【作者】 牛洪斌；

【导师】 翟虎渠；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2005， 博士

【摘要】 通过对水稻胚乳cDNA文库筛选，RT-PCR扩增并结合3’RACE克隆得到3个水稻谷蛋白新基因、1个二硫键异构酶（PDI）基因，并对上述基因的结构特点、表达特性进行了研究。在此基础上，利用农杆菌介导将反义PDI基因导入水稻植株，获得经PCR验证的转基因植株，并对外源基因表达水平及其对内源靶基因表达的干扰进行研究，试图探索水稻PDI功能及其与种子贮藏蛋白形成的关系。研究结果如下： 1 谷蛋白新基因克隆、结构特点和表达特性 以³²p标记谷蛋白基因GluB-2的cDNA片段为探针，筛选水稻胚乳cDNA文库获得一个新的谷蛋白基因全长cDNA。根据cDNA序列设计引物，以粳稻品种秀水11基因组DNA为模板，通过特异性PCR扩增获得基因组序列，命名为GluB-7（GenBank注册号AY987390）。 以谷蛋白基因保守核苷酸序列为探针，搜索水稻基因组数据库，并利用基因预测软件对高度同源的基因组序列进行基因预测，寻找未知谷蛋白新基因。结果在GenBank登录号为AP005511的contig中发现存在2个未知谷蛋白新基因。根据预测基因序列设计引物，经RT-PCR扩增获得2个特异性cDNA片段。在此基础上采用3’RACE对上述两个基因片段的3’端进行扩增、序列拼接，最终获得了2个新的谷蛋白基因全长cDNA，分别命名为GluB-6（GenBank注册号AY429651）和GluC-1（GenBank注册号AY429650）。 序列分析显示，GluB-7 cDNA全长1588bp，包含一个长度为1488bp的开放读码框（Opern Reading Frame，ORF），并编码495个氨基酸残基，预期分子量为56.02kD。GluB-6和GluC-1cDNA全长分别为1570bp和1624bp，ORF长度为1488bp和1455bp，编码氨基酸数目分别为495和484个。体外表达证实GluB6和GluC-1均能按上述读码框翻译形成正确的蛋白。 氨基酸序列比对显示，GluB-7、GluB-6和GluC-1与已知谷蛋白基因在氨基酸水平上相似性分别处于61.9-97.8％、61.9-97.8％和57.1-67.6％水平之间。聚类分析表明，谷蛋白基因家族含有3个亚家族，GluA、GluB和GluC亚族，GluB-7和GluB-6属于典型的B亚族基因成员，而GluC-1为新亚家族，C亚族。 组织表达谱分析显示，GluB-7、GluB-6和GluC-1具有高度的胚乳表达特性。GluB7完整表达谱显示，谷蛋白基因表达高峰在花后15天。更多还原

【Abstract】 Rice seeds accumulate large storage proteins (seeds storage protein, SSP) in endosperm during seed development, including glutelins and prolamins which account for up to 90% of the total proeins of the seeds. Both glutelins and prolamins were deposited in protein bodis (PBs), prolamins deposit in PB-Ⅰ while glutelins in PB- Ⅱ.In this study, three glutelin genes and one protein disulfide isomerase (PDI) gene cDNA were cloned with cDNA library sellection and reverse trascription polymerase chain reaction (RT-PCR) in addation to 3’ rapid amplification of cDNA ends (3 ’RACE). Then, the characterization structure and expression pattern of genes’ were analysized. In order to identify the the function of PDI, a prokaryotic expression vector and one plant expression vector were constructed respectively.The results were as followings:(1) Cloning, expression, and charactorization of three cDNA encoding glutelin from rice. After screening the cDNA library with α -32P labeling GluB2 partial cDNA sequence obtained from RT-PCR, a full-length glutelin cDNA was isolated, named GluB-7. The genome sequence of GluB7 was obtained by PCR with the DNA template from XiuShui 11 and the sepecial primers designed with the cDNA seqeucne. The full-length cDNA of GluB-7 is 1588bp, which containing a 1488bp OREAfter BLAST research with conserved sequence of rice glutelin genes reported previously, many identical contigs were obtained. We predict these contig sequence with gene prediction software (www.softberry.com ) and analysized the putavie gene by BLAST with the database (www.ncbi.nlm.nih.gov ) . The results shows that a contig, GenBank accession number AP005511 may contain two new glutelin genes. According to the prediction sequences, we design two gene-specific primers. Two cDNA was obtained by RT-PCR using as template totaol RNA extracted from endosperm 12DAF, and the products size were 1517bp (GluB-6) and 1519bp (GluC-1) . Based on the GluB-6 and GluC-1 fragment we designed primers for 3’RACE. Both GluB-6 and GluC-1 complete更多还原