遗传 ›› 2008, Vol. 30 ›› Issue (10): 1363-1371.doi: 10.3724/SP.J.1005.2008.01363

• 研究报告 • 上一篇    下一篇

西伯利亚蓼半胱氨酸合成酶基因的克隆与表达

刘明坤; 刘关君; 魏志刚; 阎秀峰; 曲春浦; 王垠; 刘桂丰; 杨传平

  

  1. 东北林业大学林木遗传育种与生物技术教育部重点实验室, 哈尔滨 150040

  • 收稿日期:2008-04-03 修回日期:2008-06-11 出版日期:2008-10-10 发布日期:2008-10-10
  • 通讯作者: 刘关君

Cloning and expression of cysteine synthase gene from Polygonum sibiricum Laxm.

LIU Ming-Kun; LIU Guan-Jun; WEI Zhi-Gang; YAN Xiu-Feng; QU Chun-Pu; WANG Yin; LIU Gui-Feng; YANG Chuan-Ping   

  1. The Laboratory of Forest Genetics and Breeding and Biotechnology of Ministry of Education, Northeast Forestry University, Harbin 150040, China
  • Received:2008-04-03 Revised:2008-06-11 Online:2008-10-10 Published:2008-10-10
  • Contact: LIU Guan-Jun

摘要:

摘要: 半胱氨酸合成酶是植物半胱氨酸合成反应的关键限速酶。文中应用RACE技术从西伯利亚蓼中成功克隆了半胱氨酸合成酶基因(GenBank登录号: EU597481), 命名为PcCSase1, 该基因全长cDNA为1 260 bp, 编码382个氨基酸。经生物信息学分析, 初步确定PcCSase1的N端前16个氨基酸为信号肽, 并引导PcCSase1蛋白定位于胞质, 为胞质型半胱氨酸合成酶。同源序列分析表明, 此蛋白与其他植物半胱氨酸合成酶成熟蛋白序列高度保守, 氨基酸相似性达到90%左右。荧光定量RT-PCR分析表明, PcCSase1在西伯利亚蓼的叶、茎和地下茎中均有表达, 叶中表达最高, 茎和地下茎次之, 在3% NaHCO3胁迫过程中, 该基因在叶、茎和地下茎中均在第2 d表达量最高。将PcCSase1转入酿酒酵母INVSc1, 结果显示培养基中半胱氨酸和菌体中谷胱甘肽含量均有显著增加, 在10% NaHCO3和5 mol/L NaCl胁迫下, 转基因INVSc1-pYES2-PcCSase1菌株的存活率明显高于对照INVSc1-pYES2, 证明PcCSase1基因具有耐高盐的作用。

关键词: 半胱氨酸合成酶, 西伯利亚蓼, 表达分析, 定量RT-PCR

Abstract:

Abstract: Cysteine synthase is a key enzyme for restricting plant cysteine synthesis. Cysteine synthase gene, designated PcCSase1 (GenBank accession no. EU597481), was successfully isolated from Polygonum sibiricum Laxm. by RACE technique. This gene was 1 260 bp in full-length and encoded a peptide of 382 amino acids. Based on bioinformatic analysis, PcCSase1 is a cytoplasm cysteine synthesis and contains a 16 amino-terminal (N-terminal) signal peptide, which led the PcCSase1 to go to the cytoplasm. The results obtained through homologous sequence analysis indicated that PcCSase1 ma-ture protein was highly conserved in plants, which shared approximate 90% in the amino acid sequence. Expression analy-sis by RT-PCR showed that PcCSase1 gene was expressed in leaf, stem and root with the largest expression in leaf. Under 3% NaHCO3 stress, the largest expression of PcCSase1 gene was detected in leaf, stem and root at the second day following stress. PcCSase1 gene was inserted into pYES2 and transformed into yeast cells (Saccharomyces cerevisiae). The contents of the glutathione in the recombinant yeast and the cysteine in the medium were increased. INVSc1-pYES2-PcCSase1 was more tolerant to salt treatment than INVSc1-pYES2 and the former survival rate was higher than that of the later under the stress of 10% NaHCO3 and 5 mol/L NaCl. These results proved that PcCSase1 gene may confer high salt-tolerance.