: Comparison with normal dog liver catalase [J]. The International Journal of Biochemistry and Cell Biology, 2000, 2 (1): 89-98
58. Numata H, Hidaka T. Photoperiodic control of adult diapause in the bean bug,Riptortus clavatus Thunberg (Heteroptera: Coreidae) 1. Reversible induction and termination of diapause [J]. Appl Entomoml Zool, 1982, 17: 530– 538
59. Ohtsuki Y. Silkworm eggs. In: Japanese Society of Sericultural Science, Editor, A General Textbook of Sericulture, Nihon Sansei Shinbun-Sha, Tokyo, 1979, pp 156-173 (in Japanese)
60. Pittendrigh CS, Bruce VG, Kaus P. On the significance of transients in daily rhythms [J]. PNAS, 1958, 44(9): 965-973
61. Qiu J, Hardin PE. per mRNA cycling is locked to lights-off under photoperiodic conditions that support circadian feedback loop function [J]. Mol Cell Biol, 1996, 16: 4182-4188
62. Reubsaet FA, Veerkamp JH, Brückwilder M L, et al . Peroxisomal oxidases and catalase in liver and kidney homogenates of normal and di (ethylhexyl) phthalate-fed rats [J]. Int J Biochem, 1991, 23 (9): 961-967
63. Ryan B J,ó'Fágáin C. Arginine-to-lysine substitutions influence recombinant horseradish peroxidase stability and immobilization effectiveness [J]. BMC Biotechnology, 2007, 7 (1): 86-94
64. Saito H, Takcuchi Y, Takeda R et al. The core and complementary sequence for iological activity of the diapause hormone of the silkworm, Bombyx mori. Peptides, 1994, 15: 1173-1178
65. Sato Y, Oguchi M, Menjo N, et al. Precursor polyprotein for multiple neuropeptides secreted from the SG of the silk worm, Bombyx mori: Characterization of the cDNA encoding the diapause hormone precursor and identification of additional peptides. PNAS, 1993, 90(8): 3251-3255
66. Sawchyn WW, Church NS. The effects of temperature and photoperiod on diapause development in the eggs of four species of Lestes (Odonata: Zygoptera) [J]. Can J Zool, 1973, 51: 1257– 1265
67. Schneiderman HA, Williams GM. The physiology of insect diapauseⅫ: The respiratory metabolism of the cecropia silkworm during diapause and development [J]. Biol Bull, 1953, 105: 320-334
68. Singh R, Singh RK, Mahdi AA, et al. Circadian periodicity of plasma lipidperoxides and other anti-oxidants as putative markers in gynecological malignancies. In Vivo, 2003, 17(6): 593-600
69. Takeda N. Histophysiological studies on corpus allatum during prepupal diapause in Monema flavescens (Lepidop tera) [J]. Morph. 1977, 153: 245– 262
70. Tani N, Kamada G, Ochiai K, et al. Carbohydrate moiety of Time-Interval Measuring Enzyme regulates time measurement through its interaction with time-holding peptide PIN [J]. J Biol Chem, 2001, 129: 221–227
71. Tauber M J, Tauber CA. Seasonal Adaptations of Insects [M]. Landon: Oxford University Press, 1986, 202-207
72. Ti X, Tani N, Isobe M, Kai H. Time-measurement-regulating peptide PIN may alter a timer conformation of time interval measuring enzyme (TIME). Insect Physiology, 2006, 52: 461-467
73. Ti X, Tuzuki N, Tani N, et al. Demarcation of diapause development by cold and its relation so time-interval activation of TIME-ATPase in eggs of the silkworm, Bombyx mori [J]. Insect Physiol, 2004, 50: 1053-1064
74. Ti X, Tuzuki N, Tani N, et al. The peptide PIN changes the timing of transitory burst activation of timer-ATPase TIME in accordance with diapause development in eggs of the silkworm, Bombyx mori [J]. Insect Physiology, 2005, 51: 1025-1032
75. Tomás-Zapico C, Coto-Montes A, Martínez-Fraga J, et al. Effects of continuous light exposure on antioxidant enzymes, porphyric enzymes and cellular damage in the Harderian gland of the Syrian hamster. J Pineal Res, 2003, 34(1): 60-68
76. Grigorenko V, Chubar T, Kapeliuch Y, et al. New approaches for functional expression of recombinant horseradish peroxidase C in Escherichia coli [J]. Biocatalysis and Biotransformation, 1999, 17 (5): 359-379
77. Wei ZJ., Zhang QR., Kang L., et al. Molecular characterization and expression of prothoracicotropic hormone during development and pupal diapause in the cotton bollworm, Helicoverpa armigera [J]. Insect Physiology, 2005, 51: 691-700
78. Wheeler DA, Hamblen-Coyle MJ, Dushay MS, et al. Behavior in light-dark cycles of Drosophila mutants that are arrhythmic, blind, or both [J]. Biol Rhythms, 1993, 8(1): 67-94
79. Xu SQ, Kai H, Xu JL, et al. Changes of esterase A4-activity and its relationship with diapause in hot water treated eggs of silkworm, Bombyx mori [J]. Sericology, 2003, 43: 61-67
80. Xu Shi-Qing, Dai Xuan-Ying, Han Yi-Fei, et al. Research on diapause bioclock protein esterase A4 of silkworm, Bombyx mori. Sericology, 2001, 41(4) 543-549
81. Xu SQ, Kai H, Xu JL, et al. Changes of esterase A4-activity and its relationship with diapause in acid-treated eggs of silkworm, Bombyx mori. Science of Sericulture, 1998, 24: 95-99
82. Xu SQ, Kai H, Xu JL, et al. Target of hydrochloric acid in acid-treatment of diapausing eggs of Bombyx mori. Acta Entomologica Sinica, 2001, 44: 51-55
83. Xu WH, Sato Y, Ikeda M, et al. Stage-dependent and temperature-controlled expression of the gene encoding the precursor protein of DH-PBAN in the silkworm, Bombyx mori. J. Biol. Chem. 1995, 270: 3804-3808
84. Xu WH, Denlinger DL. Molecular characterization of prothoracicotropic hormone and diapause hormone in Heliothis virescens and a new role for diapause hormone. Insect Molecular Biology, 2003, 12(5): 509-516
85. Xue FS, Kallenborn HG. Summer and winter diapause in pupae of the cabbage butterfly, Pieris melete Ménétriés [J]. J Insect Physiol., 1997, 43: 701-707
86. Yamamoto K, Banno Y, Fujii H, Miake F, Kashige N, Aso Y, 2005. Catalase from the silkworm, Bombyx mori: Gene sequence, distribution, and overexp ression. Insect Biochem.Mol.B iol., 35: 277– 283
87. Yamashita O, Hasegawa K. Oocyte age sensitive to the diapause hormone from the standpoint of glycogen synthesis in the silkworm, Bombyx mori [J]. Insect Physiol ,1970, 16: 2377 - 2 383
88. Yamashita O, Yaginma T. Silkworm eggs at low temperature, implications for sericulture [M]. In: Lee R E. and Denlinger K L eds. Insect at low Temperature, New York: Chapman and Hall Press ,1991, 424– 445
89. Yamashita O, Hasegawa K. Studies on the mode of action of diapause hormone in the silkworm Bombyxmori L. IV. Effect of diapause hormone on the glycogen content in ovaries and the blood sugar level of silkworns. J Seric Sci. 1964, 33: 407– 416
90. Yamashita O, Suzuki K. Morphogenic hormones in arthropods (ed. Gupta, A.P.), New Brunswick: Rutger University Press, 1991, pp 82-128
91. Yamashita O. Diapause hormone of the silkworm Bombyx mori: structure gene expression and function [J]. Insect Physiology, 1996, 42, 669-679
92. Yin CM, Wang ZS, Chaw WD, et al. Brain neurosecretory cell and ecdysiotropin activity of the non-diapausing and diapausing D.Gradiosella [J]. J Insect Physiol., 1985, 31: 659-667
93. Yoshii T, Sakamoto M, Tomioka K. A temperature-dependent timing mechanism is involved in the circadian cycling period gene expression in Drosophila system that drives locomotor rhythms in the fruit fly Drosophila melanogaster [J]. Zoolog Sci, 2002, 19(8): 841-850
94. Yoshii T, Fujii K, Tomioka K. Induction of Drosophila behavioral and molecular circadian rhythms by temperature steps in constant light. Journal of Biological Rhythms, 2007, 22: 103-114
95. Young MW, Youngm W. Circadian rhythms-marking time for a kingdom [J]. Science, 2000, 288: 451-453
96. Yumoto I, Ichihashi D, Iwata H, et al. Purification and characterization of a catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1T exhibiting high catalase activity[J ] . J Bacteriol, 2000, 182 (7):1903-1909
97. Zámocky M, Koller F. Understanding the structure and function of catalase: Clues f rom molecular evolution and in vitro mutagenesis [J]. Progress in Biophysics and Molecular Biology, 1999, 72(1) :19-66
98. Zhang TY, Kang L, Zhang Z-F, et al. Identification of a POU factor involved in regulating the neuron-specific expression of the gene encoding DH-PBAN in Bombyx mori [J]. Biochem, 2004, 380: 255-263
99. Zhang TY, Sun JS, Kang L, et al. Structural analysis of the gene for DH-PBAN in Helicoverpa armigera and its transcriptional regulation. Biochim.Biophys. Acta. 2005, 1728 (1-2): 44-52
100. Zhang TY, Sun JS, Zhang QR et al. The DH-PBAN gene of Helicoverpaarmigera encodes multiple peptides that break, rather than induce, diapause. Insect Physiology, 2004,50(6): 547-554
101. Zhao, LC, Sima, YF, Shen, XM, et al. Metabolism of hydrogen peroxide in the course of embryonic development in silkworm. Dev. Reprod. Biol., 1999, 8, 41-48
102.陈杨,刘望.超氧化物歧化分构看其分化[J].生物化与生物物展,1996,23(5):408-413
103.陈田飞,灵.育究况[J].业,2004,41(3):12-16
104.海,徐,马杨虎,等.野桑超氧化物歧化因隆分与核表达[J].业科,2007,33(1): 234-240
105.段,翠,贺,等.血淋巴超氧化物歧化究.业科,1995,2 (2): 102-105
106.丁道远,胡汉,朴.荧谱法究某些物与DNA相作[J].谱与谱分1990,10:24
107.侯月利,花蕾.昆育诱因[J].陕业科技,2000,3: 62-67
108.胡恕,张.血中超氧化物歧化(SOD)究[J].业科,1996,22(4):338-340
109.黄伟,于涟.生物制究展[J].生科,2000,12(1): 10-13
110.蒋明星,谢,张羲.棉育诱性[J].应生态报, 1999,10(1): 60-62
111.玺,刘继兰,王选年,等.超氧化物歧化究应展[J].物医展,2007,28 (7) :70-75
112.少琴,黄.贻贝超氧化物歧化纯化部分性质究[J].天产物究与开发,1999,11 (4): 25-29
113.刘惠霞,新岗,吴.昆生物化[M]. :陕科技术版,1998
114.鲁,张英,王赉.淡色库生育经内分泌调节[J].昆报,1993,36(3): 302-307
115.萨姆鲁J,拉塞尔DW.分隆实验南(第三版)[M].北京:科版. 2002
116.山兴亚.激,别是于育激作.科技术,1969, 8(1):72-78
117.宋艳.育诱过程中近日节因period timless表达究[D].苏:苏大,2009
118.田荟.血中过氧化氢取性质究[J].食品科,2006,27 (12): 311-314
119.王.大鼠松体生物因昼表达以外血淋巴细胞因筛选与鉴[C].论,2007,5
120.王英,马杨虎,宋艳,等.昆生物因其作制究展[J].江苏业,2008,30(1): 9-14
121.王英,徐,马杨虎,等.育生物白质EA4因构功能究[C].第届中际绸会议中会第五届青年术讨会论,苏,2007,9: 366-380
122.王,徐,马杨虎,等.育生物白质EA4因cDNA隆分[J].业科,2007,33(1): 36-42
123.王志华,良.过氧化氢其模拟物究展[J] .杭师范院报(科版), 2006 , 5 (6) :465-468
124.吴明,予元.棉育诱因究[J].物保报., 1995,22(4): 331-336
125.吴,徐俊良等.血过氧化氢活力其与体抗逆性系[J].昆报,1998,41(2): 124-129.
126.谢,蒋明星,张羲.烟青育征究[J].物保报., 1997,24(3): 199-203
127.徐丽,宋艳,苏,等.育诱度与对近日节因per表达响[J].业科,2010,36(1):52-59
128.徐,戴璇,等.育生物白质酯A4究展[J].通讯,2000b,20(4): 9-15
129.徐,甲斐英则,徐俊良,等.育生物白质EA4纯化其分构分[J].中生物化与分生物报,1999a,15(2): 241-246
130.徐,甲斐英则,徐俊良,等.体外酸处与PIN对酯A4活性响系[J].业科,1999c,25(3): 149-153
131.徐,甲斐英则,徐俊良,等.酸处卵酯A4活性变化其与育性系[J].业科,1998,24(2): 95-99
132.徐,甲斐英则,徐俊良,等.酸激量对卵酯A4活性响其与卵活化系[J].业科,2000a,26(1): 20-26
133.徐,蒋元,戴璇,等.环对越年卵催青期过氧化氢代谢响[J].江苏业, 2005,27(1): 15-17
134.徐,马杨虎,陈息,等.卵汤处酯A4活性变化[J].业科, 2004,30(1): 50-54
135.徐,琴,甲斐英则,徐俊良,郑必,陈息,马杨虎.体外25℃卵酯A4 ATP活性变化其与育性系[J].业科, 2000c, 26(3): 140-145
136.徐,郑必,马杨虎,等.育性卵酸孵化法酸作靶物[J].昆报,2001,44(1): 51-55
137.徐,陈息,卫,等.桑卵孵化制法[P].中:ZL200510041062.8. 2005-12-28
138.徐,茂顶.生产操作技术南[M].北京:中科技术版,1993, 14-29
139.徐.桑育性卵酸孵化论究[D].江农业大,1997,63-67
140.徐卫华,佐洋,山兴亚.育激因隆[J].传报, 1995b,22(3): 178-184
141.徐卫华.育分[J].传报,1999,26(2): 107-111
142.徐卫华.育激-息成激活肽因表达调节[J].中生物化分生物报.,1998,14(5): 557-561
143.押部秀二,1943.实验, ,247
144.川,时连.育性卵中辅Ⅰ辅Ⅱ量变化[J].业科,2008,34(3): 447-452
145.川,兵,等.微感染对过氧化氢代谢响[J].通讯,1998,18(2):15-17
146.川,马杨虎,兵,等.即时浸酸对胎发育中H2O2代谢响[J].业科,2000,26(4):268-270
147.川,姚祥,兵.变态过程中过氧化氢代谢点外源激对其调节作[J].通讯,1999, 19(3):5-8
148.川,姚祥.五过氧化氢代谢.南京师大报, 1997, 20(4):128-130
149.川.过氧化氢高催青期水处卵孵化[J].业科., 2004, 30(4): 440-442
150.江农业大编.良繁育与育[M],北京:中农业版,1981,33
151.郑必,徐,徐孟奎,等.蓖麻育与过氧化氢代谢系[J].业科,2006b,32(2): 221-225
152.郑荣梁,1993.生科中由究鸟瞰.由生科展1:5~13
153.苏.生物因Bm-cry隆育诱发环对其表达响[D].苏:苏大,2009
2. Andrewartha HG, Miethke PM, Wells A. Induction of diapause in the pupa of Phalaenoides glycinae by a hormone from the suboesophageal ganglion. J Insect Physiol. 1974, 20: 679 - 702
3. Barynin VV, Grebenko AI. T-catalase is a nonheme catalase of extremely thermophilic bacterium Thermus thermophi lus [J]. DoklAkad Nauk SSSR, 1986 , 286 (2): 461-464
4. Beck SD, Hanec W. Diapause in the European corn borer (ECB), Ostrinia nubilalis [J]. J.Insect Physiol., 1960, 4: 304-318
5. Berry SJ, Hormones and metabolism in the pupal diapause of silkmoths (Lepidoptera: Saturniidae) [J]. Entomol Gen, 1981, 7: 233-243
6. Boothroyd CE, Wijnen H, Naef F, et al. Integration of light and temperature in the regulation of circadian gene expression in Drosophila [J]. PLoS Genet, 2007, 3(4): 0492-0507
7. Chino H. Carbohydrate metabolism in the diapause egg of the silkworm, Bombyx mori. II. Conversion of glycogen into sorbitol and glycerol during diapause [J]. J Insect Physiol, 1958, 2: 1-12
8. Chippendale GM & Yin CM. Endocrine interactions controlling the larval diapause of the southwestern Corn borer, Diatraea grandiosella [J]. J Insect Physiol, 1976, 22: 989-995
9. Chippendale GM, Yin CM. Reappraisal of proctodone involvement in the hormonal regulation of larval diapause[J] Biol Bull, 1976, 149:151-164
10. Chippendale GM. Hormonal regulation of larval diapause [J]. Annu. Rev. Entomol., 1977, 22: 121-138
11. De Bie I, Marcinkiewicz M, Malide D, et al. The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments[J] J Cell Bio, 1996,135:1261-1275
12. De Wilde J., Hormones and insect diapause. Mem.Soc. Endocrinol, 1970, 18, 487-514
13. Denlinger DL. Hormonal control of diapause in comprehensive insect physiology, biochemistry and pharmacology (Eds Kerkut GA & Gilbert LI) [M]. Oxford: Pergamon Press, 1985, 8: 353-412
14. Denlinger DL. Regulation of diapause. Annual Review of Entomology, 2002, 47: 93-122.
15. Dismukes GC. Manganese enzymes with binuclear active sites. Chem.Rev, 1996, 96 (7): 2909-2926
16. Fan JY, Muskus MJ, Price JL. Entrainment of the Drosophila circadian clock: more heat than light [J/OL]. Sci. STKE, 2007, (413): pe65. [2008-03-19]. http://www.stke.org/cgi/content/full/2007/413/pe65
17. Frank V, Eva V, James FD, et al. The role of active oxygen species in plant signal transduction. Plant Science.2001, 161: 405- 414
18. Fukuda S. The production of diapause eggs by transplanting the suboesophageal ganglion in the silkworm. Proc Jap. Acad. 1951, 27: 672– 676
19. Glaser FT, Stanewsky R. Temperature synchronization of the Drosophila circadian clock [J]. Current Biology, 2005, 15(15): 1352-1363
20. Glaser FT, Stanewsky R. Temperature synchronization of the Drosophila circadian clock [J]. Current Biology, 2005, 15(15): 1352-1363
21. Goldberg I, Hochrnan A. Three different types of catalase in Klebsiel lapneumoniae [J]. Arch Biochem Biophys, 1989, 268(1):124-128
22. Goncalves VM , Cezar de Cerqueira Leite L, Raw I, et al . Purification of catalase from human placenta [J]. Biotechnology and Applied Biochemistry, 1999, 29 (1):73-77
23. Levin G, Mendive F, Targovnik HM, et al. Genetically engineered horseradish peroxidase for facilitated purification from baculovirus cultures by cation-exchange chromatography [J]. J Biotechnol, 2005, 118 (4): 363-369
24. Hasegawa K. Studies in voltinism in the silkworm Bombyx mori I. with special reference to the organs concerning determination of voltinism. J Fac. Agric TottoriUniv 1952, 1: 83 - 124
25. Hasegawa K. The diapauses hormone of the silkworm, Bombyx mori [J]. Nature, 1957, 179: 1300-1301
26. Hiraki T, Shibayama N, Akashi S, et al. Crystal Structures of the Clock Protein EA4 from the Silkworm Bombyx mori [J]. J Mol Biol, 2008, 377: 630-635
27. Hong B, Zhang Z-F, Tang S-M, et al. Protein-DNA interaction in the promoter region of the gene encoding DH-PBAN of the cotton bollworm, Helicoverpa armigera [J]. Biochim.Biophys. Acta, 2006, 1759:177-185
28. Isobe K, Konno T, Nakazawa I, et al. Further characterization of the silkworm diapause hormone [ J ] A. J Insect Physiol, 1975, 21 :1221 1920
29. Isobe M, Kai H, Kurahashi T, et al. The molecular mechanism of the termination of insect diapause: Part 1. A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo-glycoprotein [J]. Chem Bio Chem, 2006, 7: 1590-1598
30. Isobe M, Suwan S, Kai H, et al. Amino acid sequence of PIN peptides conducting TIME(Time-Interval-Measuring-Esterase) activation for resumption of embryonic development in the silkworm, Bombyx mori. Bioorganic & Medicinal Chemistry Letters, 1995, 5: 2851-2854
31. Iwai S, Fukui Y, Fujiwara Y, et al. Structure and expressions of two circadian clock gene, period and timeless in the commercial silk moth, Bombyx mori [J]. J Insect Physiol, 2006, 52: 625–637
32. Imai K, Konno T, Nakazaw Y, et al. Isolation and structure of diapause hormone of the silkworm [J] . Bombyx mori. Proc. Jap. Acad. 1991, 67: 98– 101
33. Jacek S, Loewen PC. Diversity of properties among catalases. Archivers of biochemistry and biophysiscs.2002, 401: 145- 154
34. Kai H, Kawai T. Similar action of acid-treatment to long chilling for breaking silkworm diapause in respect esterase A activities in eggs [J]. Seric. Sci. Jpn., 1981, 50: 373-378
35. Kai H, Nishi K. Diapause development in Bombyx eggs in relation to ?esterase A‘activity [J]. Journal of Insect Physiology., 1976, 22: 1315-1320
36. Kai H, Arai T, Yasuda F. Accomplishment of time-interval activation of esterase A4 by simple removal of PIN fraction. Chronobiology International, 1999, 16: 51–58
37. Kai H, Kawai T, Kaneto A. Esterase A4 elevation mechanism in relation to Bombyx (Lepidoptera: Bombycidae) eggs diapause development [J]. Appl. Ent. Zool., 1984, 19(1): 8-14
38. Kai H, Kawai T, Kawai Y. A time-interval activation of esterase A4 by cold-relation to the termination of embryonic diapause in the silkworm, Bombyx mori [J]. Insect Biochem, 1987, 17: 367-372
39. Kai H, Kotani H, Oda Y. Presence of PIN factor(s) responsible for the Time-interval activation of esterase A4 in the diapause duration timer of Bombyx mori. Journal of Sericultural Science of Japan, 1996, 65: 31-38
40. Kai H, Kotani Y, Miao Y, et al. Time interval measuring enzyme for resumption of embryonic development in the silkworm, Bombyx mori. J Insect Physiol. 1995,41(10): 905-910
41. Kai H, Miao Y, Kawai T. Activation of esterase A4 by hydrogen peroxide. J Seric. Sci. Japan, 1988,57(6): 533-534
42. Kai H, Miao Y, Xu PX, et al. Effective acid-treatment in vitro to elevate the time measuring esterase A4 [J]. Seric Sci Jpn,1988, 57: 313-317
43. Kai H, Nishi K. Diapause development in Bombyx eggs in relation to "esterase A" activity. Journal of Insect Physiology, 1976, 22: 1315–1320
44. Kai H, Nishi K. Diapause development in Bombyx eggs in relation to ?esterase A‘activity. J. Insect Physical., 1976, 22, 1315-1320
45. Kai, H. Molecular biology of an alarm clock: a timer-protein involved in an active resumption of embryogenesis in the Bombyx silkworm. Biophysics, 2002, 42: 168-173(in Japanese)
46. Kai H., et al. Electrophoretic protein patterns and esterase zymograms in ovaries and mature eggs of Bombyx mori in relation to diapause. J. Insect Physical., 1972a, 18, 133-142
47. Kai H., et al. Studies on the mode of action of the diapause hormone with special reference to the protein metabolism in the silkworm, Bombyx mori L.Ⅲ. Effects ofacid-treatment and detergents on ?Esterase A‘in diapause eggs. J. Seric. sci. Jpn., 1972b, 41(4): 253-262
48. Kai H, Kawai T, Kawai Y. A time-interval activation of esterase A4 by cold. Insect Biochemistry, 1987, 17: 367-372
49. Kaushik R, Nawathean P, Busza A, et al. PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila [J]. PLoS Biol, 2007, 5(6): 1257-1266
50. Khansen TE, Viik MO. The content of free amino acids in wintering insects [J]. Zool. ZH., 1985, 63: 1634-1640
51. Kitagawa N, Shiomi K, Imai K, et al. Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori. Zoological Science, 2005, 22: 213-221
52. Kono Y, Fridovich I. Isolation and characterization of the pseudocatalase of L actobacillus plantarum A new manganese containing enzyme [J]. Biol Chem, 1983, 258 (10): 6015-6019
53. Kostal V, Hodek I. Photoperiodism and control of summer diapause in the Mediterranean tiger minth Cymbalophora pudica [J]. Insect Physiol, 1997, 43: 767 - 777
54. Krishnan B, Levine JD, Lynch MK, et al. A new role for cryptochrome in a Drosophila circadian oscillator [J]. Nature, 2001, 411: 313-317
55. McCord J M & Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein) [J]. Biol. Chem, 1969, 244: 6049-6055
56. McGuire JU, Schechter MS. Synthesis of macromolecules in diapausing and nondiapausing larvae of the European corn borer [J]. Econ. Entomol., 1971, 65: 676-679
57. Nakamura K, Watanabe M, Sasaki Y,et al. Purification and characterization of liver catalase in acatalasemic beagle dog: Comparison with normal dog liver catalase [J]. The International Journal of Biochemistry and Cell Biology, 2000, 2 (1): 89-98
58. Numata H, Hidaka T. Photoperiodic control of adult diapause in the bean bug,Riptortus clavatus Thunberg (Heteroptera: Coreidae) 1. Reversible induction and termination of diapause [J]. Appl Entomoml Zool, 1982, 17: 530– 538
59. Ohtsuki Y. Silkworm eggs. In: Japanese Society of Sericultural Science, Editor, A General Textbook of Sericulture, Nihon Sansei Shinbun-Sha, Tokyo, 1979, pp 156-173 (in Japanese)
60. Pittendrigh CS, Bruce VG, Kaus P. On the significance of transients in daily rhythms [J]. PNAS, 1958, 44(9): 965-973
61. Qiu J, Hardin PE. per mRNA cycling is locked to lights-off under photoperiodic conditions that support circadian feedback loop function [J]. Mol Cell Biol, 1996, 16: 4182-4188
62. Reubsaet FA, Veerkamp JH, Brückwilder M L, et al . Peroxisomal oxidases and catalase in liver and kidney homogenates of normal and di (ethylhexyl) phthalate-fed rats [J]. Int J Biochem, 1991, 23 (9): 961-967
63. Ryan B J,ó'Fágáin C. Arginine-to-lysine substitutions influence recombinant horseradish peroxidase stability and immobilization effectiveness [J]. BMC Biotechnology, 2007, 7 (1): 86-94
64. Saito H, Takcuchi Y, Takeda R et al. The core and complementary sequence for iological activity of the diapause hormone of the silkworm, Bombyx mori. Peptides, 1994, 15: 1173-1178
65. Sato Y, Oguchi M, Menjo N, et al. Precursor polyprotein for multiple neuropeptides secreted from the SG of the silk worm, Bombyx mori: Characterization of the cDNA encoding the diapause hormone precursor and identification of additional peptides. PNAS, 1993, 90(8): 3251-3255
66. Sawchyn WW, Church NS. The effects of temperature and photoperiod on diapause development in the eggs of four species of Lestes (Odonata: Zygoptera) [J]. Can J Zool, 1973, 51: 1257– 1265
67. Schneiderman HA, Williams GM. The physiology of insect diapauseⅫ: The respiratory metabolism of the cecropia silkworm during diapause and development [J]. Biol Bull, 1953, 105: 320-334
68. Singh R, Singh RK, Mahdi AA, et al. Circadian periodicity of plasma lipidperoxides and other anti-oxidants as putative markers in gynecological malignancies. In Vivo, 2003, 17(6): 593-600
69. Takeda N. Histophysiological studies on corpus allatum during prepupal diapause in Monema flavescens (Lepidop tera) [J]. Morph. 1977, 153: 245– 262
70. Tani N, Kamada G, Ochiai K, et al. Carbohydrate moiety of Time-Interval Measuring Enzyme regulates time measurement through its interaction with time-holding peptide PIN [J]. J Biol Chem, 2001, 129: 221–227
71. Tauber M J, Tauber CA. Seasonal Adaptations of Insects [M]. Landon: Oxford University Press, 1986, 202-207
72. Ti X, Tani N, Isobe M, Kai H. Time-measurement-regulating peptide PIN may alter a timer conformation of time interval measuring enzyme (TIME). Insect Physiology, 2006, 52: 461-467
73. Ti X, Tuzuki N, Tani N, et al. Demarcation of diapause development by cold and its relation so time-interval activation of TIME-ATPase in eggs of the silkworm, Bombyx mori [J]. Insect Physiol, 2004, 50: 1053-1064
74. Ti X, Tuzuki N, Tani N, et al. The peptide PIN changes the timing of transitory burst activation of timer-ATPase TIME in accordance with diapause development in eggs of the silkworm, Bombyx mori [J]. Insect Physiology, 2005, 51: 1025-1032
75. Tomás-Zapico C, Coto-Montes A, Martínez-Fraga J, et al. Effects of continuous light exposure on antioxidant enzymes, porphyric enzymes and cellular damage in the Harderian gland of the Syrian hamster. J Pineal Res, 2003, 34(1): 60-68
76. Grigorenko V, Chubar T, Kapeliuch Y, et al. New approaches for functional expression of recombinant horseradish peroxidase C in Escherichia coli [J]. Biocatalysis and Biotransformation, 1999, 17 (5): 359-379
77. Wei ZJ., Zhang QR., Kang L., et al. Molecular characterization and expression of prothoracicotropic hormone during development and pupal diapause in the cotton bollworm, Helicoverpa armigera [J]. Insect Physiology, 2005, 51: 691-700
78. Wheeler DA, Hamblen-Coyle MJ, Dushay MS, et al. Behavior in light-dark cycles of Drosophila mutants that are arrhythmic, blind, or both [J]. Biol Rhythms, 1993, 8(1): 67-94
79. Xu SQ, Kai H, Xu JL, et al. Changes of esterase A4-activity and its relationship with diapause in hot water treated eggs of silkworm, Bombyx mori [J]. Sericology, 2003, 43: 61-67
80. Xu Shi-Qing, Dai Xuan-Ying, Han Yi-Fei, et al. Research on diapause bioclock protein esterase A4 of silkworm, Bombyx mori. Sericology, 2001, 41(4) 543-549
81. Xu SQ, Kai H, Xu JL, et al. Changes of esterase A4-activity and its relationship with diapause in acid-treated eggs of silkworm, Bombyx mori. Science of Sericulture, 1998, 24: 95-99
82. Xu SQ, Kai H, Xu JL, et al. Target of hydrochloric acid in acid-treatment of diapausing eggs of Bombyx mori. Acta Entomologica Sinica, 2001, 44: 51-55
83. Xu WH, Sato Y, Ikeda M, et al. Stage-dependent and temperature-controlled expression of the gene encoding the precursor protein of DH-PBAN in the silkworm, Bombyx mori. J. Biol. Chem. 1995, 270: 3804-3808
84. Xu WH, Denlinger DL. Molecular characterization of prothoracicotropic hormone and diapause hormone in Heliothis virescens and a new role for diapause hormone. Insect Molecular Biology, 2003, 12(5): 509-516
85. Xue FS, Kallenborn HG. Summer and winter diapause in pupae of the cabbage butterfly, Pieris melete Ménétriés [J]. J Insect Physiol., 1997, 43: 701-707
86. Yamamoto K, Banno Y, Fujii H, Miake F, Kashige N, Aso Y, 2005. Catalase from the silkworm, Bombyx mori: Gene sequence, distribution, and overexp ression. Insect Biochem.Mol.B iol., 35: 277– 283
87. Yamashita O, Hasegawa K. Oocyte age sensitive to the diapause hormone from the standpoint of glycogen synthesis in the silkworm, Bombyx mori [J]. Insect Physiol ,1970, 16: 2377 - 2 383
88. Yamashita O, Yaginma T. Silkworm eggs at low temperature, implications for sericulture [M]. In: Lee R E. and Denlinger K L eds. Insect at low Temperature, New York: Chapman and Hall Press ,1991, 424– 445
89. Yamashita O, Hasegawa K. Studies on the mode of action of diapause hormone in the silkworm Bombyxmori L. IV. Effect of diapause hormone on the glycogen content in ovaries and the blood sugar level of silkworns. J Seric Sci. 1964, 33: 407– 416
90. Yamashita O, Suzuki K. Morphogenic hormones in arthropods (ed. Gupta, A.P.), New Brunswick: Rutger University Press, 1991, pp 82-128
91. Yamashita O. Diapause hormone of the silkworm Bombyx mori: structure gene expression and function [J]. Insect Physiology, 1996, 42, 669-679
92. Yin CM, Wang ZS, Chaw WD, et al. Brain neurosecretory cell and ecdysiotropin activity of the non-diapausing and diapausing D.Gradiosella [J]. J Insect Physiol., 1985, 31: 659-667
93. Yoshii T, Sakamoto M, Tomioka K. A temperature-dependent timing mechanism is involved in the circadian cycling period gene expression in Drosophila system that drives locomotor rhythms in the fruit fly Drosophila melanogaster [J]. Zoolog Sci, 2002, 19(8): 841-850
94. Yoshii T, Fujii K, Tomioka K. Induction of Drosophila behavioral and molecular circadian rhythms by temperature steps in constant light. Journal of Biological Rhythms, 2007, 22: 103-114
95. Young MW, Youngm W. Circadian rhythms-marking time for a kingdom [J]. Science, 2000, 288: 451-453
96. Yumoto I, Ichihashi D, Iwata H, et al. Purification and characterization of a catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1T exhibiting high catalase activity[J ] . J Bacteriol, 2000, 182 (7):1903-1909
97. Zámocky M, Koller F. Understanding the structure and function of catalase: Clues f rom molecular evolution and in vitro mutagenesis [J]. Progress in Biophysics and Molecular Biology, 1999, 72(1) :19-66
98. Zhang TY, Kang L, Zhang Z-F, et al. Identification of a POU factor involved in regulating the neuron-specific expression of the gene encoding DH-PBAN in Bombyx mori [J]. Biochem, 2004, 380: 255-263
99. Zhang TY, Sun JS, Kang L, et al. Structural analysis of the gene for DH-PBAN in Helicoverpa armigera and its transcriptional regulation. Biochim.Biophys. Acta. 2005, 1728 (1-2): 44-52
100. Zhang TY, Sun JS, Zhang QR et al. The DH-PBAN gene of Helicoverpaarmigera encodes multiple peptides that break, rather than induce, diapause. Insect Physiology, 2004,50(6): 547-554
101. Zhao, LC, Sima, YF, Shen, XM, et al. Metabolism of hydrogen peroxide in the course of embryonic development in silkworm. Dev. Reprod. Biol., 1999, 8, 41-48
102.陈杨,刘望.超氧化物歧化分构看其分化[J].生物化与生物物展,1996,23(5):408-413
103.陈田飞,灵.育究况[J].业,2004,41(3):12-16
104.海,徐,马杨虎,等.野桑超氧化物歧化因隆分与核表达[J].业科,2007,33(1): 234-240
105.段,翠,贺,等.血淋巴超氧化物歧化究.业科,1995,2 (2): 102-105
106.丁道远,胡汉,朴.荧谱法究某些物与DNA相作[J].谱与谱分1990,10:24
107.侯月利,花蕾.昆育诱因[J].陕业科技,2000,3: 62-67
108.胡恕,张.血中超氧化物歧化(SOD)究[J].业科,1996,22(4):338-340
109.黄伟,于涟.生物制究展[J].生科,2000,12(1): 10-13
110.蒋明星,谢,张羲.棉育诱性[J].应生态报, 1999,10(1): 60-62
111.玺,刘继兰,王选年,等.超氧化物歧化究应展[J].物医展,2007,28 (7) :70-75
112.少琴,黄.贻贝超氧化物歧化纯化部分性质究[J].天产物究与开发,1999,11 (4): 25-29
113.刘惠霞,新岗,吴.昆生物化[M]. :陕科技术版,1998
114.鲁,张英,王赉.淡色库生育经内分泌调节[J].昆报,1993,36(3): 302-307
115.萨姆鲁J,拉塞尔DW.分隆实验南(第三版)[M].北京:科版. 2002
116.山兴亚.激,别是于育激作.科技术,1969, 8(1):72-78
117.宋艳.育诱过程中近日节因period timless表达究[D].苏:苏大,2009
118.田荟.血中过氧化氢取性质究[J].食品科,2006,27 (12): 311-314
119.王.大鼠松体生物因昼表达以外血淋巴细胞因筛选与鉴[C].论,2007,5
120.王英,马杨虎,宋艳,等.昆生物因其作制究展[J].江苏业,2008,30(1): 9-14
121.王英,徐,马杨虎,等.育生物白质EA4因构功能究[C].第届中际绸会议中会第五届青年术讨会论,苏,2007,9: 366-380
122.王,徐,马杨虎,等.育生物白质EA4因cDNA隆分[J].业科,2007,33(1): 36-42
123.王志华,良.过氧化氢其模拟物究展[J] .杭师范院报(科版), 2006 , 5 (6) :465-468
124.吴明,予元.棉育诱因究[J].物保报., 1995,22(4): 331-336
125.吴,徐俊良等.血过氧化氢活力其与体抗逆性系[J].昆报,1998,41(2): 124-129.
126.谢,蒋明星,张羲.烟青育征究[J].物保报., 1997,24(3): 199-203
127.徐丽,宋艳,苏,等.育诱度与对近日节因per表达响[J].业科,2010,36(1):52-59
128.徐,戴璇,等.育生物白质酯A4究展[J].通讯,2000b,20(4): 9-15
129.徐,甲斐英则,徐俊良,等.育生物白质EA4纯化其分构分[J].中生物化与分生物报,1999a,15(2): 241-246
130.徐,甲斐英则,徐俊良,等.体外酸处与PIN对酯A4活性响系[J].业科,1999c,25(3): 149-153
131.徐,甲斐英则,徐俊良,等.酸处卵酯A4活性变化其与育性系[J].业科,1998,24(2): 95-99
132.徐,甲斐英则,徐俊良,等.酸激量对卵酯A4活性响其与卵活化系[J].业科,2000a,26(1): 20-26
133.徐,蒋元,戴璇,等.环对越年卵催青期过氧化氢代谢响[J].江苏业, 2005,27(1): 15-17
134.徐,马杨虎,陈息,等.卵汤处酯A4活性变化[J].业科, 2004,30(1): 50-54
135.徐,琴,甲斐英则,徐俊良,郑必,陈息,马杨虎.体外25℃卵酯A4 ATP活性变化其与育性系[J].业科, 2000c, 26(3): 140-145
136.徐,郑必,马杨虎,等.育性卵酸孵化法酸作靶物[J].昆报,2001,44(1): 51-55
137.徐,陈息,卫,等.桑卵孵化制法[P].中:ZL200510041062.8. 2005-12-28
138.徐,茂顶.生产操作技术南[M].北京:中科技术版,1993, 14-29
139.徐.桑育性卵酸孵化论究[D].江农业大,1997,63-67
140.徐卫华,佐洋,山兴亚.育激因隆[J].传报, 1995b,22(3): 178-184
141.徐卫华.育分[J].传报,1999,26(2): 107-111
142.徐卫华.育激-息成激活肽因表达调节[J].中生物化分生物报.,1998,14(5): 557-561
143.押部秀二,1943.实验, ,247
144.川,时连.育性卵中辅Ⅰ辅Ⅱ量变化[J].业科,2008,34(3): 447-452
145.川,兵,等.微感染对过氧化氢代谢响[J].通讯,1998,18(2):15-17
146.川,马杨虎,兵,等.即时浸酸对胎发育中H2O2代谢响[J].业科,2000,26(4):268-270
147.川,姚祥,兵.变态过程中过氧化氢代谢点外源激对其调节作[J].通讯,1999, 19(3):5-8
148.川,姚祥.五过氧化氢代谢.南京师大报, 1997, 20(4):128-130
149.川.过氧化氢高催青期水处卵孵化[J].业科., 2004, 30(4): 440-442
150.江农业大编.良繁育与育[M],北京:中农业版,1981,33
151.郑必,徐,徐孟奎,等.蓖麻育与过氧化氢代谢系[J].业科,2006b,32(2): 221-225
152.郑荣梁,1993.生科中由究鸟瞰.由生科展1:5~13
153.苏.生物因Bm-cry隆育诱发环对其表达响[D].苏:苏大,2009