甜菜M14品系表达基因M14-100、M14-265、M14-G12 cDNA全长克隆与序列分析

【作者】 崔颖；

【导师】 李海英；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 甜菜M14品系是由栽培甜菜(Beta vulgaris L.)与野生白花甜菜(Beta corolliflora Zoss.)种间杂交,进一步回交获得的甜菜无融合生殖单体附加系,其染色体组除了含有18条栽培甜菜染色体外,还附加有白花甜菜第9号染色体,附加染色体的传递率高达96.5%,如此高的传递率是由于附加的第9号染色体携带无融合相关基因所致。因此,甜菜M14品系是无融合生殖研究极其难得的材料。同时,甜菜M14品系还具有抗寒、抗病的优良性状,对M14品系特异表达基因的研究,也有助于我们挖掘和利用甜菜M14品系具有的优良基因。为了克隆甜菜M14品系中无融合生殖相关基因,实验室前期采用抑制消减杂交方法获得了298个甜菜M14品系特异表达的ESTs。本研究挑选其中3个有研究价值的ESTs作为侯选ESTs,采用SMART RACE技术获得了侯选ESTs所在基因——M14-100、M14-265和M14-G12的cDNA全长,并采用RT-PCR法进行验证,最后对三个目的基因的cDNA全长进行了生物信息学分析。M14-100 cDNA全长1 707bp,GenBank数据库接受号为EU529829,经过生物信息学分析,该基因序列与来自拟南芥(Arabidopsis thaliana)的细胞周期蛋白相关基因序列有60~70%的相似性,但该基因的功能目前还不清楚;M14-100 cDNA最长读码框编码324个氨基酸;推定编码蛋白的分子量为34.4 kD,理论等电点5.11,有较强的亲水性;为非分泌型蛋白;推定蛋白二级结构分析结果表明,含有6个α-螺旋和3个β-折叠。M14-265 cDNA全长1 006bp,GenBank数据库接受号为EU529830,经生物信息学分析认为该基因是一个新基因;缺乏明显的读码框,最长读码框仅编码81个氨基酸,可能以RNA的形式参与生命活动,也可能编码短肽;推定编码蛋白的分子量为9.3 kD,理论等电点4.69,为非分泌型蛋白;推定蛋白的二级结构预测表明,该蛋白含有1个α-螺旋和2个β-折叠。M14-G12 cDNA全长873bp,GenBank数据库接受号为EU529828,与来自拟南芥(Arabidopsis thaliana)的钙调蛋白基因的氨基酸序列相似性达到99%,可以认定该基因即是来自甜菜M14的钙调蛋白基因;该基因编码149个氨基酸,分析结果显示其编码蛋白分子量为16.8 kD,理论等电点4.11,总平均亲水性为-0.620;二级结构预测发现该蛋白质是一个富含α-螺旋的蛋白质。三级结构预测,该蛋白包含4个螺旋-环-螺旋结构,与钙调蛋白的功能特征相符合。更多还原

【Abstract】 Apomictic monosomic addition line M14 of Beta corolliflora Zoss,was the progeny from the hybridization of Beta vulgaris L. and Beta corolliflora Zoss. in sugar beet. Constituted of the normal 18 Beta vulgaris L. chromosomes with the No.9 chromosome of Beta corolliflora Zoss, M14 was found having a chromosome transmission frequency 96.5%. The reason of apomixis in M14 was interpreted as possible existence of apomixis gene(s) in the additional chromosome, thus M14 is a precious material for research of apomixis. Meanwhile M14 was cold and disease resistant. Study of the specifically expressed genes in M14 will provide direct molecular evidence to reveal the special reproductive process of M14, and will help us to discover and use excellent genes of M14.In order to isolate and identify the apomixis gene(s) of M14, we have obtained 298 differential expression ESTs by the method of SSH and DDRT-PCR, three of which were selected as candidate ESTs in this study. SMART RACE was played to obtained full-length cDNA sequences of gene M14-100, M14-265 and M14-G12, which carry the three ESTs respectively. The sequences were then identified by RT-PCR, and bioinformatic analysis was also carried out.The results showed that M14-100 (GenBank accession No. EU529829) was 1 707bp in length, and shared 60~70% similarity with cyclin related genes from Arabidopsis thaliana by the method of bioinformatic analysis. But the function of cyclin was not clear. The longest ORF of M14-100 contained 324 amino acids. Molecular weight of its putative protein was 34.4 kD, and theoretical isoelectric point was 5.11. It has strong hydrophilicity, and is non-secretion protein. Secondary structure prediction of M14-100 putative protein indicated that it contained 6α-helices and 3β-strands.M14-265(GenBank accession No. EU529830) was 1 006bp in length. By the method of bioinformatic analysis we didn’t find any genes which share similarity with it, thus we deduced that M14-265 was a new gene. There is no significant ORF in M14-265, and the longest ORF contained 81 amino acids. M14-265 maybe encode a noncoding RNA or short-peptide. Molecular weight of its putative protein was 9.3 kD, and theoretical isoelectric point was 4.69. It is non-secretion protein. Secondary structure prediction of M14-100 putative protein indicated that it contained 1α-helices and 2β-strands.M14-G12 (GenBank accession No. EU529828) was 873bp in length, and shared 99% similarity with calmodulin gene from Arabidopsis thaliana. It was suggested that M14-G12 was calmodulin gene of M14, and its ORF contained 149 amino acids. Molecular weight of its coding protein was 16.8 kD. Theoretical isoelectric point was 4.11. Its total average hydrophilicity was -0.620. Secondary structure prediction of M14-G12 coding protein indicated that it was anα-helix rich protein. Three-dimensional structure prediction of M14-G12 coding protein showed that it contained four helix-spring-helix structures, which kept accordance with the function of calmodulin.更多还原