中国水仙水杨酸甲酯合成酶基因克隆与表达分析

Cloning and Expression Analysis of NtSAMT1 Gene from Narcissus tazetta var. chinensis

摘要:

为了解中国水仙(Narcissus tazetta var. chinensis)花香气形成的分子机理,以花发育形成相关基因的SSH文库获得的cDNA片段为基础,利用RACE技术从中国水仙花朵中克隆了水杨酸甲酯合成酶基因,命名为NtSAMT1 (GenBank No.JX273470),其cDNA全长1323 bp,包含1个1131 bp完整阅读框架,编码376个氨基酸,具有Methyltransf_7 superfamily蛋白保守区,与仙女扇(Clarkia breweri)SAMT蛋白(1m6eX)的三维结构相似。系统进化树分析表明,中国水仙与粳稻的SAMT蛋白亲缘关系最近。原核表达结果表明NtSAMT1在大肠杆菌中能高效表达。半定量RT-PCR分析表明,NtSAMT1在花蕾期就有表达,第1天完全开放时各部位均有表达,以雄蕊与雌蕊的表达量最高,第8天已检测不到表达。

Abstract:

In order to understand molecular mechanism of floral scent formation of Narcissus tazetta var. chinensis, based on a fragment cDNA from different florescence suppression subtractive hybridization (SSH), the salicylic acid carboxyl methyltransferase gene was cloned from N. tazetta var. chinensis by RACE and RTPCR, named NtSAMT1 (GenBank No. JX273470). The full length of NtSAMT1 cDNA was 1323 bp with an 1131 bp ORF, which encoded a protein of 376 amino acids with a conservative domain of methyltransf_7 superfamily. NtSAMT1 is similar to SAMT of Clarkia breweri (1m6eX) in three-dimensional structure. The NtSAMT1 is predicted an unstable protein. Phylogenetic analysis showed that SAMT in Narcissus tazetta var. chinensis had close relationship to that in Oryza sativa Japonia (BAD12867). Prokaryotic expression showed that recombinant pGEX-NtSAMT1 had highly expression in NE. coil BL21. Semi-quantitative RT-PCR revealed that NtSAMT1 could expression in all tissues of buds, the expression in stamens and pistils was the highest at first day after blooming and its expression disappeared at the eighth day after blooming. The expression of NtSAMT1 could be detected in scape, petal, corolla, stamen and pistil of the first day of blooming, expression of NtSAMT1 in the first day flowers was in order as stamen, pistil > scape > petal, corolla.