黄黑籽甘蓝型油菜类黄酮途径基因SNP位点检测分析

SNP Detection and Analysis of Genes for Flavonoid Pathway in Yellow- and Black-Seeded Brassica napus L.

类黄酮物质在植物花、叶、果实和种子颜色变化的过程中起着至关重要的作用，本研究以不同黄黑籽种皮材料为研究对象，采用基因同源克隆方法，获得17个类黄酮基因全长ORF序列，在核酸和蛋白水平上分别序列差异比较表明，这些基因在不同黄黑籽材料中共存在41个不同拷贝成员。在核苷酸水平上，检测到 BnTT3、BnTT18、BnTTG1和BnTTG2的单核苷酸位点数目介于16-52之间，且BnTTG2在3个不同的位置上还存在多个碱基的连续性缺失现象（119-121 bp、183-189 bp和325-330 bp），但在蛋白水平上仅存在2-16个氨基酸位点差异，说明BnTT3、BnTT18、BnTTG1和BnTTG2在不同甘蓝型黄黑籽材料中存在单核苷酸位点差异，而单核苷酸位点突变不一定导致氨基酸位点的变异。在不同黄黑籽材料中仅BnTT3和BnTT18存在一致性的氨基酸突变位点（252和87），推测BnTT3和BnTT18可能在黄黑籽甘蓝型油菜种皮颜色差异形成过程中发挥至关重要的作用。通过这些位点的等位特异PCR可以区分材料间透明种皮基因，为特异基因芯片的开发及阐明甘蓝型油菜种皮色泽性状的基因及其作用位点奠定基础。

Flavonoids as the secondary metabolites play a crucial role in colour changing process of flower, leaf, fruit and seed. In this research, primers for amplifying full-length ORF sequences of the genes involved in the favonoid biosynthesis pathways were designed according to conserved nucleotide regions from the public databases. Using the homology-based cloning strategy, 41 gene copies were obtained from 13 genes using 17 pairs of specific primers in different yellow-and black-seeded seed coats of B. napus. Each of full-length ORF sequences was sequenced and analyzed in the levels of both nucleic acid and protein. The results showed that the SNPs in four flavonoid pathway genes （BnTT3, BnTT18, BnTTG1, and BnTTG2）, ranged from 16 to 52, but there were olny 2 to 16 amino acid mutations detected in the protein level, indicating that the mutation of SNPs may not be involved in the mutation of amino acid. In addition, continuous bases deletion existed in different positions of sequence of BnTTG2 （119 to 121 bp, 183 to 189 bp, and 325 to 330 bp）, and two consistent amino acid mutation sites were detected in BnTT3 and BnTT18 among different materials, inferring that BnTT3 and BnTT18 may play an important role in difference of seed coat colour formation in yellow-and black-seeded B. napus. Therefore, these genes involved in flavonoid pathway could be distinguished by the allelic-specific PCR in yellow-and black-seeded B. napus. These results could help to develop specific seed coat gene chips and elucidate the genes and their action sites for the seed coat colour in B. napus.