摘要： 乙烯是调控果实成熟衰老的内源激素,有效地调控乙烯的生物合成便是控制果实成熟的关键,而ACC氧化酶是植物乙烯生物合成途径中的限速酶之一。本研究以成熟的白粉桃为材料提取果实总RNA,克隆ACC氧化酶基因,旨在利用反义技术抑制乙烯合成,延迟桃果实成熟,提高硬度。将克隆片段与pMD18-T载体连接并转化到E.coliDH5α,经PCR、酶切和测序鉴定。分析表明该基因全长为1246bp,其中编码区长960bp,共编码319个氨基酸,与已登陆的ACC氧化酶基因(AF319166)核苷酸序列和氨基酸序列的同源性分别为99%和98%,表明成功的克隆了ACC氧化酶基因。经生物信息学分析,推测该蛋白的分子式为C1626H2542N420O485S12,相对分子质量为36.12kDa,等电点为5.27;该蛋白为亲水性非分泌型蛋白,无信号肽存在;其蛋白质二级结构以α-螺旋、β-折叠和无规卷曲为主,是一个紧凑型球状结构域。

关键词: 桃, ACC氧化酶, 克隆, 生物信息学分析

Abstract: Ethylene regulation of fruit ripening and senescence is an endogenous hormone.Therefore,regulating the biosynthesis of ethylene effectivly is the key to controlling fruit ripening.1-Aminocyclopropane-1-Carboxylic acid oxidase(ACO)gene is one of the rate-limiting enzyme in ethylene biosynthesis of plant.So total RNA was extracted from mature Baifen peach in this study,and clone ACO gene.The use of antisense technology is to inhibit the formation of ethylene,delay fruit ripening and improve the hardness of peach.The cloned fragment connected with pMD18-T vector and transformed into E.coli DH5α with PCR,restriction enzyme digestion and sequencing.Sequence analysis showed that the gene had the length of 1 246 bp,which contained a 960 bp coding region,encoded a total of 319 amino acids,and compared with nucleotide sequence and amino acid sequence homology of ACC oxidase gene(AF319166)landed were 99% and 98%,which showed the successful cloning of the ACC oxidase gene.With the bioinformatics analysis,the predicted ACC oxidase protein's molecular formula was C1626H2542N420O485S12,whose relative molecular mass was 36.12 kDa and isoelectric point was 5.27;It is predicted that this protein was hydrophilicity and non-secretory protein,and there was no signal peptide.Characteristic prediction results indicated that the secondary structure of ACC oxidase protein mainly included α-helix,β-sheet and random coil,and this protein belonged to a kind of a compact globular domain.

Key words: Peach, ACC oxidase gene, Clone, Bioin formatics

中图分类号: 

• Q785