捻转血矛线虫滞育相关基因daf-22和hsp70全基因的获得及其功能的研究
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摘要
捻转血矛线虫病是寄生于牛、羊等反刍动物的一种重要的寄生虫病,因引起感染动物贫血、消瘦甚至死亡而给畜牧业造成极大的经济损失。该病是由感染性三期幼虫感染宿主引起,具有明显的季节性。当外界环境进入寒冷的冬季时,幼虫及虫卵难以存活,进入滞育阶段是捻转血矛线虫在宿主体内越冬的唯一方式,也是春季反刍动物爆发该病的主要原因。因而控制线虫的滞育、探明产生该现象的生物化学和分子生物学机制很可能是解决该病周期性流行和季节性爆发的关键问题。鉴于此,本研究以捻转血矛线虫为研究对象,以秀丽隐杆线虫为模式生物,通过mRNA差异显示技术获得捻转血矛线虫滞育阶段表达差异性基因片段,进一步利用RACE技术和基因步移技术获得滞育相关基因Hc-daf-22基因和Hc-hsp70基因全序列,并采用RNA干扰技术和拯救技术确定这些基因对线虫滞育的影响,研究结果为阐明寄生性线虫的滞育机制奠定了良好基础。
1.捻转血矛线虫三期感染性幼虫培养方法的改进
参考国外已报道的蛇形毛圆线虫和普通奥斯特他(氏)线虫虫体的培养技术,建立了捻转血矛线虫幼虫的培养方法。收集自然感染羊体内的捻转血矛线虫雌虫子宫内的虫卵,以含有Earle's液和酵母粉的琼脂平板为培养基,对捻转血矛线虫的自由生活阶段虫体进行了体外培养和观察,并对所获得的感染性三期幼虫进行了感染性验证,结果表明,该方法能获得纯度较高的捻转血矛线虫自由生活各阶段虫体,为感染实验及开展分子生物学研究提供基础。
2.利用mRNA差异显示技术获得捻转血矛线虫滞育阶段幼虫与正常虫体差异基因,通过比对进行初步分析
参考Kooyman(1994)发表的文献,将虫体置于15。C盛有自来水的平皿中5周,计数后,挑取5000条具有活力的虫体攻入无线虫感染的羊体内,感染后第35天剖杀羊只,剖检真胃,挑取虫体,根据虫体形态特征辨别获得滞育期虫体。同时以体外培养的四期幼虫为正常虫体对照。提取滞育期虫体和正常虫体的RNA,分别采用随机引物和锚定引物对其进行反转录反应,获得cDNA。将获得的cDNA进行聚丙烯酰胺凝胶电泳,银染,回收银染后的差异条带,连接至pMD18-T载体,测序,共获得了74个差异片段。将所获得的片段分别在NCBI与寄生性线虫库中进行比对,结果显示,有14个片段与已公布的线虫序列有同源性,有29个片段与已发表的捻转血矛线虫基因组序列具有较高的同源性。
3.以秀丽隐杆线虫为模式生物,选取部分同源性基因进行初步功能验证,筛选差异片段
分析所获得的差异片段中与已知序列有同源性的片段,选取合适的酶切位点,连接至RNAi专用载体L4440上,鉴定阳性后,转化至大肠杆菌HT115中,重新鉴定。将阳性克隆菌液均匀涂布于含有IPTG的NGM平板中,室温干燥后,放置过夜。挑取秀丽隐杆线虫同期虫体,饲喂构建好的重组载体,同时,选取秀丽隐杆线虫hsf-1基因作为阳性对照,空载L4440为阴性对照。通过观察RNAi后虫体表型、产卵量、以及滞育形成能力筛选出差异片段中有滞育功能的片段。实验结果显示：RNAi后,片段A342出现了明显的表型,饲喂A182与E181片段的秀丽隐杆线虫的产卵量显著低于阴性对照组L4440,片段A342、A382及E211对秀丽线虫的滞育形成起到了明显的干扰作用。
4. Hc-daf-22基因的获得,利用秀丽隐杆线虫进行启动子及功能研究
以获得的A382(541bp)为模板,设计引物,通过5’和3'RACE技术获得了该EST的全序列。该基因全长1602bp,编码533个氨基酸,与秀丽线虫的daf-22基因具有很高的同源性,故命名为Hc-daf22。根据已获得的cDNA设计引物获得该基因的基因序列6939bp,分析显示,该基因包含有16个外显子,15个内含子。通过基因步移的方法获得该基因的5’侧翼区1548bp,连接至载体pPD95.77,利用显微注射验证其启动子活性,结果证实该区域具有启动子活性,可以启动GFP在幼虫及成虫阶段虫体咽部、分泌细胞、肠道内表达。同时扩增秀丽隐杆线虫daf-22基因上游5’侧翼区2061bp,连接至载体pPD95.77,将所获得的载体进行显微注射验证其启动子活性,结果发现该启动子能够高强度的启动GFP的表达,表达部位为肠道。将所获得的Hc-daf-22全基因设计引物,使其包含有该基因的所有功能域,连接至已验证具有秀丽隐杆线虫启动子活性的重组载体上,并显微注射,观察荧光,检验捻转血矛线虫daf-22基因是否可以在秀丽线虫体内表达；结果显示：通过荧光检测,Hc-daf-22基因可以在秀丽隐杆线虫野生株体内大量表达,表达方式与秀丽线虫启动子有所不同,表达部位主要集中在肠道,但肠细胞表达更为明显；将验证具有活性的重组载体显微注射入秀丽线虫daf-22突变株(OK693)内,通过Oil-Red-O染色突变株虫体肠道内脂肪,观察脂肪沉积的多少及颗粒的大小,分析该基因对秀丽线虫的拯救情况。基因拯救结果显示：通过拯救,突变株脂肪颗粒明显减少或消失,拯救后的虫体长度、宽度以及产卵数比突变株明显增加,但与野生株相比还有显著差异,虫体的发育阶段并没有显著变化。该结果证明捻转血矛线虫daf-22基因对秀丽隐杆线虫daf-22基因突变株进行了部分拯救
同时,根据所得基因设计引物,获取捻转血矛线虫daf-22基因包含功能域的845bp,连接至L4440载体,转化HT115,通过饲喂秀丽隐杆线虫进行RNAi,观察虫体脂肪沉积的变化,分析该基因对秀丽隐杆线虫的干扰效果。研究结果显示,饲喂捻转血矛线虫daf-22基因的虫体,脂肪沉积明显增多,且具有比正常株及阴性对照组大的脂肪颗粒,结果证明,该基因对秀丽隐杆线虫起到了明显的干扰作用。
5.捻转血矛线虫hsp70基因全基因的获得,利用秀丽隐杆线虫进行启动子及表达分析
根据已获得的序列A342(709bp)设计引物获得部分基因组序列,通过基因步移技术和长距离反转录PCR扩增出捻转血矛线虫热激蛋白基因hsp70全基因序列3925bp及cDNA序列1941bp,基因分析显示该基因含有7个外显子,6个内含子,编码646个氨基酸,包含三个hsp70家族蛋白特征区,且与其他已知物种的hsp70具有很高的同源性；同时,通过基因步移的方法,本研究还扩增得到该基因上游2239bp的5'flanking区。通过软件分析显示,该区具有假定的热激因子结合位点、TATA box、 GAGA结合位点、CAC结合位点、CACCC结合位点、CCAAT box等启动子重要元件；将该片段连接至pPPD95.77载体,显微注射至秀丽隐杆线虫体内,通过荧光观察确定其是否具有启动子活性,研究结果发现：该启动子可以启动GFP在秀丽隐杆线虫各个生活阶段的肠道内表达,荧光表达强度随着虫体的发育不断增强,且存在两种表达方式。
将所获得的捻转血矛线虫hsp70基因首先连接至原核表达载体pET28b中,在其C末端加入6xHis标签,鉴定正确后,IPTG诱导表达,经SDS-PAGE与Western blot分析,研究结果显示,二者均可在70KD处检测到明显的特异性条带；以连接好的原核表达载体为模板,重新设计引物,使得捻转血矛线虫的hsp70基因下游带有6×His标签,将该片段连接至秀丽隐杆线虫热激专用载体pPPD49.78和pPD49.83,显微注射至秀丽隐杆线虫体内,通过His单抗进行VVestern blot分析：在秀丽隐杆线虫热激2,4,6小时后均可检测到特异性条带,且条带大小与原核表达条带位置一致；同时,本研究还通过荧光定量PCR的方法,检测了秀丽隐杆线虫hsp-1基因在显微注射捻转血矛线虫hsp70基因后,在mRNA水平上的变化,结果显示：显微注射后,秀丽隐杆线虫hsp-1基因表达水平热激后同N2相比显著下调。该结果证明捻转血矛线虫hsp70基因对秀丽隐杆线虫hsp-1热激后的mRNA水平具有显著影响,这为Hc-hsp70可能具有秀丽隐杆线虫hsp-1基因相似的功能提供了依据。
综上所述,本研究首次获得了捻转血矛线虫滞育期虫体差异表达基因,通过生物信息学分析及在秀丽隐杆线虫中的RNAi试验筛选出具有明显表型及对滞育形成相关功能具有显著影响的差异基因；通过RACE技术及基因步移技术,首次获得了两个差异基因Hc-daf-22及Hc-hsp70的全序列；利用秀丽隐杆线虫验证了Hc-daf-22基因5’侧翼区启动子活性,实现了基因功能域在秀丽隐杆线虫野生株和突变株体内的表达及对突变株的部分拯救,成功完成了Hc-daf-22对秀丽隐杆线虫的RNA干扰实验；；通过秀丽隐杆线虫转基因技术体系,Hc-hsp705'侧翼区启动子活性得到分析,cDNA全序列在秀丽隐杆线虫体内得到表达,该基因的表达使得转基因虫体内hsp-1基因明显下调。本研究为寄生性线虫滞育机制的研究提供了重要的参考和依据。
The gastrointestinal nematode Haemonchus contortus is a major blood feeding pathogen of sheep and goats, which inhabits the mucosal surface of the abomasum in these ruminants and causes haemonchosis. Its infections may be manifested as anemia, weight loss or even death in some cases, causing a great economic loss to the agricultural industry worldwide. The disease caused by infective third larval was seasonality, and the eggs and larval cannot live when the temperature is too low. The dauer formation is the only way for the larval to overcome the winter which is the reason for the outbreak in spring. Therefore, controlling, investing the mechanism of the dauer formation is crucial to resolving the problem. The studies carried were focused on the Haemonchus contortus, taking Caenorhabditis elegans as a model, the different expression genes between dauer and negative control worms were harvested by DDRT-PCR. The full length of Hc-daf-22and Hc-hsp70were obtained using RACE and genome walking. The impacts on dauer formation were verified by RNAi and rescue experiments. The results will be to play the foundation for clarifying the mechanism of arrest formation.
1A new method for culture of Haemonchus contortus larvae
A study was carried out in order to culture Haemonchus contortus third stage larvae according to the published data which was mainly used to culture Trichostrongylus colubriformis and Ostertagia circumcincta. The eggs were extracted from intrauterine of femal from infected sheeps and the larvae were grown in a more defined medium (agar plate fortified with Earle's medium and yeast extract). All stages of the larvaes were observed; meanwhile the sheep were infected by L3larvaes to verify the L3larvaes were infective. A lot of Haemonchus contortus every stage larvae in free living stage were gotten through this method.
2Specific genes from dauers of Haemonchus contortus were acquired by mRNA DDRT-PCR, and analyzed by comparing with genes from normal worms
Sheep free of parasitic nematodes infective were challenged with infective third larvae which were treated in tap water for5weeks according to the paper published by Kooyman, and sacrificed in35days to harvest the dauer worms in abomasums which were identified by its morphology. The fourth larval were cultured in vitro as negative control. The RNA from dauer worms and control worms were used to make cDNA by using random primers and anchorage primers. Specific cDNA were harvested and ligated into pMD18-T vector for sequencing.74pieces of genes were compared with the data in NCBI and parasite EST library.14pieces showed similarity with sequences of parasitic nematode having been published, and29of them showed high similarity with the genome of Haemonchus contortus, and the rest may be the new genes.
3Screening of specific genes by RNAi using Caenorhabditis elegans as a model
Sequences which showed similarity with other species were digested by proper enzymes for ligation into L4440and transformed into HT115after they were identified. The recombinant bacilli were coated on the NGM plates which contained IPTG and placed overnight. The hermaphrodites Caenorhabditis elegans were feeding on the recombinant vectors and hsf-1of Caenorhabditis elegans was used as a positive control and L4440as negative control. After RNAi, by observing phenotypes, brood size and dauer formation, the effective genes can be isolated. The results showed that:A342showed obvious phenotype; the brood size of A182and E181was significantly lower than that of negative control L4440; A342、A382and E211showed obvious effect in dauer formation.
4Characterization of daf-22gene from Haemonchus contortus and function analysis and promoter analysis using Caenorhabditis elegans
The complete sequence of A382was identified employing3'RACE and5'RACE using primers designed based on the sequence A382(541bp). The open reading frame of the gene encodes533a.a, which had80.3%similarity with the Caenorhabditis elegans daf-22, designed as Hc-daf-22. The full length of the genome is6939bp,16exons and15introns were included. The5'flanking region was acquired employing genome walking. The1548-bp5'-flanking region of Hc-daf-22was cloned upstream of GFP gene in ppd95.77vector and microinjected into the gonads of Caenorhabditis elegans. The results showed that the transformed animals exhibited fluorescence in the pharynx, intestine, in all the larvae stages and adult worms.2061-bp5'-flanking of Ce-daf-22was cloned to the upstream of GFP gene in ppd95.77vector and microinjected into the gonads of Caenorhabditis elegans again. The result indicated that the transformed worms expressed fluorescence in the intestine, hypodermis and body wall muscle at all development stages.
In order to determine whether the Hc-daf-22could be translated in Caenorhabditis elegans, the specific primers were designed based on the cDNA sequence of Hc-daf-22which all of the functional regions were included to obtain expression vector Cedaf22P-pPD95.77-Hc-daf-22. The recombinant vector was microinjected into Caenorhabditis elegans N2strain and showed fluorescence in the intestine in all stages. Next, the vector was injected into the Caenorhabditis elegans mutant (OK693), and the construct was expressed in daf-22(OK693) worms in intestine. Oil-Red-O staining, body size and brood size measurement, growth rate were carried out to ascertain whether Hc-daf-22can be used to rescue C. elegans mutant. The results showed transgenic worm conferred partial rescue of the mutant.
The construct containing805bp of Hc-daf-22(RNAi) experiment was carried out to assess whether the endogenous mRNA can be rescued by parasite gene in N2C. elegans worms. The worms grown on Hc-daf-22(RNAi) plates had more fat storage compared with worms grown on control plates.
5Characterization of heat shock protein70gene from Haemonchus contortus and its expression and promoter analysis using Caenorhabditis elegans
In the present study, the complete genome sequence of A342of Haemonchus contortus was identified by genome walking and long-PCR, and a cDNA sequence was obtained by reverse transcriptase PCR. Bioinformatic analysis revealed that the open reading frame of this gene the cDNA encoded a646-amino acid peptide, which had a high degree of sequence similarity with the Caenorhabditis elegans hsp-land hsp70with other species (designated as Hc-hsp70). Genome analysis indicated that7exons and6introns were contained. The5'-flanking sequence of the Haemonchus contortus hsp70was analyzed and3putative heat shock factors (HSF) and several transcriptional elements were identified. The5'-flanking region was subcloned into the vector upstream of green fluorescence protein (GFP) reporter gene and microinjected into the Caenorhabditis elegans. The fluorescence was expressed in the intestine in all larvae stages and adult with two expression patterns.
Recombinant Hc-hsp70(designed as pET28b-hsp70) with6-His-tag at the C-terminal was expressed in Escherichia coli and was verified by analyses using SDS-PAGE and western blot. The recombinant Hc-hsp70(designed pPD49.78/49.83-hsp70) with6-His-tag at the C-terminal could also expressed in Caenorhabditis elegans. However, its expression induced down-regulation of hsp-1of Caenorhabditis elegans in mRNA level. These results suggested that the H. contortus hsp70might have similar function to that of C. elegans hsp-1.
In summary, specific genes from dauers of Haemonchus contortus were firstly acquired, and specific genes which could induce phenotype and influence the dauer formation by bioinformatics analysis and RNAi in C. elegans were screened; the full length of two specific genes Hc-daf-22and Hc-hsp70were firstly obtained by RACE and genome walking respectively; the5'flanking sequence was analyzed in C. elegans and showed the promoter was included; Hc-daf-22can be expressed in N2and daf-22mutant (OK693) and conferred partial rescue of the mutant, meanwhile, Hc-daf-22induced RNAi in C. elegans successfully; the transcriptional activity of Hc-hsp70was confirmed, and it can be expressed in C. elegans and induced the down-regulation of hsp-1.This research will be particularly important for exploring the mechanism of dauer formation of parasitic nematodes.
1.捻转血矛线虫三期感染性幼虫培养方法的改进
参考国外已报道的蛇形毛圆线虫和普通奥斯特他(氏)线虫虫体的培养技术,建立了捻转血矛线虫幼虫的培养方法。收集自然感染羊体内的捻转血矛线虫雌虫子宫内的虫卵,以含有Earle's液和酵母粉的琼脂平板为培养基,对捻转血矛线虫的自由生活阶段虫体进行了体外培养和观察,并对所获得的感染性三期幼虫进行了感染性验证,结果表明,该方法能获得纯度较高的捻转血矛线虫自由生活各阶段虫体,为感染实验及开展分子生物学研究提供基础。
2.利用mRNA差异显示技术获得捻转血矛线虫滞育阶段幼虫与正常虫体差异基因,通过比对进行初步分析
参考Kooyman(1994)发表的文献,将虫体置于15。C盛有自来水的平皿中5周,计数后,挑取5000条具有活力的虫体攻入无线虫感染的羊体内,感染后第35天剖杀羊只,剖检真胃,挑取虫体,根据虫体形态特征辨别获得滞育期虫体。同时以体外培养的四期幼虫为正常虫体对照。提取滞育期虫体和正常虫体的RNA,分别采用随机引物和锚定引物对其进行反转录反应,获得cDNA。将获得的cDNA进行聚丙烯酰胺凝胶电泳,银染,回收银染后的差异条带,连接至pMD18-T载体,测序,共获得了74个差异片段。将所获得的片段分别在NCBI与寄生性线虫库中进行比对,结果显示,有14个片段与已公布的线虫序列有同源性,有29个片段与已发表的捻转血矛线虫基因组序列具有较高的同源性。
3.以秀丽隐杆线虫为模式生物,选取部分同源性基因进行初步功能验证,筛选差异片段
分析所获得的差异片段中与已知序列有同源性的片段,选取合适的酶切位点,连接至RNAi专用载体L4440上,鉴定阳性后,转化至大肠杆菌HT115中,重新鉴定。将阳性克隆菌液均匀涂布于含有IPTG的NGM平板中,室温干燥后,放置过夜。挑取秀丽隐杆线虫同期虫体,饲喂构建好的重组载体,同时,选取秀丽隐杆线虫hsf-1基因作为阳性对照,空载L4440为阴性对照。通过观察RNAi后虫体表型、产卵量、以及滞育形成能力筛选出差异片段中有滞育功能的片段。实验结果显示：RNAi后,片段A342出现了明显的表型,饲喂A182与E181片段的秀丽隐杆线虫的产卵量显著低于阴性对照组L4440,片段A342、A382及E211对秀丽线虫的滞育形成起到了明显的干扰作用。
4. Hc-daf-22基因的获得,利用秀丽隐杆线虫进行启动子及功能研究
以获得的A382(541bp)为模板,设计引物,通过5’和3'RACE技术获得了该EST的全序列。该基因全长1602bp,编码533个氨基酸,与秀丽线虫的daf-22基因具有很高的同源性,故命名为Hc-daf22。根据已获得的cDNA设计引物获得该基因的基因序列6939bp,分析显示,该基因包含有16个外显子,15个内含子。通过基因步移的方法获得该基因的5’侧翼区1548bp,连接至载体pPD95.77,利用显微注射验证其启动子活性,结果证实该区域具有启动子活性,可以启动GFP在幼虫及成虫阶段虫体咽部、分泌细胞、肠道内表达。同时扩增秀丽隐杆线虫daf-22基因上游5’侧翼区2061bp,连接至载体pPD95.77,将所获得的载体进行显微注射验证其启动子活性,结果发现该启动子能够高强度的启动GFP的表达,表达部位为肠道。将所获得的Hc-daf-22全基因设计引物,使其包含有该基因的所有功能域,连接至已验证具有秀丽隐杆线虫启动子活性的重组载体上,并显微注射,观察荧光,检验捻转血矛线虫daf-22基因是否可以在秀丽线虫体内表达；结果显示：通过荧光检测,Hc-daf-22基因可以在秀丽隐杆线虫野生株体内大量表达,表达方式与秀丽线虫启动子有所不同,表达部位主要集中在肠道,但肠细胞表达更为明显；将验证具有活性的重组载体显微注射入秀丽线虫daf-22突变株(OK693)内,通过Oil-Red-O染色突变株虫体肠道内脂肪,观察脂肪沉积的多少及颗粒的大小,分析该基因对秀丽线虫的拯救情况。基因拯救结果显示：通过拯救,突变株脂肪颗粒明显减少或消失,拯救后的虫体长度、宽度以及产卵数比突变株明显增加,但与野生株相比还有显著差异,虫体的发育阶段并没有显著变化。该结果证明捻转血矛线虫daf-22基因对秀丽隐杆线虫daf-22基因突变株进行了部分拯救
同时,根据所得基因设计引物,获取捻转血矛线虫daf-22基因包含功能域的845bp,连接至L4440载体,转化HT115,通过饲喂秀丽隐杆线虫进行RNAi,观察虫体脂肪沉积的变化,分析该基因对秀丽隐杆线虫的干扰效果。研究结果显示,饲喂捻转血矛线虫daf-22基因的虫体,脂肪沉积明显增多,且具有比正常株及阴性对照组大的脂肪颗粒,结果证明,该基因对秀丽隐杆线虫起到了明显的干扰作用。
5.捻转血矛线虫hsp70基因全基因的获得,利用秀丽隐杆线虫进行启动子及表达分析
根据已获得的序列A342(709bp)设计引物获得部分基因组序列,通过基因步移技术和长距离反转录PCR扩增出捻转血矛线虫热激蛋白基因hsp70全基因序列3925bp及cDNA序列1941bp,基因分析显示该基因含有7个外显子,6个内含子,编码646个氨基酸,包含三个hsp70家族蛋白特征区,且与其他已知物种的hsp70具有很高的同源性；同时,通过基因步移的方法,本研究还扩增得到该基因上游2239bp的5'flanking区。通过软件分析显示,该区具有假定的热激因子结合位点、TATA box、 GAGA结合位点、CAC结合位点、CACCC结合位点、CCAAT box等启动子重要元件；将该片段连接至pPPD95.77载体,显微注射至秀丽隐杆线虫体内,通过荧光观察确定其是否具有启动子活性,研究结果发现：该启动子可以启动GFP在秀丽隐杆线虫各个生活阶段的肠道内表达,荧光表达强度随着虫体的发育不断增强,且存在两种表达方式。
将所获得的捻转血矛线虫hsp70基因首先连接至原核表达载体pET28b中,在其C末端加入6xHis标签,鉴定正确后,IPTG诱导表达,经SDS-PAGE与Western blot分析,研究结果显示,二者均可在70KD处检测到明显的特异性条带；以连接好的原核表达载体为模板,重新设计引物,使得捻转血矛线虫的hsp70基因下游带有6×His标签,将该片段连接至秀丽隐杆线虫热激专用载体pPPD49.78和pPD49.83,显微注射至秀丽隐杆线虫体内,通过His单抗进行VVestern blot分析：在秀丽隐杆线虫热激2,4,6小时后均可检测到特异性条带,且条带大小与原核表达条带位置一致；同时,本研究还通过荧光定量PCR的方法,检测了秀丽隐杆线虫hsp-1基因在显微注射捻转血矛线虫hsp70基因后,在mRNA水平上的变化,结果显示：显微注射后,秀丽隐杆线虫hsp-1基因表达水平热激后同N2相比显著下调。该结果证明捻转血矛线虫hsp70基因对秀丽隐杆线虫hsp-1热激后的mRNA水平具有显著影响,这为Hc-hsp70可能具有秀丽隐杆线虫hsp-1基因相似的功能提供了依据。
综上所述,本研究首次获得了捻转血矛线虫滞育期虫体差异表达基因,通过生物信息学分析及在秀丽隐杆线虫中的RNAi试验筛选出具有明显表型及对滞育形成相关功能具有显著影响的差异基因；通过RACE技术及基因步移技术,首次获得了两个差异基因Hc-daf-22及Hc-hsp70的全序列；利用秀丽隐杆线虫验证了Hc-daf-22基因5’侧翼区启动子活性,实现了基因功能域在秀丽隐杆线虫野生株和突变株体内的表达及对突变株的部分拯救,成功完成了Hc-daf-22对秀丽隐杆线虫的RNA干扰实验；；通过秀丽隐杆线虫转基因技术体系,Hc-hsp705'侧翼区启动子活性得到分析,cDNA全序列在秀丽隐杆线虫体内得到表达,该基因的表达使得转基因虫体内hsp-1基因明显下调。本研究为寄生性线虫滞育机制的研究提供了重要的参考和依据。
The gastrointestinal nematode Haemonchus contortus is a major blood feeding pathogen of sheep and goats, which inhabits the mucosal surface of the abomasum in these ruminants and causes haemonchosis. Its infections may be manifested as anemia, weight loss or even death in some cases, causing a great economic loss to the agricultural industry worldwide. The disease caused by infective third larval was seasonality, and the eggs and larval cannot live when the temperature is too low. The dauer formation is the only way for the larval to overcome the winter which is the reason for the outbreak in spring. Therefore, controlling, investing the mechanism of the dauer formation is crucial to resolving the problem. The studies carried were focused on the Haemonchus contortus, taking Caenorhabditis elegans as a model, the different expression genes between dauer and negative control worms were harvested by DDRT-PCR. The full length of Hc-daf-22and Hc-hsp70were obtained using RACE and genome walking. The impacts on dauer formation were verified by RNAi and rescue experiments. The results will be to play the foundation for clarifying the mechanism of arrest formation.
1A new method for culture of Haemonchus contortus larvae
A study was carried out in order to culture Haemonchus contortus third stage larvae according to the published data which was mainly used to culture Trichostrongylus colubriformis and Ostertagia circumcincta. The eggs were extracted from intrauterine of femal from infected sheeps and the larvae were grown in a more defined medium (agar plate fortified with Earle's medium and yeast extract). All stages of the larvaes were observed; meanwhile the sheep were infected by L3larvaes to verify the L3larvaes were infective. A lot of Haemonchus contortus every stage larvae in free living stage were gotten through this method.
2Specific genes from dauers of Haemonchus contortus were acquired by mRNA DDRT-PCR, and analyzed by comparing with genes from normal worms
Sheep free of parasitic nematodes infective were challenged with infective third larvae which were treated in tap water for5weeks according to the paper published by Kooyman, and sacrificed in35days to harvest the dauer worms in abomasums which were identified by its morphology. The fourth larval were cultured in vitro as negative control. The RNA from dauer worms and control worms were used to make cDNA by using random primers and anchorage primers. Specific cDNA were harvested and ligated into pMD18-T vector for sequencing.74pieces of genes were compared with the data in NCBI and parasite EST library.14pieces showed similarity with sequences of parasitic nematode having been published, and29of them showed high similarity with the genome of Haemonchus contortus, and the rest may be the new genes.
3Screening of specific genes by RNAi using Caenorhabditis elegans as a model
Sequences which showed similarity with other species were digested by proper enzymes for ligation into L4440and transformed into HT115after they were identified. The recombinant bacilli were coated on the NGM plates which contained IPTG and placed overnight. The hermaphrodites Caenorhabditis elegans were feeding on the recombinant vectors and hsf-1of Caenorhabditis elegans was used as a positive control and L4440as negative control. After RNAi, by observing phenotypes, brood size and dauer formation, the effective genes can be isolated. The results showed that:A342showed obvious phenotype; the brood size of A182and E181was significantly lower than that of negative control L4440; A342、A382and E211showed obvious effect in dauer formation.
4Characterization of daf-22gene from Haemonchus contortus and function analysis and promoter analysis using Caenorhabditis elegans
The complete sequence of A382was identified employing3'RACE and5'RACE using primers designed based on the sequence A382(541bp). The open reading frame of the gene encodes533a.a, which had80.3%similarity with the Caenorhabditis elegans daf-22, designed as Hc-daf-22. The full length of the genome is6939bp,16exons and15introns were included. The5'flanking region was acquired employing genome walking. The1548-bp5'-flanking region of Hc-daf-22was cloned upstream of GFP gene in ppd95.77vector and microinjected into the gonads of Caenorhabditis elegans. The results showed that the transformed animals exhibited fluorescence in the pharynx, intestine, in all the larvae stages and adult worms.2061-bp5'-flanking of Ce-daf-22was cloned to the upstream of GFP gene in ppd95.77vector and microinjected into the gonads of Caenorhabditis elegans again. The result indicated that the transformed worms expressed fluorescence in the intestine, hypodermis and body wall muscle at all development stages.
In order to determine whether the Hc-daf-22could be translated in Caenorhabditis elegans, the specific primers were designed based on the cDNA sequence of Hc-daf-22which all of the functional regions were included to obtain expression vector Cedaf22P-pPD95.77-Hc-daf-22. The recombinant vector was microinjected into Caenorhabditis elegans N2strain and showed fluorescence in the intestine in all stages. Next, the vector was injected into the Caenorhabditis elegans mutant (OK693), and the construct was expressed in daf-22(OK693) worms in intestine. Oil-Red-O staining, body size and brood size measurement, growth rate were carried out to ascertain whether Hc-daf-22can be used to rescue C. elegans mutant. The results showed transgenic worm conferred partial rescue of the mutant.
The construct containing805bp of Hc-daf-22(RNAi) experiment was carried out to assess whether the endogenous mRNA can be rescued by parasite gene in N2C. elegans worms. The worms grown on Hc-daf-22(RNAi) plates had more fat storage compared with worms grown on control plates.
5Characterization of heat shock protein70gene from Haemonchus contortus and its expression and promoter analysis using Caenorhabditis elegans
In the present study, the complete genome sequence of A342of Haemonchus contortus was identified by genome walking and long-PCR, and a cDNA sequence was obtained by reverse transcriptase PCR. Bioinformatic analysis revealed that the open reading frame of this gene the cDNA encoded a646-amino acid peptide, which had a high degree of sequence similarity with the Caenorhabditis elegans hsp-land hsp70with other species (designated as Hc-hsp70). Genome analysis indicated that7exons and6introns were contained. The5'-flanking sequence of the Haemonchus contortus hsp70was analyzed and3putative heat shock factors (HSF) and several transcriptional elements were identified. The5'-flanking region was subcloned into the vector upstream of green fluorescence protein (GFP) reporter gene and microinjected into the Caenorhabditis elegans. The fluorescence was expressed in the intestine in all larvae stages and adult with two expression patterns.
Recombinant Hc-hsp70(designed as pET28b-hsp70) with6-His-tag at the C-terminal was expressed in Escherichia coli and was verified by analyses using SDS-PAGE and western blot. The recombinant Hc-hsp70(designed pPD49.78/49.83-hsp70) with6-His-tag at the C-terminal could also expressed in Caenorhabditis elegans. However, its expression induced down-regulation of hsp-1of Caenorhabditis elegans in mRNA level. These results suggested that the H. contortus hsp70might have similar function to that of C. elegans hsp-1.
In summary, specific genes from dauers of Haemonchus contortus were firstly acquired, and specific genes which could induce phenotype and influence the dauer formation by bioinformatics analysis and RNAi in C. elegans were screened; the full length of two specific genes Hc-daf-22and Hc-hsp70were firstly obtained by RACE and genome walking respectively; the5'flanking sequence was analyzed in C. elegans and showed the promoter was included; Hc-daf-22can be expressed in N2and daf-22mutant (OK693) and conferred partial rescue of the mutant, meanwhile, Hc-daf-22induced RNAi in C. elegans successfully; the transcriptional activity of Hc-hsp70was confirmed, and it can be expressed in C. elegans and induced the down-regulation of hsp-1.This research will be particularly important for exploring the mechanism of dauer formation of parasitic nematodes.
引文
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