丹参晚期胚胎富集蛋白基因的克隆及表达模式分析

Molecular cloning and expression analysis of a late embryogenesis abundant protein gene(SmLEA2) from Salvia miltiorrhiza

目的克隆丹参晚期胚胎富集蛋白(late embryogenesis abundant protein,LEA)基因,并进行生物信息学和表达模式分析。方法对丹参转录组数据库进行BLAST同源性比对,发现一条新的与LEA基因家族同源性较高的序列,命名为Sm LEA2,Gen Bank登录号为HQ676610。进一步利用PCR和RT-PCR技术从丹参g DNA以及c DNA水平克隆得到了Sm LEA2基因全长。采用软件分析蛋白质结构性质和系统进化关系。利用实时荧光定量PCR的方法对不同部位、不同发育时期以及不同处理丹参的Sm LEA2表达量进行检测。结果获得Sm LEA2 g DNA长1 961 bp,由1个外显子和1个内含子组成,并且内含子位于ATG的上游;开放阅读框长为960 bp,共编码319个氨基酸。生物信息学分析显示,Sm LEA2是存在于细胞质中的亲水蛋白,无跨膜结构域和信号肽,其相对分子质量为35 340,等电点为4.77。该基因在丹参的根、茎、叶中均有表达,并且在茎中的表达量最高。随着花的成熟和种子的发育,该基因表达量逐渐升高,并且该基因的表达受茉莉酸甲酯(Me JA)以及脱落酸(ABA)的诱导作用影响。结论克隆得到的丹参Sm LEA2可能参与种子发育过程,并在丹参防御反应中发挥作用。

Objective To clone and characterize a late embryogenesis abundant protein SmLEA2 with its promoter region from Salvia miltiorrhiza, and to predict its probable function. Methods SmLEA2 was cloned by PCR and RT-PCR from genomic DNA and cDNA. Protein structure and phylogenetic relationships were carried out by bioinformatic analysis. Gene expression in different organs and different development periods was detected by qPCR. Gene expression was also detected under different treatments. Results By analyzing the cDNA library for S. miltiorrhiza with BLAST program, one of these sequences showed a high homology with late embryogenesis abundant （LEA） protein and was named as SmLEA2 （GenBank： HQ676610）. We obtained 1 961 bp gene sequences of SmLEA2, which contained an intron and a single opening reading frame （ORF） of 960 bp encoding 319 amino acid pepfides. Bioinformatic analysis showed that the putative SmLEA2 protein was a hydrophilic protein without signal peptides and transmembrane domains. The SmLEA2 protein was predicted with a molecular weight of 35 340 and a theoretical isoelectric point of 4.77. SmLEA2 was expressed in the roots, stems, and leaves of S. miltiorrhiza, and the most abundant in the stems. With the development of the flowers and seeds, its expression increased gradually. Expression of SmLEA2 could be induced by methyl jasmonate （MeJA） and abscisic acid （ABA）. Conelusion Thus, we speculate that SmLEA2 may be involved in seed development and plant defenses,