摘要
L-抗坏血酸是一种抗氧化剂,能够保护植物免受活性氧的伤害,GDP-甘露糖-3′,5′-异构酶基因(GME)是L-抗坏血酸合成途径中的关键基因。根据其他植物GME的氨基酸保守序列设计简并引物,通过RT-PCR结合RACE扩增技术,从柠条叶片中克隆了1个编码GME的基因,命名为CkGME,Genebank登录号为FJ603689。基因全长1604bp,开放阅读框1134bp,编码377个氨基酸,氨基酸同源性比对分析表明,该基因编码的氨基酸与其他植物GME基因具有较高的同源性。进化树分析表明,该基因编码的氨基酸序列与豆科植物大豆和截型苜蓿的亲缘关系最近,与十字花科植物拟南芥的亲缘关系次之,与禾本科植物水稻和玉米的亲缘关系较远。利用4℃和GA3处理不同时间的柠条植株进行半定量RT-PCR分析结果表明,该基因在4℃处理不同时间后表达量没有明显变化,在GA3处理不同时间后表达量显著下降。利用pCAMBIA-1300质粒构建含35S启动子的正义表达载体,酶切图谱和PCR检测结果证明该表达载体构建成功。
L-ascorbic acid is a kind of antioxidant, which could protect the plants from ROS. GDP- mannose-3＇, 5＇-epimerase（GME） gene is a key gene in the biosynthesis pathway of L-ascorbic acid. Using a pair of degenerate primer designed based on the sequences of the homologous gene from other plants and the RACE technology, a full-length cDNA-encoding GME gene was isolated and characterized from the leaves of Caragana korshinskii. The 1604bp full-length CkGME （accession number FJ603689） had a 1134bp ORF, which encodes a peptide of 377 amino acids. The deduced amino acid sequence of CkGME protein shared high identity with other GMEs. Phylogenetic analysis indicated that CkGME is closely related with Glycine max and Medicago truncatula, then with Arabidopsis thaliana, but far away from Oryza sativa and Zea mays. Semi-quantitive RT-PCR analysis indicated that gene expression of CkGME had no difference compared with control after treated with 4℃ for a during time, but was down-regulated after treated with GA3. Subsequently, the over-expression vector of CkGME gene was constructed with the aid of pCAMBIA-1300.
出处
《中国草地学报》
CSCD
北大核心
2009年第5期75-83,共9页
Chinese Journal of Grassland
基金
"十一五"科技支撑项目(2008BADB3B01
2007BAD56B02)
国际科技合作与交流专项(2008DFR30200)
"973"项目(2007CB108905-2)
