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Acta Agron Sin ›› 2010, Vol. 36 ›› Issue (10): 1634-1641.doi: 10.3724/SP.J.1006.2010.01634

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and SNP Analysis of TT1 Gene in Brassica juncea

YAN Ming-Li1,2,LIU Xian-Jun2,GUAN Chun-Yun2,LIU Li-Li1,LU Ying1,2,LIU Zhong-Song2,*   

  1. 1 School of Biology, Hunan University of Science and Technology, Xiangtan 411201, China; 2 Oilseed Research Institute, Hunan Agricultural University, Changsha 410128, China
  • Received:2010-04-05 Revised:2010-05-03 Online:2010-10-12 Published:2010-08-04
  • Contact: LIU Zhong-song,E-mail:zsliu48@sohu.com;Tel:0731-84617381

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Abstract: 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 homology-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 purple-leaf mustard. The allele-specific PCR of the TT1 gene could distinguish purple-leaf mustard from Sichuan Yellow.

Key words: Brassica juncea, TT1, Cloning, Single nucleotide polymorphism

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