杀象耳豆根结线虫Pro-21fa6克隆子产活性蛋白酶研究

本文提取土壤样品总DNA,以Fosmid为载体,构建宏基因组文库。以脱脂奶为底物,象耳豆根结线虫为靶标,筛选杀象耳豆根结线虫的产蛋白酶基因克隆子。以克隆子Pro-21fa6为研究对象,从酶活、蛋白含量变化、p H变化对发酵过程进行了研究。结果表明,构建31 104个克隆子,库容1.067 Gb,获得产蛋白酶阳性克隆子111株,其中9株对象耳豆根结线虫具有毒杀作用,Pro-21fa6克隆子毒杀效果最好。培养条件温度35℃、转数200 r/min、以及脱脂奶与诱导剂共同作用可以使酶活提高,是基础培养基的1.33倍。Pro-21fa6蛋白酶克隆子初步研究,为蛋白酶基因基因片段亚克隆、测序、活性蛋白酶...

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Bibliographic Details
Published in西南农业学报 Vol. 28; no. 5; pp. 2129 - 2135
Main Author 张怀军 赵志祥 陈绵才
Format Journal Article
LanguageChinese
Published 海南大学环境与植物保护学院,海南海口571101 2015
海南省农业科学院植物保护研究所,海南省病虫害防控重点实验室,海南海口571100%海南省农业科学院植物保护研究所,海南省病虫害防控重点实验室,海南海口571100
Subjects
Fosmid文库
克隆子
基因组文库
活性蛋白酶
象耳豆根结线虫
Genomic library
Active protease
克隆子
象耳豆根结线虫
活性蛋白酶
Fosmid library clones
Fosmid文库
基因组文库
Meloidogyne enterolobii
Online AccessGet full text
ISSN1001-4829
DOI10.16213/j.cnki.scjas.2015.05.053

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Summary:本文提取土壤样品总DNA,以Fosmid为载体,构建宏基因组文库。以脱脂奶为底物,象耳豆根结线虫为靶标,筛选杀象耳豆根结线虫的产蛋白酶基因克隆子。以克隆子Pro-21fa6为研究对象,从酶活、蛋白含量变化、p H变化对发酵过程进行了研究。结果表明,构建31 104个克隆子,库容1.067 Gb,获得产蛋白酶阳性克隆子111株,其中9株对象耳豆根结线虫具有毒杀作用,Pro-21fa6克隆子毒杀效果最好。培养条件温度35℃、转数200 r/min、以及脱脂奶与诱导剂共同作用可以使酶活提高,是基础培养基的1.33倍。Pro-21fa6蛋白酶克隆子初步研究,为蛋白酶基因基因片段亚克隆、测序、活性蛋白酶分离纯化奠定了基础。
Bibliography:51-1213/S
Total DNA were extracted from soil samples,taken Fosmid as a carrier to construct metagenomic library. Skim milk was used as a substrate and Meloidogyne enterolobii as a target,and screening kill Meloidogyne enterolobii production protease gene was cloned. Then the pro-21fa6 clone was used as tested object,its fermentation process was studied under the changes of protease activity,protein content and p H. The result showed that building 31104 clones,the capacity of 1. 067 Gb,get positive clones produced protease 111,of which nine had a toxic effect on Meloidogyne enterolobii,and pro-21fa6 clone poison worked the best. Under the conditions of the temperature of 35 ℃ and revolutions of 200 r / min,the combined effect of skim milk and inducers could increase the activity and was 1. 33 times than that of the basal medium. It was concluded that the preliminary study on pro-21fa6 protease clone would provide the foundation for protease genes subcloned,sequenced,the separation and purification of protease a
ISSN:1001-4829
DOI:10.16213/j.cnki.scjas.2015.05.053