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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 the unigene sequence of the transcriptome, the full-length cDNA encoding key enzyme 1-aminocyclopropane-1-carboxylate synthase 1 (ACS1) was cloned from total RNA of Siraitia grosvenorius by RACE technique. Meanwhile, hi TAIL PCR Full length DNA of Sg ACS1 was obtained. The relative expression level of this gene in leaves, stems, buds, buds and flowers of male and female plants and Ag + -treated female plants before and after pollination / harvesting was detected by real-time fluorescence quantitative PCR. The full-length cDNA of 1 749 bp was cloned and the longest open reading frame was 1 530 bp encoding a protein of 509 amino acids. The sequence was named Sg ACS1 (GenBank accession number KX620760) Is 86%, which has a pyridoxal phosphate transferase domain and a transaminase binding domain and belongs to the family of aspartate aminotransferase; the full-length DNA of Sg ACS1 obtained is 3 245 bp which contains 3 introns, Intron and 5’UTR contain a number of high-level transcriptional regulatory elements, hormone response elements and environmental stress-related elements, suggesting that the gene involved in transaminase, biological synthesis of ethylene metabolism and other biological processes, and other hormones Work together to maintain the body’s normal life activities. Real-time PCR results showed that Sg-ACS1 was expressed in all parts and periods of Luo Han Guo. Among them, the relative expression level of Sg-ACS1 was highest in the flower buds of female plants after pollination, followed by the flowers of female plants before and after pollination and the shoots of male plants before and after pollination, However, the relative expression level of Agr + in the bud and bud after pollination was the lowest, which indicated that Sg ACS1 was involved in flower bud formation, stamen primordium differentiation, stamen development and female flower formation in Sg ACS1 Strain by pollination or silver ion-induced up-expression.