芥菜型油菜TT1基因的克隆和SNP分析

Cloning and SNP Analysis of TT1 Gene in Brassica juncea

拟南芥的TT1基因(编码含有WIP结构域的锌指蛋白)对种皮的发育和颜色的形成具有重要的调控作用。本研究利用同源克隆和RACE技术分离了芥菜型油菜TT1基因,在芥菜型油菜黄黑籽材料的种皮中进行转录水平的分析,比较了黑籽油菜与黄籽油菜基因序列的差异,并采用等位基因特异(allele-specific)PCR技术对可能存在的单核苷酸多态位点进行验证。结果表明,芥菜型油菜TT1基因的DNA序列全长为2197bp,包含1个内含子,与甘蓝型油菜TT1-1基因的DNA序列的相似性为99%,与拟南芥的TT1基因DNA序列的相似性为85%;推导的TT1蛋白序列为300氨基酸残基,理论分子量为33.97kD,等电点为6.99;TT1在所有材料的种皮中均检测到表达;比较紫叶芥、四川黄籽、NILA和NILB的TT1基因序列,共发现8个核苷酸变异位点,均在基因的外显子区域,其中紫叶芥和NILA的序列相同,四川黄籽和黒籽近等基因系NILB的序列相同。与紫叶芥相比,黒籽近等基因系NILB有8个核苷酸差异,但种皮颜色与紫叶芥一样,均为黑色,TT1基因这些位点的突变并不影响芥菜型油菜种皮的颜色。通过等位特异PCR可以区分来自四川黄籽与紫叶芥的TT1基因。

The quality of rapeseed can be further improved through the development of yellow-seeded cultivars, which are known to contain less lignin and more proteins, both enhancing the dietary feed value. The TT1 gene encodes a WIP domain protein with Zn-finger, which is essential for seed coat development and organ color in Arabidopsis, its mutation causes transparent testa. In order to study molecular mechanism of seed coat color in rapeseed, TT1 gene was cloned from Brassica juncea using homol- ogy-based cloning and RACE （rapid-amplification of cDNA ends） strategy. A modified allele-specific PCR procedure developed for assaying single nucleotide polymorphisms of TT1 gene in Brassica juncea. In this study, cloning and characterization of the orthologous TT1 gene from Brassica juncea were reported. The full length DNA sequence of TT1 gene was 2 197 bp with one intron. The TT1 cDNA sequence was 1 412 bp in length, 903 bp of which was open reading frames （ORF） encoding a deduced polypeptide of 300 amino acids with a predicted molecular weight of 33.97 kD and an isoelectric point of 6.99. The above-mentioned genomic sequences showed 99% identity with the TT1 gene from Brassica napus, and 85% identity with the TT1 gene from Arabidopsis. RT-PCR analysis showed that TT1 expressed in the seed coats of both the black- and the yellow-seeded B. juncea lines. Comparison of sequences of Sichuan Yellow （yellow seed） and purple-leaf mustard （black seed） revealed nucleotide variation at the eight sites which all are located within the coding region of the gene. However, the mutations of the TT1 gene at these sites had no effect on the seed coat color in B. juncea through comparing the sequences of NILB and pur- ple-leaf mustard. The allele-specific PCR of the TT1 gene could distinguish purple-leaf mustard from Sichuan Yellow.