茶树CsASMT基因的克隆和表达分析

Cloning and Expression Analysis of CsASMT Gene in Tea Plant(Camellia sinensis)

该研究在茶树转录组测序的基础上,以茶树品种‘舒茶早’为试验材料,采用SMARTTM RACE技术,克隆了茶树褪黑素合成途径中关键限速酶N-乙酰基-5-羟色胺甲基转移酶基因(CsASMT)的cDNA全长序列。CsASMT基因序列全长1 282bp,其中包含1 065bp完整开放阅读框(ORF),编码354个氨基酸,预测等电点5.37,蛋白分子量38.98kD。系统进化分析表明,CsASMT与珠美海棠(Malus zumi)ASMT9的亲缘关系最近。将目的基因分别与pET-32a(+)和pMal-c2X载体重组,然后再转化入原核表达菌株BL21(DE3)中,pET-32a(+)/CsASMT在28℃用0.5mg/mL IPTG诱导6h后,以包涵体蛋白的形式表达,而pMal-c2X/CsASMT经诱导后在上清中可获得分子量为79kD大量可溶性融合蛋白。实时定量PCR分析表明,茶尺蠖取食抑制CsASMT基因表达;褪黑素、ABA、MeJA和SA均能够诱导CsASMT基因显著上调表达。该研究结果为N-乙酰基-5-羟色胺甲基转移酶功能研究奠定了一定的基础。

Based on the tea plant transcriptome database,we cloned the full-length cDNA sequences of the key rate-limiting enzyme-N-acetylserotonin methyltransferase(CsASMT)in the melatonin synthesis pathway using SMART TM RACE from the tea plant cultivars‘Shuchazao’.The full length cDNA was 1 282 bp,with an ORF of 1 065 bp,encoding 354 amino acids.The predict protein molecular and theoretic isoelectric point of CsASMT are 38.98 kD and 5.37,respectively.Phylogenetic tree analysis showed that CsASMT had the closest genetic relationship with Malus zumi ASMT9.The target gene was cloned into pET-32a(+)and pMal-c2X vector to construct a recombinant vector,respectively,and then transformed into E.coli BL21(DE3).After they are induced by 0.5 mg/mL IPTG at 28℃for 6 h,pET-32a(+)/CsASMT is expressed as inclusion body protein.But,pMal-c2X/CsASMT is expressed as a large amount of soluble protein with molecular weight of about 79 kD in supernatant.Quantitative real-time PCR analysis showed that the expression of CsASMT was down-regulated under the treatment of Ectropis oblique feeding.Exogenous application of melatonin,ABA,MeJA and SA could significantly up-regulated the CsASMT.The obtained conclusion would provide a basis for elucidating further the function of CsASMT.