【作者】 李蕊；

【导师】 李雅轩； 胡英考；

【作者基本信息】 首都师范大学 ， 遗传学， 2007， 硕士

【摘要】 电子克隆（in silico cloning）是随着基因组计划和EST计划实施而发展起来的利用生物信息学手段进行基因克隆的新方法。相对于传统的基因克隆方法具有投入低、速度快、技术要求不高和针对性强等优点。随着人类基因组计划的实施，研究人员利用电子克隆的方法已经克隆了许多人的功能基因。但由于受序列资料的限制，植物领域的电子克隆还鲜有报道。随着EST数据库的不断丰富和完善，利用生物信息学的方法进行植物基因的电子克隆已经成为可能。本文以拟南芥基因cDNA序列为信息探针，对大豆EST数据库进行同源检索筛选，克隆了大豆维生素B₆合成过程中关键酶基因——吡哆醛激酶基因和吡哆醇生物合成蛋白基因，并将吡哆醇生物合成蛋白基因对马铃薯进行了遗传转化。本研究旨在根据物种间基因序列的同源性，建立一条跨物种电子克隆新基因的有效途径，并采用代谢工程的方法对马铃薯的品质进行改良。研究结果如下：1大豆吡哆醛激酶基因（PK）的克隆与分析以拟南芥吡哆醛激酶基因的cDNA序列为信息探针，对大豆（Glycine max）EST数据库进行同源搜索和序列拼接，获得了全长为1102bp的大豆吡哆醛激酶的基因序列（GenBank登陆号为DQ006813）。该cDNA序列的开放阅读框（ORF）位于第119-1045位，推测编码308个氨基酸。为了进一步验证电子克隆序列的正确性，在起始密码子和终止密码子区域设计特异引物，经RT-PCR克隆和序列分析验证，结果表明与电子克隆序列一致。将大豆（Glycine max）PK基因编码的蛋白序列与马铃薯（Solanum tuberosum，AAY85186）、拟南芥（Arabidopsis thaliana，AAK94021）、水稻（Oryza sativa，ABA99785）、小麦（Triticum aestivum，AAR00318）和油菜（Brassica napus，ABE73472）的吡哆醛激酶蛋白序列进行比对，发现其一致性分别为：81％、75％、78％、79％和76％。比对结果说明在植物中该基因编码的蛋白质存在显著的相似性。根据蛋白比对结果绘制的系统发育树基本代表了它们在经典分类上的地位。采用半定量RT-PCR方法分析光照时间对大豆PK基因表达量的影响，结果显示，PK基因的表达量随着光照时间的增加基本没有变化，证明大豆PK基因不受强光调控。组织表达谱分析结果表明PK基因在大豆根茎叶中都有表达。2大豆吡哆醇生物合成蛋白基因（PDX）的克隆与分析以拟南芥吡哆醇生物合成蛋白基因的cDNA序列为信息探针，对大豆（Glycine max）EST数据库进行同源搜索和序列拼接，获得了全长为1280bp的大豆吡哆醇生物合成蛋白的基因序列（GenBank登陆号为DQ139265）。该cDNA序列的开放阅读框（ORF）位于第20-955位，推测编码311个氨基酸。为了进一步验证电子克隆序列的正确性，在起始密码子和终止密码子区域设计特异引物，经RT-PCR扩增、基因组PCR扩增、分子克隆和序列分析验证，结果表明与电子克隆序列一致。将大豆（Glycine max）PDX基因编码的蛋白序列与日本百脉根（Lotus corniculatus var．japonicus，AAZ67141）、烟草（Nicotiana tabacum，AAS92255）、拟南芥（Arabidopsis thaliana，AAM66972）、截形苜蓿（Medicago truncatula，AAZ67140）、小麦（Triticum aestivum，AAZ94411）和水稻（Oryza sativa，EAZ38383）的吡哆醇生物合成蛋白序列进行比对，发现其一致性分别为：93％、92％、92％、92％、85％和70％。比对结果说明在植物中该基因编码的蛋白质存在显著的相似性。根据蛋白比对结果绘制的系统发育树基本代表了它们在经典分类上的地位。半定量RT-PCR结果显示，PDX基因的表达量随着光照时间的增加基本没有变化，证明大豆PDX基因不受强光调控。组织表达谱分析结果表明PDX基因在大豆根茎叶中都有表达。3大豆PDX基因对马铃薯的遗传转化将PDX基因通过农杆菌介导法转化马铃薯茎段和叶片，PCR检测结果显示，从42株抗性植株中得到6株转基因阳性植株；将阳性植株移栽至土壤中，进一步的工作正在进行中。更多还原

【Abstract】 In silico cloning is developing with the project of genomic and EST. It is a new method of gene cloning, which is applying with bioinformatics. Lower cost, rapid, simple and clear objective are all advantages of in silico cloning, compared with traditional strategies. With the human genomic project implement, scientists have cloned many human functional genes by in silico cloning, but it is rarely used in plants because of less sequences.In this paper, we used the EST Blast method to clone Glycine max pyridoxal kinase (PK) and pyridoxine biosynthesis protein (PDX) genes. Also, we took Glycine max PDX gene into potato by genetic transformation. The objective of this study is to establish available cloning gene approach based on the high similarity of homologous gene sequences in different species and improve the quality of potato through metabolic engineering.1 cloning and analysis of Glycine max PK geneAccording to the relative conservation of homologous gene, using Arabidopsis thaliana pyridoxal kinase cDNA sequence as a query probe to blast Glycine max EST database, a soybean PK gene cDNA of 1102bp was obtained (GenBank Accession, DQ006813). It contained a complete ORF, from 119bp to 1045bp, encoded 308 amino acids. To examine the accuracy of in silico cloning cDNA sequence, this sequence was cloned by RT-PCR and sequenced. The result confirmed that the in silico cloning cDNA sequence was accurate.The deduced amino acid sequence of Glycine max PK was aligned against related PK sequences of other species such as Solanum tuberosum, Arabidopsis thaliana, Oryza sativa, Triticum aestivum and Brassica napus. It showed the identity of 81% 75% 78% 79% and 76%, respectively. The results revealed the peptide encoded by Glycine max PK is significant similarity in plants. Phylogenetic tree of plant PKs is mainly consistent with the classification system of these plants.Using Semiquantitative RT-PCR, we found out the effect of the illumination time impacting the expression of PK. It revealed that the expression of PK was showing no difference with the illumination time increasing. In another word, PK gene was not regulated by illumination. Another conclusion was PK expressed in Glycine max leaves stems and roots.2 cloning and analysis of Glycine max PDX geneAccording to the relative conservation of homologous gene, using Arabidopsis thaliana pyridoxinebiosynthesis protein cDNA sequence as a query probe to blast Glycine max EST database, a soybean PDX gene cDNA of 1280bp was obtained (GenBank Accession, DQ139265). It contained a complete ORF, from 20bp to 955bp, encoded 311 amino acids. To examine the accuracy of in silico cloning cDNA sequence, this sequence was confirmed by RT-PCR, genomic PCR, molecular cloning and sequenced. The result confirmed that the in silico cloning cDNA sequence was accurate.The deduced amino acid sequence of Glycine max PDX was aligned against related PDX sequences of other species such as Lotus corniculatus var. japonicus, Nicotiana tabacum, Arabidopsis thaliana, Medicago truncatula, Oryza sativa and Triticum aestivum. It showed the identity of 93% 92% 92% 92% 85% and 70%, respectively. The results revealed the peptide encoded by Glycine max PDX is significant similarity in plants. Phylogenetic tree of plant PDXs is mainly consistent with the classification system of these plants.Semiquantitative RT-PCR proved that the expression of PDX was showing no difference with the illumination time increasing. Another conclusion was PAX expressed in Glycine max leaves stems and roots.3 potato genetic transformation of Glycine max PDX geneGlycine max PDX gene was transformed into potato leave discs and stem segments by Agrobacterium tumefaciens LBA4404. 42 explants were regenerated on selective media with Kanamysin, and 6 of them were confirmed by PCR as transgenic plants. Put the transgenic plants in the soil and further works are in process.更多还原