CN108359673A - 一种高效杀食用菌眼蕈蚊的Bt cry11基因、编码蛋白及其应用 - Google Patents
一种高效杀食用菌眼蕈蚊的Bt cry11基因、编码蛋白及其应用 Download PDFInfo
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- CN108359673A CN108359673A CN201810429888.9A CN201810429888A CN108359673A CN 108359673 A CN108359673 A CN 108359673A CN 201810429888 A CN201810429888 A CN 201810429888A CN 108359673 A CN108359673 A CN 108359673A
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Abstract
本发明涉及一种对食用菌眼蕈蚊高效的Bt cry11基因,分离自苏云金芽孢杆菌菌株,并从此菌株克隆了对食用菌眼蕈蚊高毒力的cry11基因,其编码的蛋白质序列如:SEQ ID No.2所示的氨基酸序列。本发明公开的基因,对食用菌眼蕈蚊有明显的杀虫作用,同时不会对食用菌菌丝及子实体的生长有影响,可降低食用菌栽培中农药的使用量,减少农药残留和药害的发生,具有重要的经济价值和应用前景。
Description
技术领域
本发明涉及一种对食用菌眼蕈蚊高毒力的Bt cry11基因,以及该基因编码的蛋白,属于分子生物学技术领域。
技术背景
食用菌作为可供人类食用或药用的大型真菌,不仅是一项集经济效益、生态效益和社会效益于一体的发展项目,而且是一类符合农业可持续发展需要的绿色食品。中国已知的食用菌已有350多种,但随着食用菌产业的迅速发展,其病虫害也引起诸多学者的关注。眼蕈蚊(Sciaridae)是食用菌栽培中危害较广的一类虫害,有6属27种。幼虫直接潜入食用菌菌袋,取食菌丝和子实体,且排出粪便滋生病菌。成虫飞行携带的病原菌、线虫、螨虫等导致二次侵染。目前在食用菌栽培中针对眼蕈蚊的防治方法主要有物理防治和化学防治,但由于物理防治多针对成虫,对幼虫无效,化学防治会不同程度抑制菌丝生长,且存在3R问题,生物防治成为研究食用菌虫害防控的热点。
苏云金芽孢杆菌(Bacillus thuringiensis,Bt)属好氧型革兰氏阳性细菌,是当今应用最广且最有效的一种微生物农药。Bt菌株的收集主要来源于土壤,也可从昆虫、粪便、储藏品及尘埃、污水和植被等中分离。1901年,苏云金芽孢杆菌被日本石渡首次发现。1976年,Goldberg等首次发现了新的对蚊虫具有强杀虫活性的Bt ONR260菌株,并命名为以色列亚种(subsp.israelensis),在其包涵体中除了含有3种晶体蛋白Cry4A、Cry4B和Cry11A,还含有一种溶细胞蛋白成分Cyt1A。随后出现有关杀双翅目害虫的苏云金芽孢杆菌及其晶体蛋白的研究。
苏云金芽孢杆菌在形成芽孢时可以产生具有杀虫活性的杀虫晶体蛋白(insecticidal crystal protein,ICPs),这种晶体蛋白可作用于昆虫中肠,最终引起昆虫死亡。其主要包括晶体蛋白(crystal,cry)和溶细胞素(cytolytic,cyt)两种蛋白,晶体蛋白具有特异的杀虫活性,溶细胞素具有溶解细胞的功能,同时作为cry蛋白的受体减缓抗性的产生。研究表明Bt的杀虫谱较广,从节肢动物门中的双翅目、鳞翅目、鞘翅目等9个目的昆虫到线形动物门、原生动物和扁形动物门中的某些有害种类均在Bt的杀虫活性范围内。Bt虽对目标性害虫具有特异性的毒杀作用,但对非目标性生物安全,因其专一、高效和对人畜无害的优点,倍受人们的青睐。
利用苏云金芽孢杆菌防治双翅目类害虫的研究已有较多报道,如Bt对韭菜眼蕈蚊、库蚊、伊蚊、瓜实蝇等的防治效果达70%以上,但有关Bt防治食用菌眼蕈蚊的研究鲜有报道。
发明内容
本发明的目的在于解决现有技术的不足,从苏云金芽孢杆菌菌株中分离克隆了基因cry11,这种基因的表达产物对食用菌眼蕈蚊有杀虫活性而不影响食用菌菌丝及子实体的生长。
技术方案
一种高效杀食用菌眼蕈蚊的Bt cry11基因,核苷酸序列如SEQ ID No.1所示。
一种cry11基因编码的蛋白质,该蛋白质是由cry11基因表达得到的,蛋白质的氨基酸序列如SEQ ID No.2所示。
cry11基因的获得方法:以Bt菌株基因组DNA为模板,分别进行PCR扩增,得到cry11基因的全长序列;
反应体系为(20μl):Mix 10μl,引物(10μmol/L)各1μl,模板1μl,超纯水补至20μl。
反应程序为:98℃2min;98℃10s,56℃10s,72℃30s,35个循环;72℃2min。
引物如下:
11F:5’-CTCTGTTTCCTCGTCAATAA-3’
11R:5’-TTGCGATGTGAGCATTGAACCA-3’
cry11基因编码的蛋白质的获得方法:通过相应的DNA片段在大肠杆菌中的表达得到,表达的引物为:
cry11F:5’-CTCTAGAGATGAATTATATGGAAGATAGT-3’
cry11R:5’-CAAGCTTGCTACTTTAGTAACGGATTAA-3’
上述cry11基因编码的蛋白质可可抑制双翅目的食用菌眼蕈蚊,可以很好的应用于食用菌的栽培过程中。
本发明具有的优点或有益效果:本发明分离了一个对食用菌眼蕈蚊具有毒性的苏云金芽孢杆菌,并从该菌株中克隆了编码Cry11毒蛋白的基因,本发明可以减少使用防治食用菌眼蕈蚊的农药,保护环境,本发明可以扩大含有该基因的生物防治菌株的杀虫谱,提高生物防治菌株对食用菌眼蕈蚊的效果。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
(一)菌株筛选及鉴定过程
实验菌株由发明人(采集地址:江苏省南京市,采集人:马林,联系方式:南京市钟灵街50号)于江苏省南京市的土壤中分离得到。分离方法为醋酸钠-抗生素法,经形态鉴定及16S rDNA分析,初步确认为苏云金芽孢杆菌(Bacillus thuringiensis)。通过菌株对食用菌眼蕈蚊的室内毒力测定及与平菇对峙试验表明,该菌株不仅对食用菌眼蕈蚊具有极高的毒力,且不影响食用菌菌丝的正常生长。
Bt菌株的采集与分离方法:
采用棋盘式法进行采集,先铲去表层土1-2cm,再取样品50g,土样放入自封袋备用。用醋酸钠-抗生素法进行菌株分离,挑选与Bt菌株形态相近的菌落进行镜检确认。
室内毒力测定方法:
Bt菌株在LB液体培养基中30℃、190r/min振荡培养过夜,按10%的菌液量转接至液体发酵培养基中30℃、190r/min振荡培养72h,至晶体产生。不同菌液分别浸泡大小一致的平菇子实体10min,用滤纸吸干表面多余菌液后,将菇片放置于铺有湿滤纸片的培养皿中。每皿挑30头幼虫,Bti制剂(苏云金芽孢杆菌以色列变种可湿性粉剂,武汉科诺生物科技股份有限公司)中分离的菌株为阳性对照,清水处理的菇片作为空白对照,每处理3次重复。培养72h后观察并记录死亡率。
(二)利用PCR扩增对Bt菌株进行Cry11毒蛋白鉴定
提取Bt菌株的基因组DNA,以此为模板,用表1中的引物进行PCR扩增。PCR反应体系为(20μl):Mix 10μl,引物(10μmol/L)各1μl,模板1μl,超纯水补至20μl。反应程序为:98℃2min;98℃10s,54℃10s,72℃20s,35个循环;72℃2min。将所得扩增产物交送南京擎科生物工程有限公司进行测序。测序结果在Genbank中进行同源性比较,结果发现该菌株含有cry11毒蛋白基因型。
表1杀虫蛋白基因型鉴定引物序列
(三)Cry11毒蛋白基因型片段的获得
以Bt菌株基因组DNA为模板,分别进行PCR扩增,反应体系为(20μl):Mix 10μl,引物(10μmol/L)各1μl,模板1μl,超纯水补至20μl。反应程序为:98℃2min;98℃10s,56℃10s,72℃30s,35个循环;72℃2min。得到cry11基因的全长序列,引物如下:
11F:5’-CTCTGTTTCCTCGTCAATAA-3’
11R:5’-TTGCGATGTGAGCATTGAACCA-3’
cry11基因序列如SEQ ID No.1所示,序列全长为1941bp,分析表明,GC含量为33.95%,编码的蛋白含有64个氨基酸。
(四)Cry11毒蛋白的获得
cry11基因编码的蛋白获得的方法,通过相应的DNA片段在大肠杆菌中的表达得到,表达的引物为:
cry11F:5’-CTCTAGAGATGAATTATATGGAAGATAGT-3’
cry11R:5’-CAAGCTTGCTACTTTAGTAACGGATTAA-3’
经测定,其氨基酸序列如SEQ ID No.2所示。
实施例2:cry11基因的表达及室内毒力测定
cry11基因克隆载体的构建:
根据该基因开放阅读框两端序列,设计并合成一对含Sma I和Sal I酶切位点的特异性引物。以该菌株的基因组DNA为模板,使用高保真酶进行扩增,扩增产物回收后,使用pEASY-Blunt Cloning Kit试剂盒进行克隆载体的构建。构建完成后进行PCR验证和双酶切验证。
cry11基因表达载体的构建:
克隆载体构建完成后,用Sma I和Sal I进行双酶切,酶切产物与同样进行双酶切后的表达载体(pGEX-KG)连接,转化入受体菌E.coli DH5α。同样进行PCR验证和双酶切验证。同时,用SDS-PAGE的方法进行该基因表达产物的验证。
室内毒力测定:
室内毒力测定方法与实施例1中的方法相同,Bt菌株和不含杀虫蛋白基因的受体菌E.coli DH5α为阴性对照,清水为空白对照,测定cry11基因的表达产物对食用菌眼蕈蚊的杀虫活性。杀虫结果表2所示。
表2四种基因表达产物的杀虫活性
DH5α | Bt | DH-cry11 | |
校正死亡率 | 19.57% | 74.57% | 61.28% |
以上实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
序列表
<110> 江苏省农业科学院
<120> 一种高效杀食用菌眼蕈蚊的Bt cry11基因、编码蛋白及其应用
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Gln Asn Pro Asn Asn Glu Pro Ile Ala Pro Arg Asp Ile Ile Asn Gln
355 360 365
Ile Leu Thr Ala Pro Ala Pro Ala Asp Leu Phe Phe Lys Asn Ala Asp
370 375 380
Ile Asn Val Lys Phe Thr Gln Trp Phe Gln Ser Thr Leu Tyr Gly Trp
385 390 395 400
Asn Ile Lys Leu Gly Thr Gln Thr Val Leu Ser Ser Arg Thr Gly Thr
405 410 415
Ile Pro Pro Asn Tyr Leu Ala Tyr Asp Gly Tyr Tyr Ile Arg Ala Ile
420 425 430
Ser Ala Cys Pro Arg Gly Val Ser Leu Ala Tyr Asn His Asp Leu Thr
435 440 445
Thr Leu Thr Tyr Asn Arg Ile Glu Tyr Asp Ser Pro Thr Thr Glu Asn
450 455 460
Ile Ile Val Gly Phe Ala Pro Asp Asn Thr Lys Asp Phe Tyr Ser Lys
465 470 475 480
Lys Ser His Tyr Leu Ser Glu Thr Asn Asp Ser Tyr Val Ile Pro Ala
485 490 495
Leu Gln Phe Ala Glu Val Ser Asp Arg Ser Phe Leu Glu Asp Thr Pro
500 505 510
Asp Gln Ala Thr Asp Gly Ser Ile Lys Phe Ala Arg Thr Phe Ile Ser
515 520 525
Asn Glu Ala Lys Tyr Ser Ile Arg Leu Asn Thr Gly Phe Asn Thr Ala
530 535 540
Thr Arg Tyr Lys Leu Ile Ile Arg Val Arg Val Pro Tyr Arg Leu Pro
545 550 555 560
Ala Gly Ile Arg Val Gln Ser Gln Asn Ser Gly Asn Asn Arg Met Leu
565 570 575
Gly Ser Phe Thr Ala Asn Ala Asn Pro Glu Trp Val Asp Phe Val Thr
580 585 590
Asp Ala Phe Thr Phe Asn Asp Leu Gly Ile Thr Thr Ser Ser Thr Asn
595 600 605
Ala Leu Phe Ser Ile Ser Ser Asp Ser Leu Asn Ser Gly Glu Glu Trp
610 615 620
Tyr Leu Ser Gln Leu Phe Leu Val Lys Glu Ser Ala Phe Thr Thr Gln
625 630 635 640
Ile Asn Pro Leu Leu Lys
645
Claims (5)
1.一种高效杀食用菌眼蕈蚊的Bt cry11基因,其特征在于,核苷酸序列如SEQ ID No.1所示。
2.权利要求1所述高效杀食用菌眼蕈蚊的Bt cry11基因的获得方法,其特征在于,以Bt菌株基因组DNA为模板,进行PCR扩增,即得;
反应体系为(20μl):Mix 10μl,引物(10μmol/L)各1μl,模板1μl,超纯水补至20μl;反应程序为:98℃2min;98℃10s,56℃10s,72℃30s,35个循环;72℃2min;
引物为:
11F:5’-CTCTGTTTCCTCGTCAATAA-3’
11R:5’-TTGCGATGTGAGCATTGAACCA-3’。
3.一种权利要求1所述cry11基因编码的蛋白质,其特征在于,该蛋白质是由cry11基因表达得到的,蛋白质的氨基酸序列如SEQ ID No.2所示。
4.权利要求3所述cry11基因编码的蛋白质的获得方法,其特征在于,通过相应的DNA片段在大肠杆菌中的表达得到,表达的引物为:
cry11F:5’-CTCTAGAGATGAATTATATGGAAGATAGT-3’
cry11R:5’-CAAGCTTGCTACTTTAGTAACGGATTAA-3’。
5.权利要求3所述cry11基因编码的蛋白质用于抑制双翅目的食用菌眼蕈蚊的应用。
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CN114149494A (zh) * | 2021-11-23 | 2022-03-08 | 中国科学院水生生物研究所 | 一种柱状黄杆菌毒力蛋白在病原检测和弱毒株制备中的应用 |
CN114149494B (zh) * | 2021-11-23 | 2023-09-15 | 中国科学院水生生物研究所 | 一种柱状黄杆菌毒力蛋白在病原检测和弱毒株制备中的应用 |
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