【作者】 牛宝龙；

【导师】 吕顺霖； 孟智启；

【作者基本信息】 浙江大学 ， 特种经济动物学， 2005， 博士

【摘要】 生物信息学(Bioinformatics)是近年来在生命科学领域由分子生物学和计算机信息处理技术相结合的一个交叉学科,它的基本出发点是从核酸和蛋白质序列出发,分析序列中表达的结构功能的生物信息。 随着家蚕基因组序列的完全解读,家蚕分子生物学数据库信息积累将越来越多,家蚕EST数据库中已有近12万条序列,利用已经获得的大量EST、cDNA克隆和基因组序列等生物信息资源对家蚕基因组中所包含的约2万多个基因的功能进行研究,是家蚕功能基因组研究的主要任务,而建立在生物信息数据库基础之上的电子克隆技术是进行功能基因研究的有效手段。 本文通过对家蚕EST数据库的检索筛选,克隆了家蚕生命活动过程中的两个关键基因,预测了它们的功能;并利用它们各自的生物信息资源对它们的表达调控和染色体定位进行了研究。研究结果如下: 1 家蚕磷酸甘油醛异构酶基因(Bmtpi)的克隆与染色体定位分析 以长翅蝶磷酸甘油醛异构酶(tpi)基因cDNA序列为探针,对家蚕EST数据库进行同源检索筛选,克隆了家蚕磷酸甘油醛异构酶(Bmtpi)基因的cDNA序列,基因全长1,051bp。通过NCBI的ORF finder服务器对所获得的cDNA序列进行开放阅读框架(ORF)分析,结果发现其开放阅读框架位于第95～839位,推测编码248个氨基酸。为了验证所克隆Bmtpi基因cDNA序列的正确性,在其起始密码子区域和终止密码子区域设计特异引物,经RT-PCR克隆、序列分析验证,结果表明与电子克隆序列完全一致。 通过设计特异引物,克隆到全长为2,875bp的家蚕Bmtpi基因组序列(GenBank登记号为AY734490)。通过与cDNA序列进行比对,发现家蚕Bmtpi基因组序列内有5个内含子,且剪切位置均符合“GT-AG法则”,大小分别为580bp、297bp、103bp、1,187bp和333bp;还有6个外显子大小分别为66bp、120bp、112bp、110bp、207bp和319bp,其ORF横跨5个外显子。分析家蚕Bmtpi基因ORF上游700bp基因组DNA序列的启动子区域,结果发现存在1个启动子,位于第267～317位,可能性为0.81,没有发现典型的TATA-box和CAAT-box。 家蚕Bmtpi基因编码248个氨基酸的蛋白质(BmTPI),在蛋白质数据库中进更多还原

【Abstract】 In recent years, the bioinformatics has become one of the focuses in genetics and biology. It is an interdiscipline formed from molecular biology and computer science. It is based on biological databases and information. It aims to reveal the relationship between structure and function of nucleic acid and protein sequences by analysis these data and alignment these sequences.With the fulfilment of silkworm genome sequencing, the documentation in the silkworm biological databases increase such as there are nearly 120 thousands ESTs in silkworm EST database. How to use these information is very important for advance the progress of the silkworm genomic research. Analysing the function of near 20 thousands genes will become the main goal of the silkworm functional genomic research. In silica cloning the novel genes based on the EST database is an important principle in the field.In this thesis, we used the EST blast to in silico clone two novel silkworm genes: triosephosphate isomerase （Bmtpi） and transformer-2 （Bmtra-2）. Moreover, we predicted the function of the putative peptides encoded by the genes. We also studied the copies of Bmtpi in the silkworm genome by comparing fluorogenic real-time quantitative PCR technique with Bmkettin and DH-PBAN as the reference gene and the model of Bmtra-2 pre-mRNA alternative splicing pathway during processing by analysis its relative EST sequence polymorphism.1 cloning and analysis the copies of silkworm Bmtpi gene in silkwormgenomeSilkworm Bmtpi gene full-length cDNA sequence of 1,051 bp deduced encoding triosephosphate isomerase was cloned by blasting the silkworm EST database with the butterfly, Heliconius tpi gene cDNA sequence information as a querying probe. Searching the cDNA sequence for potential coding regions by ORF finder （NCBI）, It has a complete open reading fragment （ORF: 95⁸39） and putative encodes 248 aa protein. To examine the accuracy of in silico cloning cDNA sequence, we cloned byRT-PCR and sequenced a cDNA fragment covering the coding region of Bmtpi gene. The result confirmed that the in silico cloning cDNA sequence was very accurate.The full-length genomic sequence of silkworm Bmtpi gene was cloned and sequenced （GenBank accession, AY734490）. The result of comparing DNA sequence to cDAN sequence showed there are 5 introns with length of 580bp, 297bp, 103bp, 1,187bp, and 333bp, respectively and 6 exons with length of 66bp, 120bp, 112bp, HObp, 207bp and 319bp, respectively, all the boundary of extrons/introns have GT/AG conserved sequences, the ORF spans 5 extrons. It was predicted that there is one potential promoter sequence with 0.81 score located at the position of 267th³17th site but no TATA-box and CAAT-box in 700bp upstream silkworm Bmtpi transcript site.Blasting the deduced amino acid sequence of silkworm Bmtpi gene against the protein motif database in NCBI GenBank showed very high similarity to that of triosephosphate isomerase. It was aligned against related TPI sequences of other species such as Drosophila melanogaster （GenBank accession, CAA40804）, Anopheles gambiae （XP₃21467）, Tenebrio molitor （CAD43178）, Helicoverpa armigera and Heliothis virescens （AAA79847）, and the identity were 70%, 70%, 76%, 80% and 80%, respectively. It was revealed that the putative peptide encoded by silkworm Bmtpi is significant similarity in sequence and function to TPIs of other insects. Phylogenetic tree of insect TPIs is mainly consistent with the classification system of these insects.According to the above analysis results, we can predict the deduced encoding protein by silkworm Bmtpi is similar function to triosephosphate isomerase （TPI, EC 5.3.1.1） for catalysis of conversion dihydroxyacetone phosphate into glyceraldehydes 3-phosphate in glucose glycolysis and it plays a vital role for silkworm life-support system.In butterfly Heliconius, tpi gene has been located on the Z chromosome. We defined the technical conditions for real-time quantitative PCR to co-amplify Bmtpi and reference genes using fluorogenic probes in one PCR tube and the comparative cycle threshold method to detect the copies of Bmtpi in the silkworm genome. Bycomparing the copy of Bmlpi gene with that of reference genes DH-PBAN, located on 11th chromosome （GenBank accession, D16230）, the copy ratio of Bmtpi to DH-PBAN were 0.5 and 1 in female and male silkworm respectively such as the ratio of Z chromosome to antosome set in female and male silkworm. The copy ratio of Bmtpi to Bmkeltin, mapped on Z chromosome （GenBank accession, AB079865）, was1 in both sex silkworm genome. It means that the copy of Bmtpi in silkworm genome is equal to that of Bmkettin. So we concluded that Bmtpi is linked with Bmkettin and located on the Z chromosome.2 cloning and analysis pre-mRNA alternative splicing pathway of silkworm Bmtra-2 geneThe cDNA sequence of the Bombyx mori that encoded amino acid peptide containing the RNA recognition motif （RRM） was selected as a probe to blast the Bombyx mori EST database for homologous clones. A novel silkworm gene {Bmtra-2) full-length cDNA of l,275bp with a complete ORF （129-984） and encoded an identical 284-aa protein was cloned and verified by RT-PCR.The full-length genomic sequence of 7,838bp of silkworm Bmlra-2 gene was cloned and sequenced （GenBank accession, AY626066）. The result of comparing its DNA sequence to cDAN sequence showed there are 7 introns with length of 127bp, l,413bp, 999bp, 556bp, l,220bp, l,020bp, and l,230bp, respectively and 8 exons with length of 143bp, 134bp, 205bp, 129bp, 173bp, 48bp, 141bp and 302bp respectively, all splice sites of exons/introns conformed to the GT/AG rule, the ORF spans 8 extrons.The deduced amino acid sequence of silkworm Bmtra-2 gene contains one RRM motif, two arginine （R）- and serine （S）-rich domains （RS domain） at each end and a seven-glycine （G）-rich region located between RRM and the C-terminus of RS domain. The overall organization of this protein is similar to many insects TRA-2 and other RNA binding proteins. It showed 80% of homology to Apis mellifera TRA-2（GenBank accession, XP₃96858）, 65% to Drosophila virilis TRA-2 （AAB58113）, 64% to Musca domestica TRA-2 （AAW34233）, 60% to Drosophila melanogaster TRA-2 （AAA28953） and 58% of Anopheles Anopheles TRA-2 （EAA13826）. The conserved regions were the RRM and RRM-linker junction region. Based on the organizational and sequence similarities of this silkworm TRA-2 to other insects TRA-2, we tentatively designated this gene as the silkworm Bmtra-2. Although similarity extended throughout the entire protein, the RS domains were of low alignment, it should be noted the glycine （G） rich region was not contained in other insects known TRA-2 protein, BmTRA-2 has RRM motif and can interact with specific silkworm pre-mRNAs to affect their splicing patterns in a manner analogousto the way as other insectsTRA-2.According to the sequence polymorphism of ESTs selected from the blasting output ofBombyx mori EST database there are multiple transcripts from Bmtra-2 gene. By comparing these ESTs to Bmtra-2 DNA sequence, it was found that there are six mRNA produced from Bmtra-2 pre-mRNA using alternative splicing pathway. The model of Bmtra-2 pre-mRNA alternative splicing pathway during processing was verified by RT-PCR. All six isoforms deduced from six alternative spliced mRNA of Bmtra-2 pre-mRNA have the same second structure and all contain one RRM and two RS domains at each end. There are differences of phosphorylation site among them. Whether these differences of phosphorylation site bring about different roles will be analyzed in the future.In this article, we successfully cloned silkworm Bmtpi gene and Bmtra-2 gene through in silico cloning gene approach together with experimental verification. Their functions were predicated by blasting protein database and comparing with other insect homologues. We located Bmtpi on Z chromosome by comparing the copies ratio of Bmtpi to DH-PBAN and Bmkettin between male and female silkwormgenome. We also found that Bmtra-2 gene can produce six mRNAs by alternative splicing pathways during its pre-mRNA processing just like that of tra-2 gene of Drosophila. But the functions of two genes should be studied with the more effective approach in the future. The effective approach established in this study should contribute to study of large-scale functional genes in silkworm genome.更多还原