一种新的抗真菌几丁质酶基因的分离及其植物表达载体的构建

以西瓜尖镰孢菌诱导、提纯的豇豆抗真菌 I类几丁质酶 N端前 1 0个氨基酸序列测定的基础上 ,设计合成了引物 ,运用 PCR等分子生物学技术 ,从豇豆基因组中分离克隆了该特异几丁质酶成熟蛋白基因 ,测定分析了其全序列。该新基因全长 894bp,无内含子 ;具 Aat I、Aat II、Bgl I、Dpn I、Dpn II、Eco R II、Hae I、Hae II、Hae III、Hinf I、Hpa II、Mae II、Mae III、Nba I、Oxa I和 Sst IV酶切位点 43个 ;豇豆、Vigna unguiculata、菜豆、豌豆、烟草、小麦、水稻的同源性依次递减。扩增克隆了菜豆几丁质酶信号肽基因 ,并将其与豇豆几丁质酶成熟蛋白基因连接 ,再与 p BI1 2 1重组 ,成功构建了特异几丁质酶基因的植物表达载体 ,为进一步培育抗真菌病转基因西瓜新品种打下了坚实基础。

Isolation of a novel gene encoding the chitinase with antifungal activity and construction of the plant expression vector

On the basis of the sequence of the first ten N-terminal amino acids from the Vigna sesquipedalis antifungal chitinase Class I,induced with Fusarium oxysporum f. niveum,purified and determined by our laboratory,certain primers for PCR had been designed and synthesized. With PCR and other techniques used in molecular biology,the gene encoding the specific chitinase mature protein was isolated from V. sesquipedalis genome,transferred into Escherichia coli and cloned. And the complete sequence for the specific gene was analyzed. The full length of the novel gene covers 894 bp,and there are no introns in it. It contains 43 restriction sites for Aat I,Aat II,Bgl I,Dpn I,Dpn II,EcoR II,Hae I,Hae II,Hae III,Hinf I,Hpa II,Mae II,Mae III,Nba I,Oxa I and Sst IV. The sequence homology for the chitinase(Class I)mature protein genes between V. sesquipedalis and V. unguiculata,Phaseolus vulgaris,Pisum sativum,Nicotiana tabacum,Triticum aestivum and Oryza sativa is decreased respectively. Equally,the Phaseolus vulgaris chitinase signal peptide gene was amplified and cloned, and combined with the V. sesquipedalis chitinase mature protein gene,and further recombined with pBI 121 vector. Finally,the plant expression vector with the specific chitinase gene was successfully constructed,which enables researchers to create novel antifungal germ plasm by transferring the recombined gene encoding the specific chitinase into Citrullus lanatus.