【作者】 刘显军；

【导师】 刘忠松；

【作者基本信息】 湖南农业大学 ， 作物遗传育种， 2008， 硕士

【摘要】 大量研究表明,在相同的遗传背景下,黄籽油菜与黑籽油菜相比,黄籽油菜具有含油量高、种皮薄、色素少、纤维素含量低、油清澈透明、蛋白质含量高等优点,培育高产优质的黄籽油菜成为重要的育种目标。研究表明,芥菜型油菜黄籽形成涉及类黄酮合成基因。本研究通过RT-PCR技术,对12个不同地区来源的芥菜型油菜品种授粉后20天的种皮分析类黄酮合成相关基因的表达情况,结果表明:结构基因PAL、C4H、CHI、F3H、F3′H、TT10、TT12、GST和调节基因TT2、TT8、TT16、TTG1、TTG2在12个黄、黑籽材料种皮间都有表达,而C4H、TT12、LAC和TT1在不同的黄、黑籽材料中的表达有量的差异。但是DFR和ANS在12个黄、黑籽种皮中有表达差异,这2个基因在5个黑籽材料种皮中有表达,而在7个黄籽材料种皮中没有检测到表达;而BAN基因在5个黑籽材料都有较高的表达,在3个黄籽材料中有较弱的表达,而其他的黄籽材料不表达。由此表明DFR,ANS,BAN这3个基因是油菜黄黑籽差异的关键基因。选用芥菜型油菜紫叶芥为材料,通过SMART^TM-RACE技术,从BjANS的保守区出发,分别获得BjANS基因的3’和5’端序列,利用DNAMAN4.0将保守区、3’和5’端序列拼接后得到BjANS基因全长cDNA序列。通过生物信息学分析,获得一个开放阅读框,编码359个氨基酸。构建了启动子为napinA的植物干扰载体pRNAi-ANS,并通过冻融法将重组质粒成功地导入根癌农杆菌菌株EHA105中,为进一步研究该基因的功能奠定了基础。更多还原

【Abstract】 The yellow-seeded rapeseed has such advantages as thinner seed coat, higher oil content, clearer oil, higher protein and lower fiber content compared with the black-seeded one under the same genetic background. Thus, it will have a bright future to develop high quality rapeseed with yellow-seeded character. Development of high yielding, high quality and yellow-seeded rapeseed is a current major aim of breeding. Formation of a yellow seed coat is showed to be associated with the expression of the genes for flavonoid biosynthesis in Brassica juncea.RT-PCR analysis was used to study the gene expression in the 20-days-after-pollination (20-DAP) seed coat of 12 Brassica juncea accessions. The expression of the structural genes PAL, C4H, CHI, F3H, F3 ’H, LAC, TT12 and GST and the regulatory genes 777, TT2, TT8, TTI6, TTG1 and TTG2 was detected in all the accessions studied. However, difference in level of expression of the four genes C4H, TT12, LAC and 777was found between the yellow and the black seed coat. The expression of the genes DFR and ANS was detected in all the five black-seeded accessions studied, while no expression of DFR and ANS was detected in the yellow-seeded accessions. The high expression of BAN was detected in all the five black-seeded accessions studied, but the weak expression in the three yellow-seeded accessions or no expression in the remaining yellow-seeded ones. It is suggested that DFR, ANS and BAN be the key genes responsible for differences in seed coat color.A Braasica juncea accession Purple Mustard(PM) was used for cloning of the gene ANS by RACE technology. 3’- and 5’-end fragments of the gene BjANS were cloned and joined with the conserved region using the software DNAMAN4.0. Full-length cDNA sequence of BjANS was obtained. Bioinformatics analysis showed that the gene BjANS encodes an open reading frame consisting of 359 amino acids.A plant RNA interference vector pRNAi-ANS under the promoter of a seed-specifically expressed gene napinA was constructed and subsequently introduced into the Agrobacterium tumefaciens strain EHA105 with freeze-thaw method, which provided foundations for analysis of the function of the gene.更多还原