CN104388349B - 苏云金芽胞杆菌分泌杀虫基因sip1A,表达蛋白及其应用 - Google Patents
苏云金芽胞杆菌分泌杀虫基因sip1A,表达蛋白及其应用 Download PDFInfo
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Abstract
本发明涉及“苏云金芽胞杆菌sip1A基因,表达蛋白及其应用”,属于生物防治技术领域。杀虫蛋白,具有如SEQ ID NO 2所示的氨基酸序列,及编码该杀虫蛋白的基因,优选所述基因的核苷酸序列如SEQ ID NO1所示。上述基因对鞘翅目害虫具有高毒力,以应用于转化微生物和植物,使之表现出对相关害虫的毒性,并克服、延缓害虫对工程菌和转基因植物的抗药性产生。
Description
技术领域
本发明涉及生物防治技术领域,特别是进一步,本发明涉及对鞘翅目农业害虫具有高毒力的Bt分泌杀虫基因及由该基因所编码的蛋白质。
背景技术
苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是一种分布广泛的***,是一种对害虫毒力强且对天敌无毒性的昆虫病原微生物,对高等动物和人无毒性。它是目前研究最为深入、使用最为广泛的微生物杀虫剂,对16个目3000多种害虫有活性。Bt在芽胞形成期可形成杀虫晶体蛋白(Insecticidal CrystalProteins,ICPs),也称δ-内毒素(delta-endotoxin),它的形状、结构和大小均与其毒力有着密切关系[Schnepf.E,Crickmore.N,Van Rie.J.,Lereclus.D,Baum.J,Feitelson.J,Zeigler.D.R.,Dean.D.H.Bacillus thuringiensis and its pesticidal crystalproteins.Microbiol.Mol.Biol.Rev,1998,62(3):775-806.]。自1981年Schnepf等克隆了Bt的第一个ICPs基因,并于1985年发表了它的DNA碱基序列及其编码蛋白的氨基酸序列起,目前(2014年4月)共776个,其中cry基因738个,cry模式基因289个;cyt基因38个,cyt模式基因11个。当今,采用喷施化学农药防治手段固然可以减轻害虫对农作物的为害,但化学农药造成环境污染,长期以来,大量喷施化学杀虫剂,不仅会增强害虫的抗药性,使益虫及其它生态区系遭受破坏,而且严重污染环境,提高生产成本,破坏生态平衡。苏云金芽胞杆菌杀虫晶体蛋白因其杀虫效果好、安全、高效等优点而被广泛的应用于虫害防治。苏云金芽胞杆菌除了直接作为生物农药以外,1996年全世界第一例转基因抗虫植物在美国获准应用,它使用的基因来自Bt cry1Ac。在接下来的几年里,转cry1Ab基因的抗虫玉米,转cry3Aa基因的抗虫土豆等相距问世。在中国,自1998年开始正式推广含有cry1Ac/cry1Ab基因的抗虫棉以来,已经被普遍种植。在转基因作物商业化的第一个12年(1996-2007)中,由于能得到持续稳定的收益,农民种植转基因作物量逐年增加。2013年,27个国家的1800万农民种植了1.752亿公顷(4.33亿英亩)的转基因作物,比2012年持续增长了3%,即500万公顷(1200万英亩)。1800万农民从转基因作物中获益,其中90%为资源匮乏的小农户。前五个种植转基因作物的发展中国家是亚洲的中国和印度、拉丁美洲的巴西和阿根廷以及非洲的南非,共种植8270万公顷的转基因作物,占全球转基因作物种植面积的47%,并且这五个国家的人口约占全球70亿人口的41%。中国750万资源匮乏的小农户种植了420万公顷的Bt棉花,采用率为90%,平均每户农民种植0.5公顷的Bt棉花。转基因作物商业化给工业化国家和发展中国家的农民都带来了经济和环境效益。苏云金芽胞杆菌及其基因发掘已成为可持续发展农业中重要课题。
由于目前商品化的转基因抗虫作物的抗虫基因种类比较单一,如此大面积推广种植存在害虫避难所减少与害虫抗药性上升的风险。因此需要不断分离高毒力的或者新的基因组合来避免害虫抗药性上升的风险。因此,筛选分离克隆新的、高毒力的Bt杀虫基因,可以丰富杀虫基因资源,为转基因作物与工程菌株提供新的基因来源,提高Bt转基因产品的抗虫效果,并且可以降低害虫对Bt毒蛋白的抗性风险,避免新的生态灾难降临,具有重要的经济、社会和生态效益。
发明内容
本发明提供一种对鞘翅目害虫大猿叶甲高毒力的苏云金芽胞杆菌QZL38,及其杀虫新基因分泌基因sip1A和其晶体杀虫蛋白,以应用于转化微生物和植物,使之表现出对相关害虫的毒性,并克服、延缓害虫对工程菌和转基因植物的抗药性产生。
苏云金芽胞杆菌菌株QZL38,其保藏编号为:CGMCC No.9641。
苏云金芽胞杆菌菌株QZL38在杀灭鞘翅目农业害虫中的应用。
杀虫分泌蛋白Sip1A,其氨基酸序列如SEQ ID NO 2所示。
sip1A基因,编码杀虫蛋白Sip1A。
优选其核苷酸序列如SEQ ID NO1所示。
一种表达载体,其特征是含有sip1A基因。
所述表达载体为pEB-sip1A,其骨架载体为pEB,其结构如图6所示;或者所述表达载体为pET21b-cry1Ca,其骨架载体为pET21b,其结构如图7所示。
一种微生物转化体,其特征是含有sip1A基因。
sip1A基因在杀灭大猿叶甲等鞘翅目农业害虫中的应用。
所述应用是将sip1A基因表达的蛋白作为生物杀虫剂的有效成分。
本发明从辽宁千山圆通观附近土壤中分离得到一株苏云金芽胞杆菌菌株QZL38,其保藏编号为CGMCC No.9641,该菌株生物学特性为在生长周期中可以产生芽胞,并且同时产生有毒杀鞘翅目害虫作用的伴胞晶体,其对大猿叶甲具有很强的杀灭能力;从该菌株中得到一个新基因的阳性克隆,即pEB-sip(见图6),对其进行测序分析,发现克隆Sip1A中含有1188个碱基,见SEQ ID NO1,编码395个氨基酸,见SEQ ID NO2。从上述阳性克隆提取质粒,转入受体菌,得到表达菌株,测定基因的表达蛋白的活性,基因表达的Sip1A蛋白对大猿叶甲的初筛生测结果为校正死亡率为100%,见表1。
Sip1A基因可按生物技术的常规方法转化微生物、植物,表现出对相关翘翅目害虫的毒性。
将上述基因转化菌株,表达得到的蛋白可以制成生物农药用于杀死鞘翅目害虫。同时,可以转入植物构建抗虫转基因植物,用于害虫的防治。
本发明分离克隆的Bt sip基因序列及其基因表达产物能够对鞘翅目大猿叶甲,榆蓝叶甲害虫产生强毒力,特别是对大猿叶甲有高活性,是很好的生物杀虫基因,有很广泛的应用前景。通过该sip基因与cry1Ac、cry1Ab、cry1Ba、cry2Ab等基因表达产物组合,可扩大对鞘翅目害虫的杀虫谱。通过应用于转化微生物和植物,使它们表现出对相关害虫的毒性,可克服或延缓昆虫对工程菌和转基因植物抗药性的产生。
菌株保藏信息:
菌种分类命名:苏云金芽胞杆菌(Bacillus thuringensis)
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏日期:2014年09月5日
保藏编号:CGMCC No.9641
附图说明
图1光学显微镜下观察菌株QZL38菌体的形态,
图2电镜下菌株QZL38菌体形态,
图3含有目的基因的基因组PCR鉴定,
M:Marker(100、200、500、750、1000、2000、3000、5000bp)
图4sip1Aa基因全长PCR结果,
M:Marker(100、200、500、750、1000、2000、3000、5000bp),
图5sip1Aa基因在大肠杆菌中表达蛋白的SDS-PAGE,
其中a为pEB-sip1A重组质粒的表达,b为pET-sip1A重组质粒的表达,
1:IPTG诱导的pEB-sip1A蛋白;2:IPTG诱导的pEB空载体蛋白;
3:IPTG诱导的pET21b-sip1A蛋白;4:IPTG诱导的pET21b空载体非可溶组分;
图6重组载体pEB-sip1A的结构图,
图7重组载体pET21b-sip1A的结构图。
具体实施方式
实施例1、分离得到苏云金芽胞杆菌菌株QZL38
本申请人的实验室工作人员从辽宁省千山土壤中分离的得到一株苏云金芽胞杆菌,苏云金芽胞杆菌的芽胞由外向内依次为芽胞外壁、芽胞衣、皮层、芽胞内壁,原生质膜和原生质体。其中皮层的主要成分是肽聚糖,不含营养细胞的多聚糖磷壁酸,它保持着芽胞的脱水状态和耐热性,另一方面,芽胞形成过程中,会产生大量DPA-Ca鳌合物,使得芽胞中的生物大分子形成耐热凝胶,在80℃下热处理20min,苏云金芽胞杆菌芽胞也不会死亡并且休眠的芽胞在75℃的亚致死温度下处理15min,活化效果最好,不但促其快速萌发,还可提高芽胞的成活率(喻子牛1990)。依据这一特性,可实施温度筛选(Knowles B H,Ellar DJ.Colloid-osmotic lysis is a general feature of the mechanism of action ofBacillus thuringiensis d-endotoxins with different specificity[J].Biochimicaet biophysica acta,1987,924:509-518.;戴莲韵,王学聘等.中国八个自然保护区森林土壤中苏云金芽胞杆的分布[J].微生物学报,1994,30(2)117-121)。
1、1菌株的分离
1)取分装的土样加入到50ml大离心管中,至锥形管锥形处。
2)加灭菌水至15ml处,放入玻璃珠5~10颗。
3)用振荡器将土样打碎。
4)放入水浴锅中80℃,20分钟。
5)取1.5ml的EP管,每个管中加1ml灭菌水,再从50ml管中取10微升菌液加入到EP管中混匀。
6)从EP管中取100微升喷到1/2LB培养基中,涂匀。
7)放入到30℃温箱中培养2~3天。
8)镜检观察。
晶体观察
光学显微镜:
将胞晶混合液滴于载玻片上,涂抹均匀,烘干固定,石炭酸复红染液染色3min,清水冲洗,100x油镜进行镜检,石炭酸复红染液配制方法参见文献(Baroy F,Lecadet M M,Deleluse A.Cloning and sequencing of three new putative toxin genes fromClostridium bifermentans[J].Gene,1998,211:293-295)。见图1所示。在1/2LB培养基上培养48h后形成单菌落,光学显微镜下观察到菌株QZL38菌体为长杆状,芽胞为长圆棒状,晶体为双锥形。
电镜显微观察:
扫描电镜制样:孢晶混合液滴于玻璃片上,干燥,经锇酸固定,而后经酒精梯度脱水,临界点干燥,离子溅射喷金(2nm厚),New Bio-TEM H-7500扫描电镜观察拍照。如图2所示。
生物学测定表明,对小菜蛾、甜菜夜蛾的初筛生测结果为校正死亡率为100%。
将该菌株保藏,其保藏编号CGMCC No.9641。
实施例2.获得新基因
2.1利用sip类基因通用引物检测菌株QZL38,引物如下
SP5 GTTGCTCTATAATATGGATTAGCAC
SP3 CTGGTAAACCAATAAATATGCAAG
扩增循环:94℃变性1分钟,56℃退火1分钟,72℃延伸4分钟,25个循环,最后72℃延伸10分钟。
结果如图3所示,菌株QZL38进行基因型的PCR鉴定,用sip类基因鉴定引物sp5/sp3获得了大小为750bp的PCR产物。
2、1QZL38菌株中sip1A类基因的克隆
采用快速克隆方法对该菌株中的新sip1A基因进行分离克隆。
参考GenBank中公布的sip1A的基因编码区的5’端和3’端序列,引物序列如下:
上游引物Sip5:5′-ATGAAATACAAGTTTTCAAAAGTCGTTAAG-3′
下游引物Sip3:5′-TTAATTTCCACTTAAAATCTTTGTT-3′
用pfuDNA聚合酶,用如下体系进行PCR扩增。
超纯水补至50μL,混匀离心。
扩增循环:94℃变性1分钟,54℃退火1分钟,72℃延伸1分钟,25个循环,最后72℃延伸10分钟。
2.2连接方案
用超纯水补足体积到10μL,充分混匀,16℃连接4h或4℃连接过夜。
设计sip1A类基因的全长引物Sip5/Sip3,扩增得到sip1A 1188bp的全长基因,将其与载体pEB、pET(公开载体,本所实验室有保存,可以对外公开发放)载体进行连接,转化入感受态JM109中,经抗性筛选、PCR鉴定分析,筛选出含有sip1A基因的阳性重组质粒。图4PCR鉴定结果。将纯化片段与载体pEB,载体pET21b连接转化大肠杆菌JM109,得到阳性转化子。对***片断进行测序分析,得到序列SEQ ID NO 1,序列全长1188bp,编码395个氨基酸组成的蛋白。经测定,其氨基酸序列为SEQ ID NO 2所示。
2.3转化方案
2.3.1大肠杆菌转化
1.挑取单菌落于5ml LB震荡培养过夜;
2.按1%接种量接种于LB液体培养基中,37℃,230rpm培养2-2.5hr,(OD600=0.5-0.6);
3.4℃,4,000rpm离心10min;
4.弃上清,加入预冷的0.1M CaCl250ml悬浮细胞,置于冰上30min以上;
5.4℃,4,000rpm离心10min,回收细胞;
6.用2-4ml冰预冷的0.1M CaCl2重悬细胞,分装成200μl/0.5mL离心管中,于4℃保存(可保存一周)。
7.取200μl感受态细胞与5μL连接产物充分混匀,冰浴30min。
8.42℃热激1.5min,冰浴3min。
9.加入800μl LB培养基37℃培养45min。
10.取200μl涂板,加入相应的抗生素,及IPTG,X-gal,37℃培养。
实施例3、基因表达与活性测定
3.1.1在上述克隆提取质粒DNA,转入受体菌Rosetta(DE3)中,获得表达菌株。
IPTG诱导表达后,进行SDS-PAGE蛋白电泳检测。
诱导表达过程如下:
1)活化菌种(37℃、12hr);
2)10%接种于LB培养基中(37℃、2hr);
3)加入诱导物IPTG,150rpm,18-22℃低温诱导4-20h;
4)离心收集菌体,加入10mM Tris·Cl(pH 8.0)悬浮;
5)破碎菌体(超声波破碎完全);
离心12,000rpm 10min 4℃;
收集上清及沉淀各10-15μL,分别电泳检测。
聚丙烯酰胺凝胶配置如下。
上样:上样10-15μl,电泳:130-150V恒压。
染色与脱色:电泳后取出凝胶,用蒸馏水冲洗后,放入染色液中,60rpm振荡染色1hr左右,脱色液中脱色2hr左右,脱色至凝胶背景透明,清水中漂洗至蛋白带清晰。
将重组质粒pEB-sip1A,(见图6)转化到E.coli Rosetta(DE3)中,IPTG诱导表达,SDS-PAGE(12%)凝胶电泳。结果表明,sip1A基因都能通过表达载体pEB在大肠杆菌中高效表达41kD蛋白,而经IPTG诱导的转入Rosetta(DE3)的pEB空载体没有特异的目的条带产生(图5中A)。
将重组质粒pET21b-sip1A(见图7)转化到E.coli Rosetta(DE3)中,产生41kD蛋白,如图5中B。
3.2Bt菌株QZL38及sip1A基因编码蛋白的杀虫活性测定
将sip1A基因表达蛋白,用水稀释到不同浓度,对鞘翅目害虫的杀虫活性,具体方法如下,采用饲料混合法进行杀虫生物活性测定。将制备好不同浓度梯度的表达蛋白样品并分装于经过消毒的培养皿中,分别与饲料搅拌混匀,挑选活跃的初孵幼虫接于饲料上,每个处理重复3次,大猿叶甲、榆蓝叶甲、暗黑鳃金龟农业害虫每个重复为30头试虫。阴性对照为10mmol/L Tris-Cl溶液作。试虫的饲养条件为相对湿度为70%~80%、温度为27℃的光照培养箱中培养,大猿叶甲幼虫饲育48h后调查死、活虫数量,计算死亡率。采用POLO软件计算95%置信区间和LC50值。
表1QZL38菌株对大猿叶甲和大黑晒金龟杀虫活性测定结果
生测样品 | CK | QZL38 | 校正死亡率 |
大猿叶甲 | 0 | 30 | 100% |
榆蓝叶甲 | 0 | 30 | 100% |
暗黑鳃金龟 | 0 | 5 | 16.7% |
表2Sip1A蛋白对大猿叶甲杀虫活性测定结果
本发明的有益效果:本发明分离克隆的Bt sip1A基因序列及其基因表达产物能够对鞘翅目产生毒力,特别对于大猿叶甲和榆蓝叶甲,校正死亡率100%,可扩大对鞘翅目害虫的杀虫谱,通过应用于转化微生物和植物,使它们表现出对相关害虫的毒性,可克服或延缓昆虫对工程菌和转基因植物抗药性的产生。
Claims (8)
1.苏云金芽胞杆菌菌株QZL38,其保藏编号为:CGMCC No.9641。
2.苏云金芽胞杆菌菌株QZL38在杀灭鞘翅目害虫中的应用。
3.杀虫蛋白Sip1Aa,其氨基酸序列如SEQ ID NO 2所示。
4.sip1Aa基因,其核苷酸序列如SEQ ID NO1所示。
5.一种表达载体,其特征是含有权利要求4所述的sip1Aa基因。
6.一种微生物转化体,其特征是含有权利要求4所述的sip1Aa基因。
7.权利要求4所述的sip1Aa基因在杀灭大猿叶甲,榆蓝叶甲,金龟子害虫中的应用。
8.权利要求7所述应用,是将权利要求4所述的sip1Aa基因表达的蛋白作为生物杀虫剂的有效成分。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4797276A (en) * | 1985-03-22 | 1989-01-10 | Mycogen Corporation | Control of cotton boll weevil, alfalfa weevil, and corn rootworm via contact with a strain of Bacillus thuringiensis |
CN101984045A (zh) * | 2010-09-29 | 2011-03-09 | 东北农业大学 | 苏云金芽孢杆菌cry8Nal基因,表达蛋白及其应用 |
CN102559554A (zh) * | 2012-01-09 | 2012-07-11 | 东北农业大学 | 苏云金芽胞杆菌cry1Ca基因,表达蛋白及其应用 |
CN102718872A (zh) * | 2012-06-20 | 2012-10-10 | 中国农业科学院植物保护研究所 | 苏云金芽胞杆菌基因组合、其工程菌及其制备方法 |
-
2014
- 2014-11-21 CN CN201410675353.1A patent/CN104388349B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4797276A (en) * | 1985-03-22 | 1989-01-10 | Mycogen Corporation | Control of cotton boll weevil, alfalfa weevil, and corn rootworm via contact with a strain of Bacillus thuringiensis |
CN101984045A (zh) * | 2010-09-29 | 2011-03-09 | 东北农业大学 | 苏云金芽孢杆菌cry8Nal基因,表达蛋白及其应用 |
CN102559554A (zh) * | 2012-01-09 | 2012-07-11 | 东北农业大学 | 苏云金芽胞杆菌cry1Ca基因,表达蛋白及其应用 |
CN102718872A (zh) * | 2012-06-20 | 2012-10-10 | 中国农业科学院植物保护研究所 | 苏云金芽胞杆菌基因组合、其工程菌及其制备方法 |
Non-Patent Citations (6)
Title |
---|
Bt新型基因sip的克隆、表达和生物信息学分析;刘艳杰 等;《生物技术通报》;20121231(第12期);101-105 * |
Bt菌株DQ89的sip基因的克隆、表达及杀虫活性分析;张金波 等;《中国生物防治学报》;20150831;第31卷(第4期);598-602 * |
Discovery and characterization of Sip1A: a novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae;William P. Donovan等;《Appl Microbiol Biotechnol》;20060218;第72卷;第713页摘要,第715页图2,第716页右栏第2段 * |
EEM31129.1;Zwick,M.E.等;《GenBank》;20090430;蛋白注释以及氨基酸序列 * |
新型cry7Ab基因的鉴定、克隆、表达与杀虫活性;邓淑 等;《农业生物技术学报》;20091231;第17卷(第5期);908-913 * |
桂林喀斯特地貌分布区苏云金芽抱杆菌菌株的分离鉴定;孙晓东 等;《中国生物防治》;20090531;第25卷(第2期);第138页摘要,141页2.5节 * |
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