Cloning and SNP Analysis of TT1 Gene in Brassica juncea

TT1 gene encodes a WIP domain protein with Zn-finger, which is essential for seed coat development and organ color in Arabidopsis. The mutation of this gene causes transparent testa. The BjTT1 gene was cloned from B. juncea using homology-based cloning and rapid-amplification of cDNA ends (RACE) strategy. A modified allele-specific PCR procedure was developed for assaying single nucleotide polymorphisms (SNP) of the BjTT1 gene. The full length of BjTT1 sequence was 2197 bp with only one intron. The cDNA sequence of BjTT1 was 1412 bp in length including 903 bp of open reading frames. This gene encodes a deduced polypeptide of 300 amino acids with a predicted molecular weight of 33.97 kD and an isoelectric point of 6.99. The genomic sequence of BjTT1 showed 99% and 85% identity with that of BnTT1 and AtTT1, respectively. The reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the expressions of BjTT1 in the seed coats of both black-seeded near-isogenic lines NILA and NILB. Based on sequence comparisons between BjTT1 genes from parents with different seed coat colors and between the NILB mutant and its wide type parent, nucleotide variations at 8 sites inside the BjTT1 coding region were detected, but mutations at these sites had no effect on seed coat color. The allele-specific PCR of BjTT1 could distinguish the yellow-seeded and black-seeded parents.

摘 要拟南芥的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2 197 bp, 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33.97 kD, 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。