【作者】 王更先；

【导师】 徐世清；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 鳞翅目(Lepidoptera)昆虫是昆虫中的第二大目,与人类生产和生活关系密切,其中许多是极具危害性的农业害虫。家蚕(Bombyx mori)是国际无脊椎动物协会确定的鳞翅目模式昆虫,也是唯一完成全基因组测序的鳞翅目生物,这为鳞翅目害虫的功能基因研究提供了一个便利的技术平台。利用显性致死基因来防治害虫技术(RIDL)自2000年被提出以来,受到人们的广泛重视。其原理是人工饲养携带显性致死基因的雄虫,释放到自然界和野生害虫交配,遗传致死基因给后代,致其死亡,从而达到控制害虫的目的。该技术具有物种特异性、环保等特点,对农业害虫的防治具有重大的意义。目前RIDL技术已在果蝇中试验成功,在鳞翅目害虫棉铃虫防治方面也取得了一些成果。为了发掘鳞翅目昆虫的lethal相关基因,本研究分别以黑腹果蝇(Drosophila melanogaster)lethal蛋白序列(P-因子诱导致死突变)、Triplo-lethal(三倍体或单倍体致死)基因座中Osiris基因家族的蛋白序列和温敏性致死突变基因Pros(Pros261和Prosβ21)的蛋白序列为质询序列,利用生物信息学、RT-PCR、RACE、表达和RNA干涉等技术克隆了家蚕的相关基因,并在棉铃虫(Helicoverpa armigera)中克隆了3条Pros相关基因,进行了结构分析和功能研究。主要获得了以下结果:1.家蚕lethal(P-因子诱导致死突变)相关基因的克隆及功能研究果蝇l(3)neo18为NADH脱氢酶基因P转座子诱发的致死突变体,因此编码的蛋白质具有还原型烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶的功能。利用生物信息学、RT-PCR和RACE等方法克隆了家蚕l(3)neo18相关基因,分析了基因结构与表达谱。Bm-l(3)neo18基因的cDNA全长为868 bp,由573 bp的完整开放读码框(ORF)序列、25 bp的5′端非编码区序列(5′UTR)和251 bp的3′端非编码区序列(3′UTR)组成,编码蛋白质为190个氨基酸残基,分子量22.7 kD,pI为9.60。Bm-l(3)neo18基因由3个外显子和2个内含子组成,定位于家蚕第3染色体,位于nscaf2930的120.3knt-120.4knt。Bm-l(3)neo18蛋白质在1-185氨基酸残基位置为NADH脱氢酶的SGDH亚基保守区,在61-83氨基酸残基位置具有一个保守的跨膜区域。采用Clustal W进行多序列比对发现,Bm-l(3)neo18与埃及伊蚊等昆虫NADH脱氢酶具有50%以上的蛋白质同源性,NADH脱氢酶的保守区域高度一致。NJ法分子进化分析也显示,Bm-l(3)neo18与昆虫NADH脱氢酶进化上同源。该基因除在卵期的表达量较低外,在幼虫、蛹和蛾期均有较高表达,且存在组织差异性。果蝇lethal (1)G0334为丙酮酸脱氢酶(PDH)基因P转座子诱发的致死突变体,编码的蛋白质具有PDH的功能。利用生物信息学、RT-PCR和RACE等方法克隆了家蚕lethal (1) G0334相关基因,命名为Bm-l (1)。分析了基因结构与表达谱。Bm-l(1)基因的cDNA全长为1630 bp,由1200 bp的完整ORF序列、186 bp的5′UTR和207 bp的3′UTR组成,编码蛋白质为399个氨基酸残基,分子量43.93kD,pI为8.07。Bm-l(1)基因由8个外显子和7个内含子组成。Bm-l(1)编码的蛋白质在69-365氨基酸残基为E1-dh结构域,该结构域为硫胺素焦磷酸(TPP)依赖性脱氢酶所特有。蛋白质二级结构预测结果表明α螺旋占28.8%,β折叠占12.0%。采用Clustal W进行多序列比对发现,Bm-l(1)与赤拟谷盗等昆虫PDH具有63%以上的蛋白质同源性,保守区域高度一致。Bm-l (1)基因在整个卵期、幼虫期、蛹期以及5龄幼虫所检测的不同组织都有较高的表达量,且存在较小的组织差异性。脱氧羟腐胺赖氨酸合酶(DHS)和脱氧羟腐胺赖氨酸羟化酶(DOHH)是催化真核翻译起始因子(eIF5A)序列中羟腐胺赖氨酸(hypusine)合成的两个酶。羟腐胺赖氨酸的合成是eIF5A活化成熟的标志,对真核细胞的存活及增殖具有重要作用。果蝇lethal(3) s1921为DOHH基因P转座子诱发的致死突变体,编码的蛋白质具有DOHH的功能。利用生物信息学、RT-PCR和RACE等方法克隆了家蚕DHS和DOHH的相关基因BmDHS和BmDOHH,分析了基因结构和表达谱。BmDHS和BmDOHH的cDNA全长分别为1311 bp和1874 bp。BmDHS只有一个外显子,没有内含子,包含最大ORF为1116 bp,编码371个氨基酸,分子量为41.11 KD,pI为5.84。BmDOHH由4个外显子和3个内含子构成,包含最大ORF框为915 bp,编码304个氨基酸,分子量为34.30 kD,pI为4.86。BmDHS编码的蛋白质在47-361氨基酸残基位置为DHS的保守区域;BmDOHH编码的蛋白质分别在23-52,54-83,87-116,177-206,208-237,241-270氨基酸残基位置存在E-Z type HEAT重复区域。BmDHS和BmDOHH基因所编码的蛋白质与人、果蝇等生物均具有55%以上的蛋白质同源性,保守区域高度一致。系统发育树分析结果显示DHS和DOHH在分子进化上存在物种特异性。表达谱分析表明BmDHS和BmDOHH在所检测的组织中均没有组织和时空特异性。用4龄起蚕为材料,以桑抗冻蛋白基因dsRNA和ddH2O为对照,利用微量注射RNA干涉技术初步获得BmDHS基因沉默表型。沉默家蚕体内BmDHS基因mRNA水平平均下降了约25%,BmeIF5A基因mRNA水平平均下降了约4.8%。注射后10 d平均体重增加了42.9%,在上簇后5 d蚕茧的重量平均值增加了35.4%。初步证明BmDHS基因在家蚕生长发育中起重要作用。2.家蚕Osiris相关基因的克隆及表达研究Triplo-lethal Locus(Tpl)是果蝇基因组中唯一一个具有单倍体或三倍体致死效应的基因座。在Tpl区内存在一个Osiris基因家族,依次命名为Osiris1-Osiris20。通过BLASTP和PSI-BLAST分析,仅发现3个该家族基因位于Tpl区域外,命名为Osiris21-Osiris23。该基因家族为昆虫纲(Insecta)所特有。利用生物信息学、RT-PCR和RACE等方法克隆了家蚕6个Osiris基因家族的成员,依次命名为BmOsiris7、BmOsiris9、BmOsiris18、BmOsiris19、BmOsiris20和BmOsiris21。序列分析表明BmOsiris基因的序列ORF长度在723-882之间,所编码的氨基酸分子量大小也很接近,在26 kD-31 kD之间。pI分析表明BmOsiris21为9.10,其余在6.41-8.66之间。SMART在线保守区域预测发现,该蛋白家族属于跨膜蛋白,都含有Osiris家族特有的DUF1676保守区域,支持了克隆的所有基因序列属于Osiris基因家族的推测。Osiris家族蛋白序列进化树分析显示家蚕和黑腹果蝇的同系同源蛋白要近于同物种的旁系同源蛋白。表达谱分析表明各基因在所调查的组织中均没有组织和时空特异性,但表达量的多少存在显著差异。染色体定位结果显示BmOsiris7、BmOsiris9、BmOsiris18、BmOsiris19和BmOsiris20定位在Chr.26上,而BmOsiris21单独定位在了Chr.4上。家蚕Osiris基因家族的以上所有分析结果和黑腹果蝇中Osiris基因家族极其相似,推测家蚕Osiris家族的功能可能与果蝇相似。3.家蚕温敏性致死品系伴性赤蚁(Sch)Pros相关基因的克隆及序列比对研究果蝇Pros261和Prosβ21同属于显性温敏性致死突变基因,两者分别由蛋白酶体β6和β2亚基的单核苷酸突变造成的。家蚕大造品种Pros261和Prosβ21相关基因在NCBI上已被登录,但未见书面报道,本文分别命名为DzPros26和DzProsβ2。本实验依据登录的DzPros26和DzProsβ2的氨基酸序列,设计引物,在家蚕温敏性致死品系Sch中克隆了Pros261和Prosβ21相关基因,分别命名为SchPros26和SchProsβ2。ORF内碱基序列比对显示SchProsβ2和DzProsβ2的碱基序列完全一致。SchPros26和DzPros26的ORF内碱基序列比对显示SchPros26分别在223 bp、556 bp、621 bp、624 bp、658 bp和659 bp碱基位置发生突变。在线分析软件分析表明碱基序列的突变导致其编码蛋白质一级结构、二级结构和三级结构都发生了变化。蛋白质功能位点预测显示:家蚕Sch品系中由于SchPros26编码蛋白质的第75个氨基酸残基由甘氨酸(G)突变为丝氨酸(S)而造成其缺少蛋白酶体β亚基信号位点,而正常品系大造中DzPros26编码的蛋白质却具有蛋白酶体β亚基信号位点。这表明SchPros26可能与Sch品系的温敏致死表型有关。4.棉铃虫Pros相关基因的克隆及序列分析利用生物信息学、RT-PCR和RACE等方法克隆了棉铃虫Pros261相关基因,命名为Haproβ1。由于蛋白酶体β1-β7亚基都具有β亚基保守区域,之间具有同源性,因此我们以黑腹果蝇Prosβ21为质询序列在棉铃虫EST数据库中搜索到2条不能成为重叠群的EST序列,利用RACE和RT-PCR等方法克隆了棉铃虫Prosβ21的2个同源基因,命名为Haproβ5和Haproβ7。Haproβ1基因编码蛋白质的分子量为25.54 kD,pI为5.96,22-232氨基酸残基为蛋白酶体β1亚基的保守区域;HaProβ5基因编码蛋白质的分子量为30.87 kD,pI为6.53,74-261氨基酸残基为蛋白酶体β5亚基的保守区域;HaProβ7基因编码蛋白质的分子量为30.07 kD,pI为8.04,40-229氨基酸残基为蛋白酶体β7亚基的保守区域。经比对分析显示Haproβ1、Haproβ5和Haproβ7在不同物种间都相当保守,系统进化关系也很相似,均与已知的物种系统发生关系完全一致。基因表达谱分析结果表明:Haproβ1、Haproβ5和Haproβ7之间极其相似。都是在体壁、中肠和生殖腺中表达较高,在头部表达量最少。这可能与头部的蛋白质代谢活动没有其它所检测组织旺盛有关。更多还原

【Abstract】 Lepidoptera insects are the second biggest order in Insecta and have closely related with human in production and life. Much of Lepidoptera insects are agricultural pests. Silkworm is the model insect of Lepidoptera, which was determined by international non-vertebrate association, and is the only Lepidoptera biology that completed the whole genome sequencing. So it provided a convenient technology platform for functional genomics research.Since 2000, release of insects carrying a dominant lethal technology (RIDL) has been caught extensive attention. It’s principle is raised the male that carried conditional lethal genes, and then released into the wild.They will mate with the native population, then all the progeny of such matings will die , so it achieved the purpose of pest control. This technology has a species-specific and environmental characteristic. It has great significance to control agricultural pests. Currently, RIDL technology has been successfully tested in Drosophila melanggaster. In Lepidoptera pests, such as Helicoverpa armigera, also has yielded some results.In this study, we closely around the key word "lethal", relied on the protein sequence which encoded by Drosophila melanggaster lethal genes(P-element induced mutations), Osiris gene family that in the Triplo-lethal (triploid and haploid lethal) locus and thermo-sensitive lethal mutation Pros (Pros261 and Prosβ21) genes respectively, made use of bioinformatics, RT-PCR, RACE, expression and RNA interference technologies, cloned the relevant genes of Bombyx mori and down structural analysis and functional study. The major results are as follows:1. Cloning and expression analysus of the lethal-related genes (P-element induced lethal mutation) from Bombyx moril (3)neo18 gene of fruit fly is the lethal mutation of NADH dehydrogenase gene by inserted P element, so its’encoded protein has the function of NADH dehydrogenase. By using the methods of bioinformatics, RT-PCR and RACE technology, a full-length cDNA was isolated from silkworm, named as Bm-l(3)neo18. We had analyzed the structure and expression of Bm-l(3)neo18. It is 868 bp nucleotide long, contains an ORF (573 bp), 25 bp nucleotide sequence in 5’UTR and 251 bp nucleotide sequence in 3’UTR. It encodes 190 amino acids, with their predicted mass 22.7 kD and isoelectric point 9.60. Bm-l(3)neo18 contains 3 exons and 2 introns. Gene localization indicated that Bm-l(3)neo18 mapped within from 120.3knt to 120.4knt region of nscaf2930 in No. 3 chromosome. The deduced amino acid showed that a NADH dehydrogenase SGDH subunit domain between 1 to 185 amino-acid residue and a transmembrane region between 61-83 amino-acid residue. It has more than 50% identity to other insects such as Aedes aegypti. The NADH dehydrogenase conservative regions were much similar with each other. Molecular evolution by Neighbor Joining method indicated that Bm-l(3)neo18 had homologous with other NADH dehydrogenase of insects. Bm-l(3)neo18 was expressed lowly at egg stage, while expressed highly at larva, pupa and moth stages in our tested samples.lethal (1) G0334 gene of fruit fly is the lethal mutation of pyruvate dehydrogenase (PDH) gene by inserted P element, so its’encoded protein has the function of PDH. By using the methods of bioinformatics, RACE and RT-PCR technology, a full-length cDNA was isolated from silkworm, named as Bm-l (1). We had analyzed the structure and expression of Bm-l (1). It is 1630 bp nucleotide long, contains an ORF (1200 bp), 186 bp nucleotide sequence in 5’UTR and 207 bp nucleotide sequence in 3’UTR. It encodes 399 amino acids, with their predicted mass 43.93 KD and isoelectric point 8.07. Bm-l (1) contains 8 exons and 7 introns. The deduced amino acid showed that a E1-dh domain between 69 to 365 amino-acid residue and it is dehydrogenases (all of which use thiamine pyrophosphate as a cofactor) specificity. Secondary structure prognosticate results revealed:αhelix is 28.8%,βstrand is about 12.0%.It has more than 63% identity to other insects such as Tribolium castaneum. The conservative regions were much similar with each other. Bm-l(1) was expressed at egg stage, larva, pupa and no tissue and stage specific in our tested samples.Deoxyhypusine snyhtase (DHS) and deoxyhypusine hydroxylase (DOHH) are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A (eIF5A). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. lethal(3) s1921 gene of fruit fly is the lethal mutation of DOHH gene by inserted P element, so its’encoded protein has the function of DOHH. Here we described the cloning and expression of two full length cDNAs, encoding respectively DHS-like protein and DOHH-like protein from Bombyx mori by using the methods of bioinformatics, RT-PCR and RACE technology, named as BmDHS and BmDOHH. Sequencing results indicate that they are 1311 bp and 1874 bp in length including complete open reading frame (ORF) 1116 bp and 915 bp, which encode 371 amino acids (molecular weight is about 41.11 kD and isoelectric point is 5.84) and 304 amino acids (molecular weight is about 34.30 kD and isoelectric point is 4.86), respectively. BmDHS contains only one exon, and BmDOHH contains 4 exons and 3 introns. The deduced amino acid sequence of BmDHS contains a deoxyhypusine synthase domain from 47 to 361 amino acid residues, and the deduced amino acid sequence of BmDOHH contains six E-Z type HEAT repeat domains (23-52, 54-83, 87-116, 177-206, 208-237, 241-270). Compared to DHS and DOHH amino acid sequences from other species, such as Homo sapiens, Drosophila melanogaster, both silkworm DHS protein and DOHH protein have more than 55% identity. The conservative regions are very similar with each other. The phylogenetic tree analysis indicated that not only DHS but also DOHH from different species has genus-specific features. The expressions of BmDHS and BmDOHH are no tissue and stage specific in our tested samples.By using mulberry antifreeze protein gene’dsRNA and autoclaved distilled water as control, based on microinjection RNA interference technology, we have preliminarily got BmDHS gene silence phenotype by injecting 4th larval instar. Compared with control group, in the silent silkworm, the average level of BmDHS mRNA was reduced almost 25%; the average level of BmeIF5A mRNA was reduced about 4.8%; the average larval weight increased by 42.9% 10 days after injection; the average cocoons weighet increased by 35.4% at the fifth day after mounting. It preliminarily proofed that BmDHS gene plays an important role in silkworm’s growth and development.2. Cloning and expression analysis of the Osiris-related genes from Bombyx moriTriplo-lethal Locus (Tpl) was surveyed as the only one that both triplo-lethal and haplo-lethal gene locus in the Drosophila genome. There was an Osiris gene family named gene Osiris1 through Osiris20 in the Tpl region. Through BLASTTP and PSI-BLAST analysis, there are only three other members of this family located outside of this locus. We called them Osiris21 to Osiris23. This gene family has the Insecta specificity. Through bioinformatics, RT-PCR and RACE methods, six silkworm genes belonging to Osiris gene family have been cloned and named as BmOsiris7、BmOsiris9、BmOsiris18、BmOsiris19、BmOsiris20 and BmOsiris21, respectively. Sequence analysis indicated that BmOsiris gene’s ORF are between 728 bp and 882 bp in length. The molecular size of deduced amino acid which seemed quite similar with Drosophila Osiris protein and are 26 kD-31 kD range. The analysis of isoelectric point indicated that BmOsiris21’s isoelectric point is 9.0, others are at between 6.41 and 8.66. SMART conservative region prediction on line found that this protein family belong to transmembrane protein and have specific conservative regions for the Osiris family, called DUF1676. All above dated supported the speculation that the cloned genes belonged to Osiris gene family. Phylogenetic tree analysis demonstrated paralogs of Bombyx mori and Drosophila melanogaster are much closer to the infraspecific orthologs. Expression pattern analysis indicated that each gene in our tested samples has no tissue and space-time speciality, but expression amount had significant differences. chromosome location showed that BmOsiris7、BmOsiris9、BmOsiris18、BmOsiris19 and BmOsiris20 located on the Chr.26, however, BmOsiris21 individually located on Chr.4. This result is very similar with Osiris genes from Drosophila melanogaster.3. Cloning and sequence alignment investigation of Pros-related genes from Bombyx moriBoth Pros261 and Prosβ21 in Drosophila belong to dominant mutation of temperature-sensitive lethality, and they are caused by mononucleotide mutation of proteasomeβ6 subunit and proteasomeβ2 subunit, respectively. Pros-related genes in Dazao breed of Bombyx mori have been registered in NCBI, but have not yet reported in written. In this article we named them as DzPros26 and DzProsβ2, respectively. This experiment designed primers by amino acid sequence of DzPros26 and DzProsβ2 registered, and cloned Pros-related genes in temperature-sensitive lethal system of Sch breed, named them as SchPros26 and SchProsβ2. Sequence alignment of ORF indicates the sequence of SchProsβ2 is is in perfect accordance with DzProsβ2. Sequence alignment of SchPros26 ORF and DzPros26 ORF showed that SchPros26 has mutated at the site of 223 bp, 556 bp, 621 bp, 624 bp, 658 bp and 659 bp. The results of the online software analysis displays that the base mutation brings the changes of primary structure, secondary structure and tertiary structure of deduced amino-acid residue sequence. Predicting results of proteinic functional site shows that Sch breed runs short of signal site of proteasomeβsubunit, because the seventy-fifth residue of SchPros26 protein, Glycine(G), has changed into Serine(S). However, DzPros26 protein in normal Dazao breed has the signal site of proteasomeβsubunit. It displays that SchPros26 may be relating with temperature-sensitive lethality of Sch breed.4. Cloning and sequence analysis of Pros-related genes from Helicoverpa armigera Homologous gene of cotton bollworm Pros261 has been cloned by using bioinformatics, RT-PCR, RACE and so on, named it as Haproβ1. Proteasomeβsubunits have the same conserved region and homologous with one another, so taking Drosophila Melanogaster Prosβ21 as question sequence, we find two EST sequences in cotton bollworm EST database. But they can not being contig with each other. Two homologous genes of cotton bollworm Prosβ21’have been cloned by the way of RT-PCR and RACE, called them Haproβ5 and Haproβ7, respectively. Protein encoded by Haproβ1 is 25.54kD, pI is 5.96, and amino-acid residues from 22 to 232 is the conserved region of proteasomeβ1 subunit; Protein encoded by Haproβ5 is 30.87kD, pI is 6.53, and amino-acid residues from 74 to 261 is the conserved region of proteasomeβ5 subunit; Protein encoded by Haproβ7 is 30.07kD, pI is 8.04, and amino-acid residues from 40 to 229 is the conserved region of proteasomeβ7 subunit. Alignment analysis indicates that Haproβ1、Haproβ5 and Haproβ7 are conserved in different species. They have the similar system evolution relationship that consistent with species evolution. The results of gene expression profiling analysis showed that Haproβ1、Haproβ5 and Haproβ7 are pretty similar, and high expression in body wall, midgut, gonad, but low expression in head, which is possibly because proteinic metabolic activity of head is lower than other detected tissues.更多还原