Abstract:

Based on the reported EST from Leymus chinensis, the chitinase ClassⅡgene (GenBank accession number GQ397277) from leaves of Leymus chinensis under 100 mmol/L Na2CO3 stress, named LcChi2 gene, was cloned by RACE. The sequencing and bioinformatic analysis revealed that the opening reading-frame (ORF) of this gene was 771 bp and encoding 256 amino acid. LcChi2 protein belonged to chitinase ClassⅡ, Family 19. The molecular weight of the protein was 27.4 kDa and the isoelectric point was 8.67. LcChi2 protein was highly identified with chitinase in Triticum aestivum, Hordeum vulgare, Secale cereale and Oryza sativa, sharing 96.5%、96.5%、95.2% and 80.5% homology, respectively, indicating that they had similar biological functions. The gene was expressed in E.coli for preparation of its polyclonal antibody used for detection of transgenic plant. The data predicted by bioinformatics are helpful for further analysis of LcChi2.

Key words: Leymus chinensis, chitinase, RACE, bioinformatics, prokaryotic expression

摘要：

根据已报道的羊草几丁质酶基因的EST序列,利用RACE技术,从100 mmol/L Na2CO3胁迫的羊草叶片中克隆得到羊草几丁质酶基因cDNA序列全长(GenBank登录号为GQ397277),命名为LcChi2基因。经序列测定和生物信息学分析,结果表明该序列开放阅读框为771 bp,编码256个氨基酸,为Ⅱ类几丁质酶,属于19家族,分子量约为27.4 kDa,预测等电点为8.67,与小麦(Triticum aestivum)、大麦(Hordeum vulgare)、黑麦(Secale cereale)和水稻(Oryza sativa)等植物的几丁质酶具有高度的同源性,序列相似性分别为96.5%、965%、95.2%和80.5%,推测LcChi2蛋白与其他几丁质酶执行相似的功能。在大肠杆菌中进行了原核表达,为后期植物转化的验证准备多克隆抗体。本研究所获得的信息为今后对羊草几丁质酶基因功能的进一步研究奠定了基础。

关键词: 羊草;几丁质酶;RACE技术;生物信息学;原核表达