罗汉果乙烯合成酶SgA CS1基因的克隆与表达分析

Cloning and Expression Analysis of 1-aminocyclopropane-1-carboxylic Acid Synthase Gene (Sg ACS1) from Siraitia grosvenorii

在转录组unigene序列基础上,结合RACE技术,从罗汉果总RNA中克隆乙烯合成关键酶-1-氨基环丙烷-1-羧酸合成酶1(ACS1)全长cDNA,同时,应用hi TAIL PCR技术获得Sg ACS1的DNA全长。通过实时荧光定量PCR检测该基因在雄株、雌株以及Ag^+处理后的雌株在取/授粉前后的叶、茎、芽、花蕾、花中的相对表达量。克隆得到1 749 bp的cDNA全长,最长开放阅读框为1 530 bp,编码509个氨基酸,命名为Sg ACS1(GenBank登录号为KX620760),编码蛋白与同源物种苦瓜的相应蛋白序列相似性为86%,该蛋白具有磷酸吡哆醛转移酶结构域和转氨酶结合结构域,属于天冬氨酸转氨酶家族;获得Sg ACS1的DNA全长为3 245 bp,其中含有3个内含子,该内含子和5'UTR含有多个高水平转录调控因子、激素响应元件和环境胁迫相关的作用元件,暗示该基因参与转氨反应、生物体内乙烯的合成代谢等生物学过程,并且与其他激素共同发挥作用维持机体的正常生命活动。实时荧光定量PCR结果表明,Sg ACS1在罗汉果各个部位和时期均有表达,其中在授粉后的雌株花蕾中相对表达量最高,其次是授粉前后雌株的花和取粉前后雄株的芽,而Ag^+处理后的雌株在授粉后的花蕾和芽中的相对表达量最低,说明该暗示罗汉果Sg ACS1参与花芽形成、雄蕊原基分化、蕾中雄蕊发育以及雌花形成,Sg ACS1在雌株蕾中受到授粉或银离子诱导上调表达。

Based on transcriptome unigene sequence, a full-length cDNA of 1-aminocyclopropane-1-carboxylic acid synthase gene（ACS1） from a key enzyme in ethylene biosynthesis pathway was cloned from total RNA of Siraitia grosvenorii by rapid-amplification of cDNA ends（RACE）. Then, its full-length DNA sequence was obtained via hi thermal asymmetric interlaced PCR（hi TAIL PCR）. The relative expressions of the gene in leaves, stems, buds, alabastrums and flowers of male plants and female plants as well as Ag~＋-treated female plants before and after pollination were detected by quantitative real-time PCR. As a result, the full length cDNA of the gene（1 749 bp）with the longest open reading frame（ORF） of 1 530 bp was obtained and designated as Sg ACS1（GenBank accession number： KX620760）. The protein with 509 amino acids shared 86% similarity with Momordica charantia L.which had pyridoxal phosphate transferase domain and aminotransferase domain and classified as the aspartate aminotransferase family. The full length DNA of Sg ACS1（3 245 bp） contained three introns. The introns and the5＇UTR had a plurality of high level transcription factors, hormonal response elements and regulatory elements of environmental stress which were suggested that the gene was involved in transamination, in vivo ethylene anabolism and interaction with other hormones in maintaining normal life activities and other biological processes.Real-time PCR results showed that Sg ACS1 was expressed in various parts and phases of the plant. The relative expression of the alabastrums of the female plant after pollination was the highest, then the expressions of the flowers of the female plant and the buds of the male plant before and after pollination was higher than others, on the contrary the expressions of bud and alabastrums of the Ag~＋-treated female plant were the lowest. It revealed that Sg ACS1 was involved in the bud formation, stamen primordia differentiation, stamen development, and female flower formation which was the up-regulated expression induced by pollination or silver ion in the alabastrums of the female plant.