Cloning and SNP Analysis of TT1 Gene in Brassica juncea

Abstract

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.