Please wait a minute...
浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2012, Vol. 38 Issue (5): 535-541    DOI: 10.3785/j.issn.1008-9209.2012.03.221
生物科学与技术     
白菜上皮硫特异蛋白基因克隆与功能验证
孙海燕1， 袁高峰1,2， 汪俏梅2*
1.浙江海洋学院 食品与药学学院，浙江 舟山316004；
2.浙江大学 园艺系 农业部园艺植物生长发育与品质控制重点开放实验室，浙江 杭州 310058
Cloning and function verification of epithiospecifier protein gene from Chinese cabbage
SUN Haiyan1, YUAN Gaofeng1,2，WANG Qiaomei2*
1. College of Food and Medicine, Zhejiang Ocean University, Zhoushan, Zhejiang 316004, China; 2. Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture/Department of Horticulture, Zhejiang University, Hangzhou 310058, China
 全文: PDF(2140 KB)   HTML ( ) 
摘要： 10.3785/j.issn.10089209.2012.03.221
为阐明上皮硫特异蛋白（epithiospecifier protein）功能，对白菜上皮硫特异蛋白基因（BcESP）进行克隆并对其功能进行初步验证。结果表明：BcESP全长cDNA编码含343个氨基酸的蛋白质，BcESP与拟南芥ESP（AtESP）和青花菜ESP（BoESP）具有很高的相似性，分别达94.7%和95.6%。进一步构建BcESP CaMV35S过量表达载体并通过浸花法转化拟南芥表明，BcESP过量表达改变了芥子油苷代谢产物组成，其异硫代氰酸盐含量显著降低而腈类物质含量升高。本研究通过代谢工程方法调节BcESP表达而改变芥子油苷代谢产物组成，不仅可以为提高芸薹属蔬菜对病虫害的防御能力提供新的途径，也可为进一步研究芥子油苷及其代谢产物的生物学功能提供优良的实验体系。
Abstract: Glucosinolatemyrosinase system is one of the most important defense systems in plants, which is essential for plants to defense against herbivores and pathogens. Glucosinolates are a group of sulfur and nitrogencontaining secondary metabolites that mainly occur in crops belonging to the family of Brassicaceae. They can be hydrolyzed by myrosinases into different degradation products, which have a variety of biological activities. The chemical nature of the hydrolysis products from the glucosinolatemyrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs are specifically involved in glucosinolate degradation catalyzed by myrosinase. ESPs have been purified and identified in Crambe abyssinica, Arabidopsis thaliana, Brassica napus and Brassica oleracea. However, the gene of Chinese cabbage (Brassica campestris ssp. chinensis) ESP (BcESP) has not been cloned yet and the function of BcESP in the regulation of glucosinolate hydrolysis and its physiological role in Chinese cabbage remain unclear. To investigate  the physiological roles of ESPs in Brassica vegetables, the gene of BcESP was coloned and its function was preliminarily verified. Total RNA was extracted from Chinese cabbage used as the template to amplify 3′ sequence and 5′ sequence of cDNA of BcESP by RTPCR using degenerate primers, and then the full cDNA of BcESP was obtained. The full cDNA fragment of BcESP was subsequently cloned into pMDTM20T vector. BcESP gene was then digested completely from pMDTM20-T-BcESP by BamHⅠ and SalⅠ,
and the gene fragment was cloned into plant expression vector pCambia2301．The overexpression construct of BcESP CaMV35S was introduced into the model plant A. thaliana by Agrobacterium tumefaciens-mediated genetic transformation. The seeds of T0 plants were sown on medium containing 50 mg/L kanamycin and resistant plants were verified by PCR amplification to be transgenic A. thaliana. The transgenic T1 seeds were sown and the seedlings were harvested after 4 weeks. The hydrolysis products of glucosinolate in transgenic T1 seedlings were analyzed by gas chromatograph with flame ionization detector and the constitue of hydrolysis products in vivo in the transgenic plants were evaluated.
A full-length cDNA encoding BcESP of 343 amino acids was isolated, which showed high similarity to ESP of A. thaliana and Brassica oleracea with identity of 94.7% and 95.6%, respectively. The results of double restriction enzyme digestion of BcESP CaMV35S by BamH1 and SalⅠ showed the successful construction of plant overexpression vector BcESP CaMV35S. The transformation with the BcESP CaMV35S construct altered the hydrolysis of glucosinolates. The hydrolysis of allyl glucosinolate was redirected towards epithionitrile and nitrile formation in the BcESP-overexpressing lines. The content of simple nitrile in A. thaliana with BcESP overexpression was significantly increased, whereas that of isothiocyanates in A. thaliana with BcESP overexpression was significantly decreased, compared with the wild-type plants.
The study will provide unique tools for modulating glucosinolates metabolism in agricultural plants as a means to improve their insect and pathogen resistance and a good experimental system to study glucosinolates and their derived products.    
出版日期: 2012-09-20
CLC:  Q 943.2  
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　

引用本文:

链接本文:

http://www.zjujournals.com/agr/CN/Y2012/V38/I5/535

[1] . 青花菜上皮硫特异蛋白基因的功能[J]. 浙江大学学报(农业与生命科学版), 2012, 38(5): 529-534.