罗汉果色氨酸转氨酶基因SgTAR2的克隆及表达分析

Cloning and expression analysis of tryptophan aminotransferase of Arabidopsis 1-Related 2

关键词:  罗汉果, 色氨酸转氨酶, 克隆, 基因表达

Key words:  Siraitia grosvenorii, tryptophan aminotransferase of Arabidopsis 1-Related, cloning, gene expression

摘要:

色氨酸转氨酶基因家族,是直接参与植物生长素生物合成途径的关键酶基因。该研究在罗汉果转录组测序的基础上,结合 RACE 技术克隆罗汉果色氨酸转氨酶基因SgTAR2的全长cDNA 序列和DNA序列; 并对其进行生物信息学分析和时空表达分析。结果表明:克隆所得SgTAR2的cDNA全长序列2 078 bp,最长ORF为1 332 bp,编码 443 个氨基酸,GenBank登录号为KU949381,其编码蛋白具有2个蒜氨酸酶保守结构域和多个5'-PLP结合位点,推测其可能参与催化色氨酸转氨基作用、化学防御作用、生长素生物合成等生物学过程; SgTAR2基因DNA长为4 103 bp,含有4个内含子和5个外显子,其内含子具有多个高水平转录调控因子和多个与激素、环境等胁迫响应相关的作用元件,暗示SgTAR2基因内含子协同调控罗汉果生长素合成、抗胁迫反应、形态发育等生物学过程。实时荧光定量结果显示,SgTAR2基因在罗汉果各组织器官均有表达,在雌蕊和15 d幼果期表达量较高,暗示该基因参与罗汉果果实早期发育。该研究结果表明SgTAR2参与了生长素介导的罗汉果不同生长发育过程,特别对幼果及花的起始发育和器官形态建成等具有重要意义

Abstract:

Tryptophan aminotransferase gene family is a key gene that directly involved in auxin biosynthesis pathway in plants. The full-length cDNA sequence and DNA sequence of Siraitia grosvenorii tryptophan aminotransferase of Arabidopsis 1-Related 2(SgTAR2)gene was amplified by using transcriptome sequencing data and the technology of rapid amplification of cDNA ends(RACE)method. Subsquently, analysis of the gene structure of SgTAR2 by bioinformatics, and the expression levels of SgTAR2 in different tissues of S. grosvenorii were analysed by real-time quantitative method. The results showed that cloned full length of SgTAR2 cDNA sequence was 2 078 bp, containing a 1 332 bp open reading frame(ORF)which encoded 443 amino acids, and GenBank accession number was KU949381. The coding protein had two alliinase domains and multiple 5'-PLP binding sites, which might take part in catalytic amino acid transfer, chemical defense, auxin biosynthesis and other biological processes. The full-length DNA sequence was 4 103 bp with four introns and five extrons, and these introns contained multiple 5UTR Py-rich stretch, hormonal and environmental stress responses related cis-acting elements, which might play a role in auxin biosynthesis, stress response, morphological development and other biological processes. The result of Real Time PCR exhibited that SgTAR2 was the highest abundance in pistils and 15 d fruit, and suggested that auxin synthesis was exuberant in initial development of S. grosvenorii fruit. Therefore, SgTAR2 plays a significant role in the growth and development of S. grosvenorii by auxin mediated, especially in young fruit and flower initial development and organ morphogenesis.