一种新型的大丽轮枝菌葡聚糖-1,3-β-糖苷酶的表达和性质研究
详细信息 查看官网全文
- 英文论文题名：Expression and Characterization of a Novel Glucan 1,3-β-glucosidase from Verticillium dahliae VdLs.17
- 论文作者：孙瑶 ; 杨晓杰 ; 侯甲男 ; 唐中杰 ; 赵元明 ; 谢德意 ; 李武 ; 赵付安 ; 吕淑平 ; 聂利红 ; 段铮铮 ; 于跃波 ; 王红卫 ; 房卫平
- 英文论文作者：Sun Yao ; Yang Xiaojie ; Hou Jianan ; Tang Zhongjie ; Zhao Yuanming ; Xie Deyi ; Li Wu ; Zhao Fu'an ; Lü Shuping ; Nie Lihong ; Duan Zhengzheng ; Yu Yuebo ; Wang Hongwei ; Fang Weiping ; Economic Crop Research Institute ; Henan Academy of Agriculture Sciences
- 年：2017
- 作者机构：河南省农业科学院经济作物研究所;
- 论文关键词：棉花 ; 黄萎病 ; 杀菌剂 ; 大丽轮枝菌 ; 葡聚糖-1 ; 3-β-糖苷酶 ; 纯化
- 英文论文关键词：cotton ; Verticillium wilt ; fungicide ; Verticillium dahliae Kleb ; glucan 1 ; 3-β-glucosidase ; purification
- 会议召开时间：2017-08-07
- 会议录名称：中国农学会棉花分会2017年年会暨第九次会员代表大会论文汇编
- 语种：中文
- 分类号：S435.621.2
- 学会代码：MHXB
- 会议名称：中国农学会棉花分会2017年年会暨第九次会员代表大会
- 会议地点：中国河南郑州
- 主办单位：中国农学会棉花分会
- 学会名称：《棉花学报》编辑部
- 页数：2
- 文件大小：80k
- 原文格式：O
- 会议级别：全国
摘要
棉花黄萎病是1种严重的真菌性病害,每年给我国农业生产造成重大经济损失。大丽轮枝菌的休眠结构微菌核,是黄萎病最重要的初侵染源,它致病力强,寄主范围广,存活时间长。微菌核的萌发(膨胀与产生分支)与细胞壁的代谢过程息息相关。细胞壁是细菌、真菌和植物细胞的特有结构,以细胞壁代谢途径中的调控酶为靶标研制的杀菌剂,不会对人体和动物产生直接影响。分子生物学和基因工程研究表明:葡聚糖-1,3-B-糖苷酶(BG2)在细胞壁消解途径中起关键作用,可作为潜在的杀菌剂靶标。克隆大丽轮枝菌的BG2基因,并在大肠杆菌中进行异源表达,最终获得相对分子质量为45kDa的BG2。对BG2表达过程中,诱导剂的浓度、诱导温度、诱导时间均作了优化。IPTG浓度为0.5mmol·L~(-1),25℃诱导6 h,可以获得最大表达量的BG2。收集培养的菌体并裂解离心后,1 L培养基中生产1264 mg可溶性蛋白,BG2表达的目的蛋白主要为可溶性蛋白。经过裂解离心、亲和层析、凝胶过滤层析、超滤4个纯化步骤后,最终1 L培养基得到0.06 mg BG2,纯化效率为0.09%,蛋白纯度高达95%。BG2产量较低的原因可以归结为目的蛋白与Ni-NTA树脂亲和能力不强,导致部分目的蛋白没有与金属离子形成螯合物就直接通过层析柱流失了。另外,以pNPG为底物,测试大丽轮枝菌BG2的活性,同时对其活性测试条件进行优化。在pH7.5的缓冲溶液中,30℃温浴反应5 min,可以使大丽轮枝菌BG2活性达到最大35.2 U·mg~(-1)。大丽轮枝菌BG2的克隆、表达及其产物的纯化以及活性测试工作为其抑制剂的设计及抑制活性的测试奠定基础,为我国黄萎病新型杀菌剂的研制工作提供基础数据与关键理论。
Glucan 1,3-β-glucosidase plays an important role in fungal cell wall metabolism,and it is considered to be a potential enzymatic target for inhibition of fungal growth.The glucan 1,3-β-glucosidase gene from Verticillium dahliae Kleb.was cloned and expressed in Escherichia coli as a 45 kDa protein belonging to an intracellular enzyme.Isopropyl β-D-1-thiogalactopyranoside(IPTG) is a gratuitous inducer,and the induction concentration,temperature,and time were assessed and optimized in this study.After the addition of 0.5 mmol·L~(-1) IPTG at 25℃ for 6 h,the highest expression levels of soluble BG2 were observed.Following lysis and extraction,1264 mg protein per 1 L culture medium was obtained from the cell lysate.Though the protein yield is 0.06%,the BG2 purity is as much as 95%.It is all attributed to a low affinity for interest protein binding with Ni-NTA.Moreover,the activity of BG2 was assayed with pNPG as the substrate.The purified protein was found to exhibit specific activity of 35.2 U·mg~(-1) under pH 7.5 at 30 ℃ for 5min.Here we report on the expression and purification conditions,and enzyme activity assays.An advantage of the present method is that it enables high purity and activity,both of which may be used to study the biochemical structure of the protein.
Glucan 1,3-β-glucosidase plays an important role in fungal cell wall metabolism,and it is considered to be a potential enzymatic target for inhibition of fungal growth.The glucan 1,3-β-glucosidase gene from Verticillium dahliae Kleb.was cloned and expressed in Escherichia coli as a 45 kDa protein belonging to an intracellular enzyme.Isopropyl β-D-1-thiogalactopyranoside(IPTG) is a gratuitous inducer,and the induction concentration,temperature,and time were assessed and optimized in this study.After the addition of 0.5 mmol·L~(-1) IPTG at 25℃ for 6 h,the highest expression levels of soluble BG2 were observed.Following lysis and extraction,1264 mg protein per 1 L culture medium was obtained from the cell lysate.Though the protein yield is 0.06%,the BG2 purity is as much as 95%.It is all attributed to a low affinity for interest protein binding with Ni-NTA.Moreover,the activity of BG2 was assayed with pNPG as the substrate.The purified protein was found to exhibit specific activity of 35.2 U·mg~(-1) under pH 7.5 at 30 ℃ for 5min.Here we report on the expression and purification conditions,and enzyme activity assays.An advantage of the present method is that it enables high purity and activity,both of which may be used to study the biochemical structure of the protein.
引文