转基因抗虫、耐除草剂低酚棉近等基因系生理特性与蛋白表达差异及抗虫杂交棉生理与产量性状的遗传分析
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摘要
虫害和草害是制约棉花生产的两大重要因素,防治虫害和杂草的用工投入大,而培育抗虫、耐除草剂棉花新品种将有助于进一步简化棉花栽培体系,降低植棉投入,提高棉农收入具有重要意义。低酚棉的诞生使棉花成为“棉、油、粮、饲”四位一体的新型作物,但低酚棉因棉酚和其它萜醛缺乏而易遭虫害。因此,培育转基因抗虫和耐除草剂低酚棉品种对发展低酚棉生产具有重要的现实意义。转基因抗虫杂交棉因兼具抗虫高产优质等特点得到广泛应用,因此加速选育新的转基因抗虫杂交棉也成为热点。基于棉花生长变化和遗传发育,结合生理生化指标评估外源基因导入对低酚棉品系影响以及为选育潜力亲本和杂交组合提供生育期筛选指标。本研究通过分析比较转Bt基因抗虫低酚棉和转1EPsPS-G6基因低酚棉近等基因系在生育、生理代谢上的差异,以期从形态学、生理学及蛋白表达谱方面探讨外源基因导入对低酚棉农艺与生理性状及蛋白表达谱的影响。另外,利用转基因抗虫棉和常规品种(系)进行双列杂交研究杂种F1优势利用潜力,通过多变量条件分析来评估生理生化性状对皮棉产量贡献,寻找影响皮棉产量的关键生理生化指标,以期为抗虫棉的杂种优势利用和新品种培育提供理论依据及早期筛选指标。主要研究结果如下：
1.转Bt基因低酚棉近等基因系生理生化性状与蛋白表达差异
以一对低酚棉近等基因系为材料(仅cry1Ac差异),分析比较生育、生理代谢的差异,结果表明,与其受体相比,转Bt低酚品系有较高的株高和有效结铃率,内围铃较多；单株结铃数、单铃重和单株产量较低。与其受体相比,转Bt低酚叶绿素含量和蒸腾速率较低、Chl a/b、初始荧光(Fo)和最大荧光(Fm)较高。转Bt低酚结铃期可溶性蛋白显著高于其受体,但初花期低于其受体；初花期Ca、Mg、Cu、Zn、Mn及Fe,结铃期P和Cu显著高于其受体,但初花期P、K和B,结铃期K.S.Zn及Fe显著低于其受体；结铃期转Bt低酚系膜脂过氧化产物丙二醛(MDA)、初花结铃期超氧化物歧化酶(SOD)均显著低于其受体。蛋白质组分析在转Bt低酚系与其受体之间检测到20个显著差异蛋白点,其中4个上调蛋白点,分别属于信号转导、能量代谢、防御响应；另有16个下调点,可归结为信号转导和蛋白合成代谢。
2.转基因耐除草剂低酚棉近等基因系生理生化性状与蛋白表达差异
以一对低酚棉近等基因系为材料(仅EPSPS-G6差异),分析比较光合特性、矿质元素吸收、活性氧代谢及蛋白表达谱差异,结果表明,与其受体相比,转耐除草剂低酚棉转5629结铃期显示高净光合速率和低蒸腾速率,始絮期高气孔导度、胞间CO2浓度及蒸腾速率；结铃期和始絮期叶绿素a、b及a+b含量显著低于其受体。.EPSPS基因导入改变了元素营养吸收：与其受体相比,转5629N、Mg及K含量较低,P、Ca、Fe、K、Cu、Mn及Zn含量较高。结铃期和始絮期可溶性糖含量、盛花结铃期可溶性蛋白含量显著高于其受体,但盛花期可溶性糖和蕾期可溶性蛋白低于其受体；与其受体相比,转5629盛花始絮期SOD和过氧化氢酶(CAT)活性较高、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性较低；除盛花期外,其它三个时期转5629MDA含量显著高于其受体；蛋白质表达谱分析结果显示,转5629与其受体之间共检测到11个差异蛋白点,其中7个蛋白点上调、4个下调。转5629显示上调了核酮糖-1,5-二磷酸羧化酶/加氧酶(RuBisCO)大亚基、CP4EPSPS及ATP合成酶,下调了谷氨酸-1-半醛转氨酶-2,1氨基酸变位酶以及锰稳定蛋白。
3.转Bt抗虫棉与常规棉不完全双列杂交生理生化与产量性状的遗传效应分析
2010年以6个常规陆地棉品系(P1-6)和3个转基因抗虫棉品系(P7-9)为亲本进行不完全双列杂交设计配置16个组合,分析测定生理生化、农艺与纤维品质性状,以AD模型和条件分析方法进行数据分析,结果表明,苗蕾期和初花期功能叶Chl a和Chl b含量可以考虑用于F1组合选择,盛花始絮期可用选择亲本：苗蕾、初花期和始絮期功能叶C/N可以用于筛选F1组合,盛花期选择亲本指标：初花盛花始絮期功能叶N含量以显性为主,可以用于选择F1组合；P和K含量四个时期均以显性为主,可以用于选择F1组合；抗氧化酶系(SOD、POD、APX及CAT)及MDA含量可考虑用于选择F1组合。盛花期K含量表现正向极显著超亲优势。选择Chl a、苗蕾初花盛花期C/N和N、初花盛花始絮期K加性效应高的亲本有利于获得皮棉产量高的F1杂交组合。单株总铃数、衣分、麦克隆值及纤维长度主要受加性效应控制,可以通过选择纯系来改良。皮棉产量的加性贡献率主要来自衣分,显性贡献率最大的是单铃重。衣分对比强度、麦克隆值及纤维长度的加性贡献率最高,表明选择衣分高的亲本可以提高后代纤维主要品质。亲本1(P1,31)可以作为常规棉亲本来提高后代皮棉产量和纤维品质,亲本8(P8,B7抗)可以作为转Bt基因亲本来提高后代衣分和皮棉产量。16个杂交组合中,皮棉产量正向达显著或极显著显性效应的组合有8个：H18(P1×P8)、H28(F2×P8),H29(P2×P9),H37(P3×P7),H48(P4×P8)、H59(P5×P9)、H67(P6×P7)及H69(P6×P9)。
4.转Bt抗虫棉与常规棉双列杂交生理生化与产量性状的遗传效应分析
2011年以4个常规陆地棉品系和5个转基因抗虫棉品系为亲本进行NC Ⅱ设计配置20个组合,遗传分析表明,参试的12个生理生化指标三个时期均以显性效应为主；杂交组合K含量均有显著的平均优势和超亲优势；C/N对皮棉产量在三个时期均有显著的加性贡献率。果枝数、铃数、铃重及皮棉产量主要受加性效应控制,可以通过选择纯系来改良。单株总铃数加性贡献率主要来自外围铃和中下部铃,而显性贡献主要来自于外围铃和中部铃。皮棉产量的加性贡献率主要来自铃数和衣分,显性贡献率主要来茎粗、株高和铃重。衣分对比强度的加性贡献率最大,株高和铃数对麦克隆值的加性贡献率最高,茎粗对纤维长度的加性贡献率最大。P3(慈96-21)可以作为常规棉亲本来提高后代的皮棉产量,P9(黄岗抗)可以作为转Bt基因亲本来提高后代的皮棉产量。20个杂交组合中,皮棉产量正向达显著或极显著显性效应的组合有：H39、H16、H35、H28、H47、H45。
5.转Bt基因抗虫杂棉与常规棉正反交生理生化与产量性状的遗传效应分析
常规陆地棉品系(慈-ZJ6,P1)和5个转基因抗虫棉品系(外4、B9抗、E29、太抗、黄岗抗)进行正反交,遗传分析表明,Chl a初花始絮期以加性为主,盛花期以显性为主；Chl b初花以显性为主,盛花始絮以加性为主；Ch1a/b三个时期均以加性为主；N初花盛花以加性为主,始絮期以显性为主；P初花始絮期以显性为主,盛花以加性为主；K初花以显性为主,盛花始絮以加性为主；C/N初花始絮以加性为主,盛花以显性为主。抗氧化酶系和MDA三个时期均以加性为主。F1杂交组合参试12个生理生化指标在三个时期均有不同程度显著的平均优势,但没有正向显著的超亲优势。茎粗、外围铃数、上部铃数、单株铃重、衣分、纤维长度、麦克隆值及纤维比强度主要受加性效应控制,可以通过选择纯系来改良。对于亲本而言,铃数加性方差比率主要来自上部和外围铃。对于杂交组合而言,铃数加性方差比率主要来自外围铃及中下部铃。皮棉产量的加性贡献率主要来果枝数、茎粗、上部铃及衣分。衣分对比强度和麦克隆值的加性贡献率最大。常规棉亲本P1具有衣分、单铃重、纤维长度、比强度极显著加性效应,可以用来改良后代的衣分、单铃重、纤维长度、比强度,P5(太抗)可以作为转Bt基因亲本来提高后代的果枝数和单株总铃数,从而获得正向皮棉产量。P4(E29)可以作为转Bt基因亲本来提高后代的单株总铃数和纤维长度,也获得正向皮棉产量。10个杂交组合中,皮棉产量的正向达显著或极显著显性效应的组合有：H13、H15及H41。正反交组合中,H15和H51茎粗、纤维长度有平均优势,H12和H21有株高、内围铃、衣分平均优势,H13和H31有衣分平均优势,H16和H61有单铃重平均优势,说明常规亲本P1和5个抗虫亲本正反交存在极显著差异,其中以常规亲本P1为母本,抗虫亲本为父本的组合明显高于反交组合,说明转基因抗虫棉正交在农艺产量组分纤维品质性状方面存在细胞质遗传。
Insect and weeds are the two major factors affecting cotton production. It is one of cost-effective methods to breed insect-resistance and herbicide-tolerant cotton cultivars using transgenic methods. The development of glandless cotton makes upland cotton becoming a new economic crop with multi-utilization of fiber, oil, food and feed. However, glandless cotton is more susceptible to pests than normal cultivars due to the absence of gossypol and other terpenoid aldehydes. Therefore, breeding Bt transgenic glandless cotton is benifical and a cost effective approach for its commercial utilization. Transgenic Bt hybrid cotton is widely used with the feature of insect-resistance, high yield and good quality. Based on the change of cotton growth and development, researches are still limited concerning the use of biochemical parameters to assess exogenous genes effects on glandless cotton lines and screen the promising parents and crosses. The present work was carried out by comparing two pairs of near-isogenic lines (NILs) of Bt and non-Bt and EPSPS and non-EPSPS glandless upland cotton in mineral uptake, photosynthetic performance, active oxygen metabolism and protein expression. In addition, diallel crosses using conventional upland cotton lines with transgenic Bt upland cotton lines were conducted to analyze their difference in phenotypic and genetic contribution ratios of biochemical traits to lint yield and the heterosis of yield, yield components and fiber properties and to further screen the promising parents and hybrids among Bt hybrid crosses and their parents lines. The main results are as follows:
1. Agronomic and biochemical traits and protein expression were compared using a pair of near-isogenic lines (NILs) of glandless upland cotton differing in only by the presence of cryIAc gene (non-Bt variety-Zhong5629vs its Bt-transgenic near-isoline). The results showed that Bt isoline had higher plant height and rate of effective bolls, and more internal bolls than non-Bt isoline, while being lower in yield and number of bolls. Physiological analyses indicated that Bt isoline had lower chlorophyll contents and transpiration rate relative to non-Bt isoline, but higher in Ch1a/b, F0and Fm, and no significant difference in net photosynthetic rate (Pn), intercellular CO2concentration and stomatal conductance, and Fv/Fm. Content of soluble protein in Bt isoline was significantly higher at boll setting stage (BSS) but lower at initial flowering stage (IFS) compared with non-Bt isoline. The concentrations of Ca, Mg, Cu, Zn, Mn, and Fe at IFS, P and Cu at BSS in Bt isoline were significantly higher than those in non-Bt isoline, but reversely P, K and B at IFS, K, S, Zn, and Fe at BSS, respectively. Malonaldehyde (MDA) content at BSS and superoxide dismutase (SOD, EC1.15.1.1) activity at IFS and BSS were lower in Bt-isoline.2-DE analysis of the two NILs detected20differentially expressed protein spots, with4and16being up-and down-regulated in Bt vs. non-Bt isoline, respectively. These proteins were attributed to protein metabolism, defense response, transcription, energy metabolism, and cell structure. Among them, a core-neofusion protein spot as a selectable marker and neomycin/kanamycin resistance was specific expressed in Bt isoline.
2. Physiological traits and proteomic expression were compared between EPSPS-G6transgenic glyphosate-tolerant glandless upland cotton (GT) and its non-transgenic wild type (non-GT, cv. Zhong5629). GT showed higher net photosynthetic rate at BSS than non-GT, with higher stomatal conductance (gs), intercellular CO2concentration (Ci) and transpiration rate (Tr) at the beginning of boll open stage (BBOS), but lower in Ci at BSS. Contents of Ch1a, Ch1b and Ch1a+b in GT were uniformly significantly less than those in non-GT at BSS and BBOS. Insertion of EPSPS gene had altered uptake of mineral nutrients:GT demonstrated less concentrations in N, Mg and K, but higher in P, Ca, Fe, K, Cu, Mn and Zn compared with non-GT. Contents of soluble sugar at BSS and BBOS and soluble protein at PFS and BSS in GT were significantly higher than non-GT; but GT had lower soluble sugar content at PFS and soluble protein at squaring stage (SS). GT recorded significantly higher activities in superoxide dismutase and catalase, but lower in peroxidase and ascorbate peroxidase compared with non-GT. Proteomic alteration in leaves of GT vs. non-GT was analyzed using2-DE coupled with mass spectrometry. Eleven differentially expressed proteins were identified, of which7and4spots being up-and down-regulated, respectively. GT showed up-regulated expression of RuBisCO large subunit, CP4EPSPS, and ATP synthase, but down-regulated glutamate-1-semialdehyde-2,1-aminomutase and manganese-stabilising protein, respectively.
3. To examine the genetic contributions of biochemical parameters to lint yield at both antioxidant enzymatic activity and nutrient levels and to determine the major indicator traits for indirect selection on lint yield in Bt hybrid cotton lines, multivariable conditional analysis was conducted using a additive-dominance model. Six conventional upland cotton lines were crossed with3transgenic Bt gene lines using diallel mating design method. Nine breeding lines and derived16F1crosses were grown at the farmland of Cixi cotton research institute in Zhejiang province, China, in2010. Biochemical parameters at different developmental stages were analyzed, agronomic and economic traits were investigated and fiber quality characters were surveyed. Chi a and Chi b at SS and IFS, C/N raton at IFS and BBOS could be used as indicative physiological traits for selecting F1crosses, while Chi a and Chi b at full flowering stage (FFS) and BBOS, C/N raton at FFS for parents lines. N content at IFS, FFS, and BBOS, P and K contents at four stages were mainly affected by dominant effects, which could be used to choose F1crosses. Similarity, Parent's lines have high additive effects in Chi a content, in C/N ratio and N content at SS, IFS and FFS, in K at IFS, FFS and BBOS, their hybrids F1have more lint yield. Bolls per plant, lint percent, micronarie and fiber length were mainly affected by additive effects, which suggested they could be improved by choosing pure lines. Additive effects contribution of lint yield was mainly from lint percent, while dominant effects contribution of lint yield from boll weight. Having high contribution ratio of additive effects to fiber strength, micronarie and fiber length, lint percent in parents could be used as indicative traits for selecting fiber quality of F1crosses. In short, Parent1(31) is an acceptable non-Bt line and parent8(B7kang) is an excellent Bt transgenic cotton with insect-resistance. Among16F1crosses, there are8F1crosses (H18, H28, H29, H37, H48, H59, H67and H69) with positive and significant lint yield.
4. Four conventional upland cotton lines with five transgenic line using NCII design, and their20F1crosses were grown at the same location above in2011. genetic effects analysis showed that12biochemical parameters at three stages were mainly affected by dominant effects. K content in F1crosses at three stages had significant mean and better parents heterosis. C/N ratio had significant additive contribution ratio to lint yield at three stages. Branches, bolls, boll weight and lint yield were mainly controlled by additive effects, which suggested they could be improving by choosing pure lines. Additive variance contribution ratio of bolls for parents was mainly from external, middle and lower bolls on the plants, while for F1crosses from external and middle bolls. Additive contribution ratio of lint yield was from bolls and lint percent, while dominant contribution ratio from diameter, plant height and boll weight. Lint percent to fiber strength, plant height and bolls to micronarie, diameter to fiber length had largest additive contribution, respectively. In short, Parent3(Ci96-21) is an acceptable non-Bt line and parent9(huanggang kang) is an excellent Bt transgenic cotton with insect-resistance. Among20F1crosses, there are6F1crosses (H39, H16, H35,H28, H47, and H45) with positive and significant lint yield.
5. Reciprocal crosses between one conventional upland cotton line (Ci-ZJ6) and five transgenic Bt gene lines was made to produce10F1hybrid crosses and all lines planted in2011at the same location above. Genetic analysis showed that Chl a at IFS and BBOS, Chl b at FFS and BBOS, Chl a/b ratio at three stages, N at IFS and FFS, P at FFS, K at FFS and BBOS, C/N ratio at IFS and BBOS, anti-oxidant enzyme systems (SOD, POD, CAT, APX) and MDA, were mainly affected by additive effects. While Chl a at FFS, Chl b at IFS, N at BBOS, P at IFS and BBOS, K at IFS, C/N ratio at FFS, were mainly affected by dominant effects. All12biochemical parameters in F1crosses had significant mean parents heterosis at three stages, but no positive and significant better parents heterosis. Diameter, external bolls, middle bolls, boll weight, lint percent, fiber length, micronaire and fiber strength were mainly affected by additive effects, which suggested those traits could be improved by choosing pure lines. Bolls additive variance ratio for parents was mainly from top and external bolls on the plants, while for F1crosses from central, lower, and external bolls. Additive contribution ratio of lint yield was mainly from branches, diameter, up bolls and lint percent. Lint percent to fiber strength and micronaire had largest additive contribution. Having significant additive effects in lint percent, boll weight, and fiber length and fiber strength. Parent1(Ci-ZJ6) is an acceptable non-Bt line. Parent4(E29) with better performance in bolls, fiber length and positive lint yield, and parent5(Tai kang) with more branches, bolls and positive lint yield, are excellent Bt transgenic cotton with insect-resistance. Among10F1crosses, there are3F1crosses (H13, H15and H41) with positive and significant lint yield. Among reciprocal crosses, H15and H51in diameter and fiber length, H12and H21in plant height, internal bolls and lint percent, H13and H31in lint percent had mean parents heterosis, respectively, which suggested that there were significant differences between conventional parent P1and five transgenic Bt cotton lines. Moreover, the overall performance in the crosses with female parent P1was better than in the crosses with female parents transgenic Bt cotton lines, which confirmed the possible existance of cytoplasmic inheritance in transgenic Bt hybrid lines.
1.转Bt基因低酚棉近等基因系生理生化性状与蛋白表达差异
以一对低酚棉近等基因系为材料(仅cry1Ac差异),分析比较生育、生理代谢的差异,结果表明,与其受体相比,转Bt低酚品系有较高的株高和有效结铃率,内围铃较多；单株结铃数、单铃重和单株产量较低。与其受体相比,转Bt低酚叶绿素含量和蒸腾速率较低、Chl a/b、初始荧光(Fo)和最大荧光(Fm)较高。转Bt低酚结铃期可溶性蛋白显著高于其受体,但初花期低于其受体；初花期Ca、Mg、Cu、Zn、Mn及Fe,结铃期P和Cu显著高于其受体,但初花期P、K和B,结铃期K.S.Zn及Fe显著低于其受体；结铃期转Bt低酚系膜脂过氧化产物丙二醛(MDA)、初花结铃期超氧化物歧化酶(SOD)均显著低于其受体。蛋白质组分析在转Bt低酚系与其受体之间检测到20个显著差异蛋白点,其中4个上调蛋白点,分别属于信号转导、能量代谢、防御响应；另有16个下调点,可归结为信号转导和蛋白合成代谢。
2.转基因耐除草剂低酚棉近等基因系生理生化性状与蛋白表达差异
以一对低酚棉近等基因系为材料(仅EPSPS-G6差异),分析比较光合特性、矿质元素吸收、活性氧代谢及蛋白表达谱差异,结果表明,与其受体相比,转耐除草剂低酚棉转5629结铃期显示高净光合速率和低蒸腾速率,始絮期高气孔导度、胞间CO2浓度及蒸腾速率；结铃期和始絮期叶绿素a、b及a+b含量显著低于其受体。.EPSPS基因导入改变了元素营养吸收：与其受体相比,转5629N、Mg及K含量较低,P、Ca、Fe、K、Cu、Mn及Zn含量较高。结铃期和始絮期可溶性糖含量、盛花结铃期可溶性蛋白含量显著高于其受体,但盛花期可溶性糖和蕾期可溶性蛋白低于其受体；与其受体相比,转5629盛花始絮期SOD和过氧化氢酶(CAT)活性较高、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性较低；除盛花期外,其它三个时期转5629MDA含量显著高于其受体；蛋白质表达谱分析结果显示,转5629与其受体之间共检测到11个差异蛋白点,其中7个蛋白点上调、4个下调。转5629显示上调了核酮糖-1,5-二磷酸羧化酶/加氧酶(RuBisCO)大亚基、CP4EPSPS及ATP合成酶,下调了谷氨酸-1-半醛转氨酶-2,1氨基酸变位酶以及锰稳定蛋白。
3.转Bt抗虫棉与常规棉不完全双列杂交生理生化与产量性状的遗传效应分析
2010年以6个常规陆地棉品系(P1-6)和3个转基因抗虫棉品系(P7-9)为亲本进行不完全双列杂交设计配置16个组合,分析测定生理生化、农艺与纤维品质性状,以AD模型和条件分析方法进行数据分析,结果表明,苗蕾期和初花期功能叶Chl a和Chl b含量可以考虑用于F1组合选择,盛花始絮期可用选择亲本：苗蕾、初花期和始絮期功能叶C/N可以用于筛选F1组合,盛花期选择亲本指标：初花盛花始絮期功能叶N含量以显性为主,可以用于选择F1组合；P和K含量四个时期均以显性为主,可以用于选择F1组合；抗氧化酶系(SOD、POD、APX及CAT)及MDA含量可考虑用于选择F1组合。盛花期K含量表现正向极显著超亲优势。选择Chl a、苗蕾初花盛花期C/N和N、初花盛花始絮期K加性效应高的亲本有利于获得皮棉产量高的F1杂交组合。单株总铃数、衣分、麦克隆值及纤维长度主要受加性效应控制,可以通过选择纯系来改良。皮棉产量的加性贡献率主要来自衣分,显性贡献率最大的是单铃重。衣分对比强度、麦克隆值及纤维长度的加性贡献率最高,表明选择衣分高的亲本可以提高后代纤维主要品质。亲本1(P1,31)可以作为常规棉亲本来提高后代皮棉产量和纤维品质,亲本8(P8,B7抗)可以作为转Bt基因亲本来提高后代衣分和皮棉产量。16个杂交组合中,皮棉产量正向达显著或极显著显性效应的组合有8个：H18(P1×P8)、H28(F2×P8),H29(P2×P9),H37(P3×P7),H48(P4×P8)、H59(P5×P9)、H67(P6×P7)及H69(P6×P9)。
4.转Bt抗虫棉与常规棉双列杂交生理生化与产量性状的遗传效应分析
2011年以4个常规陆地棉品系和5个转基因抗虫棉品系为亲本进行NC Ⅱ设计配置20个组合,遗传分析表明,参试的12个生理生化指标三个时期均以显性效应为主；杂交组合K含量均有显著的平均优势和超亲优势；C/N对皮棉产量在三个时期均有显著的加性贡献率。果枝数、铃数、铃重及皮棉产量主要受加性效应控制,可以通过选择纯系来改良。单株总铃数加性贡献率主要来自外围铃和中下部铃,而显性贡献主要来自于外围铃和中部铃。皮棉产量的加性贡献率主要来自铃数和衣分,显性贡献率主要来茎粗、株高和铃重。衣分对比强度的加性贡献率最大,株高和铃数对麦克隆值的加性贡献率最高,茎粗对纤维长度的加性贡献率最大。P3(慈96-21)可以作为常规棉亲本来提高后代的皮棉产量,P9(黄岗抗)可以作为转Bt基因亲本来提高后代的皮棉产量。20个杂交组合中,皮棉产量正向达显著或极显著显性效应的组合有：H39、H16、H35、H28、H47、H45。
5.转Bt基因抗虫杂棉与常规棉正反交生理生化与产量性状的遗传效应分析
常规陆地棉品系(慈-ZJ6,P1)和5个转基因抗虫棉品系(外4、B9抗、E29、太抗、黄岗抗)进行正反交,遗传分析表明,Chl a初花始絮期以加性为主,盛花期以显性为主；Chl b初花以显性为主,盛花始絮以加性为主；Ch1a/b三个时期均以加性为主；N初花盛花以加性为主,始絮期以显性为主；P初花始絮期以显性为主,盛花以加性为主；K初花以显性为主,盛花始絮以加性为主；C/N初花始絮以加性为主,盛花以显性为主。抗氧化酶系和MDA三个时期均以加性为主。F1杂交组合参试12个生理生化指标在三个时期均有不同程度显著的平均优势,但没有正向显著的超亲优势。茎粗、外围铃数、上部铃数、单株铃重、衣分、纤维长度、麦克隆值及纤维比强度主要受加性效应控制,可以通过选择纯系来改良。对于亲本而言,铃数加性方差比率主要来自上部和外围铃。对于杂交组合而言,铃数加性方差比率主要来自外围铃及中下部铃。皮棉产量的加性贡献率主要来果枝数、茎粗、上部铃及衣分。衣分对比强度和麦克隆值的加性贡献率最大。常规棉亲本P1具有衣分、单铃重、纤维长度、比强度极显著加性效应,可以用来改良后代的衣分、单铃重、纤维长度、比强度,P5(太抗)可以作为转Bt基因亲本来提高后代的果枝数和单株总铃数,从而获得正向皮棉产量。P4(E29)可以作为转Bt基因亲本来提高后代的单株总铃数和纤维长度,也获得正向皮棉产量。10个杂交组合中,皮棉产量的正向达显著或极显著显性效应的组合有：H13、H15及H41。正反交组合中,H15和H51茎粗、纤维长度有平均优势,H12和H21有株高、内围铃、衣分平均优势,H13和H31有衣分平均优势,H16和H61有单铃重平均优势,说明常规亲本P1和5个抗虫亲本正反交存在极显著差异,其中以常规亲本P1为母本,抗虫亲本为父本的组合明显高于反交组合,说明转基因抗虫棉正交在农艺产量组分纤维品质性状方面存在细胞质遗传。
Insect and weeds are the two major factors affecting cotton production. It is one of cost-effective methods to breed insect-resistance and herbicide-tolerant cotton cultivars using transgenic methods. The development of glandless cotton makes upland cotton becoming a new economic crop with multi-utilization of fiber, oil, food and feed. However, glandless cotton is more susceptible to pests than normal cultivars due to the absence of gossypol and other terpenoid aldehydes. Therefore, breeding Bt transgenic glandless cotton is benifical and a cost effective approach for its commercial utilization. Transgenic Bt hybrid cotton is widely used with the feature of insect-resistance, high yield and good quality. Based on the change of cotton growth and development, researches are still limited concerning the use of biochemical parameters to assess exogenous genes effects on glandless cotton lines and screen the promising parents and crosses. The present work was carried out by comparing two pairs of near-isogenic lines (NILs) of Bt and non-Bt and EPSPS and non-EPSPS glandless upland cotton in mineral uptake, photosynthetic performance, active oxygen metabolism and protein expression. In addition, diallel crosses using conventional upland cotton lines with transgenic Bt upland cotton lines were conducted to analyze their difference in phenotypic and genetic contribution ratios of biochemical traits to lint yield and the heterosis of yield, yield components and fiber properties and to further screen the promising parents and hybrids among Bt hybrid crosses and their parents lines. The main results are as follows:
1. Agronomic and biochemical traits and protein expression were compared using a pair of near-isogenic lines (NILs) of glandless upland cotton differing in only by the presence of cryIAc gene (non-Bt variety-Zhong5629vs its Bt-transgenic near-isoline). The results showed that Bt isoline had higher plant height and rate of effective bolls, and more internal bolls than non-Bt isoline, while being lower in yield and number of bolls. Physiological analyses indicated that Bt isoline had lower chlorophyll contents and transpiration rate relative to non-Bt isoline, but higher in Ch1a/b, F0and Fm, and no significant difference in net photosynthetic rate (Pn), intercellular CO2concentration and stomatal conductance, and Fv/Fm. Content of soluble protein in Bt isoline was significantly higher at boll setting stage (BSS) but lower at initial flowering stage (IFS) compared with non-Bt isoline. The concentrations of Ca, Mg, Cu, Zn, Mn, and Fe at IFS, P and Cu at BSS in Bt isoline were significantly higher than those in non-Bt isoline, but reversely P, K and B at IFS, K, S, Zn, and Fe at BSS, respectively. Malonaldehyde (MDA) content at BSS and superoxide dismutase (SOD, EC1.15.1.1) activity at IFS and BSS were lower in Bt-isoline.2-DE analysis of the two NILs detected20differentially expressed protein spots, with4and16being up-and down-regulated in Bt vs. non-Bt isoline, respectively. These proteins were attributed to protein metabolism, defense response, transcription, energy metabolism, and cell structure. Among them, a core-neofusion protein spot as a selectable marker and neomycin/kanamycin resistance was specific expressed in Bt isoline.
2. Physiological traits and proteomic expression were compared between EPSPS-G6transgenic glyphosate-tolerant glandless upland cotton (GT) and its non-transgenic wild type (non-GT, cv. Zhong5629). GT showed higher net photosynthetic rate at BSS than non-GT, with higher stomatal conductance (gs), intercellular CO2concentration (Ci) and transpiration rate (Tr) at the beginning of boll open stage (BBOS), but lower in Ci at BSS. Contents of Ch1a, Ch1b and Ch1a+b in GT were uniformly significantly less than those in non-GT at BSS and BBOS. Insertion of EPSPS gene had altered uptake of mineral nutrients:GT demonstrated less concentrations in N, Mg and K, but higher in P, Ca, Fe, K, Cu, Mn and Zn compared with non-GT. Contents of soluble sugar at BSS and BBOS and soluble protein at PFS and BSS in GT were significantly higher than non-GT; but GT had lower soluble sugar content at PFS and soluble protein at squaring stage (SS). GT recorded significantly higher activities in superoxide dismutase and catalase, but lower in peroxidase and ascorbate peroxidase compared with non-GT. Proteomic alteration in leaves of GT vs. non-GT was analyzed using2-DE coupled with mass spectrometry. Eleven differentially expressed proteins were identified, of which7and4spots being up-and down-regulated, respectively. GT showed up-regulated expression of RuBisCO large subunit, CP4EPSPS, and ATP synthase, but down-regulated glutamate-1-semialdehyde-2,1-aminomutase and manganese-stabilising protein, respectively.
3. To examine the genetic contributions of biochemical parameters to lint yield at both antioxidant enzymatic activity and nutrient levels and to determine the major indicator traits for indirect selection on lint yield in Bt hybrid cotton lines, multivariable conditional analysis was conducted using a additive-dominance model. Six conventional upland cotton lines were crossed with3transgenic Bt gene lines using diallel mating design method. Nine breeding lines and derived16F1crosses were grown at the farmland of Cixi cotton research institute in Zhejiang province, China, in2010. Biochemical parameters at different developmental stages were analyzed, agronomic and economic traits were investigated and fiber quality characters were surveyed. Chi a and Chi b at SS and IFS, C/N raton at IFS and BBOS could be used as indicative physiological traits for selecting F1crosses, while Chi a and Chi b at full flowering stage (FFS) and BBOS, C/N raton at FFS for parents lines. N content at IFS, FFS, and BBOS, P and K contents at four stages were mainly affected by dominant effects, which could be used to choose F1crosses. Similarity, Parent's lines have high additive effects in Chi a content, in C/N ratio and N content at SS, IFS and FFS, in K at IFS, FFS and BBOS, their hybrids F1have more lint yield. Bolls per plant, lint percent, micronarie and fiber length were mainly affected by additive effects, which suggested they could be improved by choosing pure lines. Additive effects contribution of lint yield was mainly from lint percent, while dominant effects contribution of lint yield from boll weight. Having high contribution ratio of additive effects to fiber strength, micronarie and fiber length, lint percent in parents could be used as indicative traits for selecting fiber quality of F1crosses. In short, Parent1(31) is an acceptable non-Bt line and parent8(B7kang) is an excellent Bt transgenic cotton with insect-resistance. Among16F1crosses, there are8F1crosses (H18, H28, H29, H37, H48, H59, H67and H69) with positive and significant lint yield.
4. Four conventional upland cotton lines with five transgenic line using NCII design, and their20F1crosses were grown at the same location above in2011. genetic effects analysis showed that12biochemical parameters at three stages were mainly affected by dominant effects. K content in F1crosses at three stages had significant mean and better parents heterosis. C/N ratio had significant additive contribution ratio to lint yield at three stages. Branches, bolls, boll weight and lint yield were mainly controlled by additive effects, which suggested they could be improving by choosing pure lines. Additive variance contribution ratio of bolls for parents was mainly from external, middle and lower bolls on the plants, while for F1crosses from external and middle bolls. Additive contribution ratio of lint yield was from bolls and lint percent, while dominant contribution ratio from diameter, plant height and boll weight. Lint percent to fiber strength, plant height and bolls to micronarie, diameter to fiber length had largest additive contribution, respectively. In short, Parent3(Ci96-21) is an acceptable non-Bt line and parent9(huanggang kang) is an excellent Bt transgenic cotton with insect-resistance. Among20F1crosses, there are6F1crosses (H39, H16, H35,H28, H47, and H45) with positive and significant lint yield.
5. Reciprocal crosses between one conventional upland cotton line (Ci-ZJ6) and five transgenic Bt gene lines was made to produce10F1hybrid crosses and all lines planted in2011at the same location above. Genetic analysis showed that Chl a at IFS and BBOS, Chl b at FFS and BBOS, Chl a/b ratio at three stages, N at IFS and FFS, P at FFS, K at FFS and BBOS, C/N ratio at IFS and BBOS, anti-oxidant enzyme systems (SOD, POD, CAT, APX) and MDA, were mainly affected by additive effects. While Chl a at FFS, Chl b at IFS, N at BBOS, P at IFS and BBOS, K at IFS, C/N ratio at FFS, were mainly affected by dominant effects. All12biochemical parameters in F1crosses had significant mean parents heterosis at three stages, but no positive and significant better parents heterosis. Diameter, external bolls, middle bolls, boll weight, lint percent, fiber length, micronaire and fiber strength were mainly affected by additive effects, which suggested those traits could be improved by choosing pure lines. Bolls additive variance ratio for parents was mainly from top and external bolls on the plants, while for F1crosses from central, lower, and external bolls. Additive contribution ratio of lint yield was mainly from branches, diameter, up bolls and lint percent. Lint percent to fiber strength and micronaire had largest additive contribution. Having significant additive effects in lint percent, boll weight, and fiber length and fiber strength. Parent1(Ci-ZJ6) is an acceptable non-Bt line. Parent4(E29) with better performance in bolls, fiber length and positive lint yield, and parent5(Tai kang) with more branches, bolls and positive lint yield, are excellent Bt transgenic cotton with insect-resistance. Among10F1crosses, there are3F1crosses (H13, H15and H41) with positive and significant lint yield. Among reciprocal crosses, H15and H51in diameter and fiber length, H12and H21in plant height, internal bolls and lint percent, H13and H31in lint percent had mean parents heterosis, respectively, which suggested that there were significant differences between conventional parent P1and five transgenic Bt cotton lines. Moreover, the overall performance in the crosses with female parent P1was better than in the crosses with female parents transgenic Bt cotton lines, which confirmed the possible existance of cytoplasmic inheritance in transgenic Bt hybrid lines.
引文
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