【作者】 王艳敏；

【导师】 张国政；

【作者基本信息】 江苏科技大学 ， 特种经济动物饲养， 2009， 硕士

【摘要】 家蚕既是重要的经济昆虫,又是鳞翅目昆虫模式生物。充分利用家蚕基因组和功能基因组研究优势,发掘影响鳞翅目昆虫胚胎发育、性别决定、性细胞发育等重要生命过程相关的关键功能基因,不仅为进一步对家蚕进行分子改良和分子设计育种提供依据,也将为其它鳞翅目害虫的防治提供借鉴。家蚕个体在性别间的经济价值具有较大差异,雄蚕具有更高的茧丝转化效率。一般认为,家蚕在个体发育的早期阶段即已经完成性别的决定与分化,但是家蚕性别决定的分子机制研究目前尚不明了,尤其是家蚕早期发育阶段性别决定、分化和发育相关的功能基因还缺乏任何信息。为了探索家蚕早期胚胎性别间差异表达基因的信息,本实验首次以单性生殖技术获得的家蚕单一性别早期胚胎发育蚕卵为材料,利用抑制消减杂交技术(SSH)构建雌性与雄性早期发育卵的正向、反向抑制消减杂交cDNA文库,进行了差异表达基因的分析。主要研究结论如下:1、家蚕单性生殖卵的制备以菁松×皓月品种家蚕为材料,利用非减数分裂孤雌生殖(AMP)和双精雄核发育(BSA)单性生殖技术,分别获得家蚕单一雌性和雄性早期胚胎发育蚕卵。经调查两者转色卵率分别为76.56%和76.43%,具有较高的发育卵率且差别不大。2、家蚕单性生殖抑制消减杂交cDNA文库构建为了获得家蚕性别间差异表达的基因信息,本实验采用正、反向抑制消减杂交。正向消减以AMP ( 0～72 h )蚕卵为试验组对BSA ( 0～24 h )蚕卵为参照组进行抑制消减杂交,构建孤雌生殖消减cDNA文库。反向消减则以BSA ( 0～24 h )蚕卵为试验组对AMP ( 0～72 h )蚕卵为参照组进行抑制消减杂交,构建雄核发育消减cDNA文库。两个消减文库中的产物多集中在200～750 bp之间。在正向消减文库中随机选择62个阳性克隆进行测序,共获得43种cDNA序列,长度在88～479 bp之间;在反向消减文库中随机选择46个阳性克隆进行测序,共获得29种cDNA序列,长度为132～724 bp之间。3、家蚕单性生殖抑制消减杂交cDNA文库分析在孤雌生殖消减文库的43种cDNA序列中,12个序列在2个或2个以上测序克隆中出现,总出现频次占克隆总数的50%。26个序列有同源家蚕cDNA存在,17个序列未能找到高度同源的家蚕EST或cDNA,为新发现的家蚕cDNA序列。在NCBI、Silk DB等数据库中进行BLAST分析,有19个序列与已知功能基因(Unigenes)高度同源,7个序列与推定蛋白(Hypothetical protein)基因高度同源,17个为未知基因。已知基因多为热激同源蛋白、核糖体蛋白、逆转录酶、转座酶、转录或翻译调控因子、转运蛋白等。在雄核发育消减文库的29种cDNA序列中,11个序列在2个或2个以上测序克隆中出现,总出现频次占克隆总数的60.87%。21个序列有同源cDNA存在,8个序列未能找到高度同源的家蚕EST或cDNA,为本实验新发现的家蚕cDNA序列。在NCBI、Silk DB等数据库中进行BLAST分析, 16个序列与已知功能基因高度同源,13个为未知基因。已知基因为Cu / Zn SOD、脱氢酶类、转座酶、谷胱甘肽S-转移酶、热休克蛋白及核糖体蛋白等。4、家蚕差异表达基因的克隆与分析对孤雌生殖消减文库中的5个序列( AMP05、AMP20、AMP25、AMP29、AMP50 )分别进行了cDNA末端快速扩增。延长后的cDNA经NCBI中BLASTx分析,AMP05同埃及伊蚊( Aedes aegypti )、AMP29同衣虱( Pediculus humanus corporis )推定蛋白基因同源,AMP20在谷蛀虫( Tribolium castaneum )中有同源基因,但是未能在家蚕中找到同源基因。AMP25、AMP50序列仍未能找到同源基因序列与之相对应。对雄核发育消减文库中的5个序列( BSA12、BSA22、BSA23、BSA29、BSA30 )也分别进行了cDNA末端快速扩增。延长后的cDNA经NCBI中BLASTx分析,BSA12、BSA22同家蚕已知基因铜锌SOD、TCTP高度同源,确认是该基因的序列。BSA29经tBLASTx分析,该序列3’端与编码家蚕的细胞溶质苹果酸脱氢酶( cytosolic malate dehydrogenase,1424 bp )高度相似,但5’端140 bp并不相同。BSA23、BSA30序列仍未能找到同源的基因序列与之相匹配。5、差异表达基因的半定量RT-PCR分析从每个文库中分别选择6个序列,对家蚕孤雌生殖和雄核发育蚕卵进行半定量RT-PCR分析。结果表明,AMP01 ( BmHSC70-4 )是孤雌生殖卵相对于雄核发育卵表达差异显著的基因,BSA05、BSA30序列所代表的基因是雄核发育卵相对于孤雌生殖卵表达差异显著的基因,三者均为上调表达基因。6、差异表达基因的实时荧光定量RT-PCR分析利用实时定量RT-PCR技术分析部分消减文库序列在孤雌生殖发育卵和雄核发育卵中所代表基因的表达水平比较。在孤雌生殖消减文库中,AMP01在孤雌生殖发育卵中的表达量是雄核发育卵中的4.41倍,而AMP05、AMP20、AMP39、AMP50在雄核发育蚕卵与孤雌生殖发育蚕卵中的表达量差异值在1倍以内。在雄核发育消减文库中,BSA05、BSA30在雄核发育蚕卵中的表达量分别是孤雌生殖蚕卵的3.01倍和3.92倍,而BSA07、BSA12、BSA29仅相差1倍多,差异不明显。更多还原

【Abstract】 As it is well known that Silkworm, Bombyx mori is an important economic insect, also an important organism model of lepidoperan insects,so the research background of silkworm genome and functional genome, which were achieved recently, is significant to explore the key crucial functional genes of lepidoperan insects related to life activities such as embryonic development, sex determination, sexual cell development.They not only provide a basis for the further molecular improvement and molecular design of silkworm breeding,will also be used for reference to control other lepidoperan pests.Silkworm individuals have the differences in economic value between male and female, and the male has high silk conversion efficiency.In general, the sex determination and sex differentiation of silkworm have been completed in the early embryos stages, but the research of molecular mechanism in sex determination of Bombyx mori has not yet been resolved.In particular, the functional genes related to sex determination, sex differentiation and development of silkworm in early development stage is a lack of any information.In order to explore the differentially expressed genes in early embryo between male and female of silkworm, in this experiment,the early embryos of silkworm parthenogenesis eggs were prepared by Ameiotic Parthenogenesis ( AMP ) and Bispermic Androgenesis ( BSA ) technology and the forward and reverse direction subtracted hybridization cDNA libraries between female and male of silkworm were constructed using Suppression Subtractive Hybridization ( SSH ).We also have done validation of differentially expressed sequences obtained from the two subtracted libraries.The main conclusions are as follows:1.The preparing of parthenogenesis eggs of silkwormThe early embryos of parthenogenesis eggs were prepared by AMP and BSA parthenogenesis technology in the jingsong×Haoyue silkworm varieties . After investigation, both the percentage of pigmented eggs are 76.43% and 76.56%, with higher rates and not very different.2.The construction of silkworm parthenogenesis SSH cDNA libraryIn order to explore the differentially expressed genes in early embryo between male and female of the silkworm, in this experiment, the forward and reverse direction subtracted hybridization cDNA libraries were constructed between female and male of the silkworm using SSH.The forward is AMP( 0 ~ 72 h) silkworm eggs as the experimental group against BSA ( 0 ~ 24 h ) eggs as a reference group to carry out SSH and construct a AMP cDNA subtracted library.The reverse is BSA (0 ~ 24 h) eggs for the experimental group against AMP( 0 ~ 72 h ) eggs as a reference group to carry out SSH and construct a BSA subtracted cDNA library.The products of two subtracted libraries are more between 200 bp and 750 bp.In the forward subtracted library 62 positive clones were selected to sequence and obtained 43 kinds of cDNA sequences between 88 bp and 479 bp.In the reverse subtracted library 46 positive clones were selected to sequence and obtained 29 kinds of cDNA sequences between 132 bp and 724 bp.3.Sequence analysis of cDNAs in SSH cDNA subtracted libraryThere are 12 sequences in the AMP subtracted library of 43 kinds of cDNA sequences appear two or more than two clones, with a total frequency of emergence of the total clones accounted for 50% . There are 26 sequences existing silkworm homology cDNA sequences,17 sequences could not be found silkworm high homology ESTs or cDNAs are the new cDNA sequences of Bombyx mori in our experiment.Through the BLAST analysis in NCBI and Silk DB database, there are 19 sequences have a high homology with the known functional genes (Unigenes), 7 sequences have a high homology with the Hypothetical protein genes, 17 sequences are unknown genes.The unigenes are Heat shock cognate protein, Ribosomal protein, Reverse transcriptase, Transposase enzyme, Transcription or Translation regulatory factors and Ransporter and so on.There are 11 sequences in the BSA subtracted library of 29 kinds of cDNA sequences appear two or more than two clones, with a total frequency of emergence of the total clones accounted for 60.87%.There are 21 sequences existing silkworm homology cDNA sequences.8 sequences could not be found silkworm high homology ESTs or cDNAs are the new cDNA sequence of Bombyx mori in our experiment.Through the BLAST analysis in NCBI and Silk DB database, there are 16 sequences have a high homology with the unigenes, 13 sequences are unknown genes . The unigenes are Cu / Zn SOD, Dehydrogenase class, Transposase, Glutathione S-transferase, Heat shock protein and Ribosomal protein and so on.4.Molecular cloning and analysis of Bombyx mori differentially expressed genesRapid Amplification of cDNA Ends ( RACE ) were separately carried out on the five sequences ( AMP05, AMP20, AMP25, AMP29, AMP50 ) of AMP subtracted cDNA library.Through the BLASTx analysis in NCBI of cDNA extended, AMP05 with Aedes aegypti and AMP29 with Pediculus humanus corporis have homology with the Hypothetical Protein gene.AMP20 have a homology gene in Tribolium castaneum , but failed to find in Bombyx mori.AMP25 and AMP50 don’t have homology sequences to match.RACE also were separately carried out on the five sequences ( BSA12, BSA22, BSA23, BSA29, BSA30 ) of BSA subtracted cDNA library.Through the BLASTx analysis in NCBI of cDNA extended, BSA12 and BSA22 have high homology with silkworm Cu / Zn SOD and TCTP, which are confirmed these genes.The 3 ’end of BSA29 is highly similar to the Cytosolic malate dehydrogenase (1 424 bp) by tBLASTx analysis, but the 5’ end (140 bp) are not the same which worth exploring.BSA23 and BSA30 don’t have homology sequences to match.5.Analysis of differentially expressed genes by semi-quantitative RT-PCRSix sequences from each subtracted library were chosen to do semi-quantitative RT-PCR in AMP and BSA silkworm egg samples.The results show that, AMP01 ( BmHSC70-4 ) is represented significantly differentially expressed genes in AMP silkworm egg.BSA05 and BSA30 are represented significantly differentially expressed genes in BSA silkworm egg.They are up-regulated expression.6.Analysis of differentially expressed genes by real-time fluorescence quantitative RT-PCRThe genes expression levels from the parts of differentially expressed sequences in the two subtracted library ware compared by real-time fluorescence quantitative RT-PCR in AMP and BSA silkworm eggs.The results show that, the expression of AMP01 in AMP silkworm eggs is 4.41 times different comparing to that in BSA silkworm eggs.But the expression of AMP05, AMP20, AMP39 and AMP50 in BSA silkworm eggs is not significant than in AMP eggs in 1 time difference.The expression of BSA05 and BSA30 in BSA silkworm eggs is 3.01 and 3.92 times different comparing to in AMP silkworm eggs.But the expression of BSA07, BSA12 and BSA29 ware not significant only 1 time difference.更多还原