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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): 529-534    DOI: 10.3785/j.issn.1008-9209.2011.11.131
生物科学与技术     
青花菜上皮硫特异蛋白基因的功能
袁高峰1,2， 汪俏梅1*
（1.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；
2.浙江海洋学院 食品与药学学院，浙江 舟山316004）
Function of epithiospecifier protein gene from broccoli
YUAN Gaofeng1,2，WANG Qiaomei1*
1. Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture / Department of Horticulture, Zhejiang University, Hangzhou 310058, China; 2. College of Food and Medicine, Zhejiang Ocean University, Zhoushan, Zhejiang 316004, China
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摘要： 为探明上皮硫特异蛋白（epithiospecifier protein，ESP）在芸薹属蔬菜中对芥子油苷代谢的调控作用及其功能，通过构建青花菜上皮硫特异蛋白基因（BoESP）CaMV35S过量表达载体，并通过浸花法转化模式植物拟南芥，对BoESP基因进行功能验证。结果表明：BoESP的过量表达改变了芥子油苷的代谢产物组成，与野生型植株相比，BoESP CaMV35S过量表达的拟南芥植株4-methoxy-indole-3-acetonitrile含量增加，而1-isothiocyanato-4-methanesulfinyl-butane显著减少。这为青花菜BoESP在体内对芥子油苷水解代谢的调控提供了直接的证据。
Abstract: Glucosinolates are a group of sulfur and nitrogencontaining secondary metabolites that occur in crops belonging to the family of Brassicaceae. Glucosinolates are chemically stable until they become in contact with the enzyme myrosinase. Upon tissue damage, glucosinolates are released from plant vacuoles and rapidly hydrolyzed by myrosinase to glucose and other unstable thiohydroximate-O-sulfonate intermediates, which, as dictated by chemical conditions, spontaneously rearrange to isothiocyanates, thiocyanates, or nitriles. 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 the glucosinolate degradation catalyzed by myrosinase. ESPs have been purified and identified in Crambe abyssinica, Arabidopsis thaliana, Brassica napus and Brassica oleracea that appear responsible for the formation of epithionitriles. However, the function of ESP in the regulation of glucosinolate hydrolysis and its physiological role in Brassica vegetables remain unclear.
The objective of the present study was to analyze the function of BoESP and investigate the regulation mechanism of ESP in glucosinolate hydrolysis of Brassica vegetables.
Total RNA was extracted from broccoli (Brassica oleracea L. ssp. italica) used as the template to amplify the full length of cDNA of broccoli ESP  gene (BoESP) by RT-PCR, and the gene fragment
was subsequently cloned into pMDTM20-T vector．BoESP gene was digested completely from pMDTM20-T-BoESP vector by KpnⅠ and BamHⅠ, and the gene fragment was cloned into plant expression vector pCambia2301．The overexpression construct of BoESP was then 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 the resistant plants were verified by PCR amplification to be transgenic A. thaliana. The transgenic T1 seeds were sown and seedlings were harvested after 3 weeks. The hydrolysis products of glucosinolate in transgenic T1 seedlings were analyzed by gas chromatograph with flame ionization detector and the compositions of hydrolysis products in vivo in the transgenic plants were evaluated.
The results from the electrophoresis of PCR-amplified products confirmed that the cDNA of BoESP was successfully cloned. The results of double restriction enzyme digestion of BoESP CaMV35S by KpnⅠand BamHⅠ and PCR detection of BoESP CaMV35S in A. tumefaciens EHA105 showed the successful construction of plant overexpression vector BoESP CaMV35S. The transformation with the BoESP CaMV35S construct altered the constituents of glucosinolate hydrolysis products in A. thaliana. The content of 4-methoxy-indole-3-acetonitrile in A. thaliana with BoESP overexpression    was significantly increased by 3.49%, whereas that of 1-isothiocyanato-4-methanesulfinyl-butane in A. thaliana with BoESP overexpression was significantly decreased by 17.63%, compared with the wild-type plants.
As a result, it is provided the direct evidence for the regulatory  role of BoESP in glucosinolate metabolism. The research on the function of ESP in the regulation of glucosinolates hydrolysis can provide unique tools in agricultural crops as a strategy to improve their insect and pathogen resistance as well as nutrition value. Our findings are of great theoretical significance and potential in crop improvement.  
出版日期: 2012-09-20
CLC:  Q 943.2  
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[1] . 白菜上皮硫特异蛋白基因克隆与功能验证[J]. 浙江大学学报(农业与生命科学版), 2012, 38(5): 535-541.