CN110093301B - 一种苏云金芽孢杆菌及其在防治鳞翅目类害虫中的应用 - Google Patents
一种苏云金芽孢杆菌及其在防治鳞翅目类害虫中的应用 Download PDFInfo
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- CN110093301B CN110093301B CN201910463196.0A CN201910463196A CN110093301B CN 110093301 B CN110093301 B CN 110093301B CN 201910463196 A CN201910463196 A CN 201910463196A CN 110093301 B CN110093301 B CN 110093301B
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Abstract
本发明公开了一种苏云金芽胞杆菌及其在防治鳞翅目类害虫中的应用。该苏云金芽胞杆菌命名为CS72‑2,保藏于中国典型培养物保藏中心,保藏单位地址:中国.武汉.武汉大学,保藏日期为2018年12月27日,保藏编号为CCTCC NO.M2018930。该菌株对对鳞翅目产生毒力,尤其是玉米螟和棉铃虫有一定的毒杀作用。本发明还提供了一种新的、高毒力的Bt杀虫基因,可以应用于植物的遗传改造,提高其对害虫的抗性,具有较好的生物防治作用,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。本发明提供的杀虫剂制备方法简单,易于工业化生产,防治效果良好,具有良好的开发应用前景。
Description
技术领域
本发明属于生物防治技术领域,具体涉及一种苏云金芽胞杆菌及其在防治鳞翅目类害虫中的应用。
背景技术
在人类生产过程中,虫害是造成农业生产损失及影响人类健康的重要因素。据FAO统计,全世界农业生产每年因虫害造成的经济损失高达14%,病害损失达12%,草害损失达11%。损失额高达1260亿美元,相当于中国农业总产值的一半。为了减少这些损失,多年来,对农作物害虫及蚊虫普遍采用化学防治手段进行防治,但由于化学农药的长期、大量使用,造成了对环境的污染,农副产品中农药残留量增加,给人类的生存和健康带来了危害。此外,化学农药在杀灭害虫的同时,也杀伤了天敌及其它有益物,破坏了生态平衡。与化学防治相比,生物防治具有安全、有效、持久的特点。并且避免了化学防治带来的一系列问题。因此,生物防治技术成了人们研究的热点。在生物杀虫剂中,苏云金芽胞杆菌是目前世界上用途最广、产量最大的一类微生物杀虫剂。
苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是一种革兰氏阳性细菌,它的分布极为广泛,在芽胞形成的同时可形成具有杀虫活性的由蛋白质组成的伴胞晶体,又名杀虫晶体蛋白(Insectididal crystal proteins,简称ICPs),ICPs主要由cry和cyt基因编码,具有特异的杀虫活性,cyt类杀虫晶体蛋白发现了3类,分别是cyt1类、cyt2类和cyt3类,cyt类蛋白中,第一等级蛋白共3类,其中最多的一类为cyt2类,共25个蛋白,cyt类蛋白分子量一般在27~30kDa。Bt具有高效专一、无毒、不污染环境等特点,被广泛作为微生物杀虫剂应用在农林、果蔬以及卫生等害虫防治,近年来Bt杀虫晶体蛋白编码基因已大量应用在转基因玉米、棉花、大豆等农作物中,取得了良好的防治效果。
自1981年Schnepf从菌株HD-1中克隆了第一个能表达杀虫活性的基因以来,人们已经分离克隆了500多种编码杀虫晶体蛋白的基因,根据编码的氨基酸序列同源性它们被分别确定为不同的群、亚群、类和亚类。最近研究表明,cyt类蛋白可能成为控制害虫对cyt类杀虫晶体蛋白产生抗性的一种新策略,因为它与广泛应用的cry类蛋白相比,具有不同的蛋白结构和独特的作用机制。它除了具有杀虫特性以外,还具有在体外广泛溶细胞的能力。这些特性使它成为控制害虫对苏云金杆菌的cry类毒素蛋白的抗性的一种最有潜力的毒蛋白。此外,Federici发现cyt1Aa不但对棉花叶甲(Chrysomela scripa)有较高的毒性,而且可以抑制此叶甲对Cry3Aa的抗性达5000倍。可见,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。
以Bt杀虫晶体蛋白为基础的杀虫剂的使用已有50多年的历史,最初一直没有检测到昆虫对Bt的抗性,但是,上世纪80年中期开始,抗性问题不断在实验室及田间试验中得到证实,原因主要是持续使用单品种及亚致剂量的Bt以及Bt转基因抗虫植物的应用造成昆虫种群长期受到杀虫剂的选择压力。1985年,McGaughey报道仓库谷物害虫印度谷螟(Plodiainterpunctella)在Dipel(Bt subsp.kurstaik HD-1的商品制剂)的选择压力下,繁殖15代后,抗性增加97倍;在高剂量选择压力下,抗性可增加250倍。1990年,在夏威夷首次证实大田中的小菜蛾对Bt杀虫剂产生了明显的抗性(Tabashnik,B.E.,etal.Proc.Natl.Acad.Sci.USA,1994;91:4120-4124),上世纪90年代以来,在我国应用Bt杀虫剂时间较长的深圳、广州、上海等地,发现Bt杀虫剂对小菜蛾防治效果明显下降,意味着抗性已经形成。目前发现在实验室及田间至少有十几种昆虫对Bt及其杀虫晶体蛋白产生了抗性,用选择压力数学模型预测到,在Bt转基因抗虫植物选择压力的条件下,昆虫将会产生抗性。另外,有研究证明Bti在大田的使用中尚未发现抗性问题,但是蚊虫对其抗性问题不断在实验室中得到证实,这种情况也可能会在大田中出现。
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。因此,寻找新的cyt基因资源对我国的生物防治有着十分重要的意义。
发明内容
针对现有技术中的上述不足,本发明提供了一种苏云金芽胞杆菌及其在防治鳞翅目类害虫中的应用,该菌株可有效毒杀农作物中的鳞翅目类害虫,且不会对农作物产生不利影响。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:
本发明提供了一种苏云金芽胞杆菌,该菌株命名为CS72-2,保藏于中国典型培养物保藏中心,保藏单位地址:中国.武汉.武汉大学,保藏日期为2018年12月27日,保藏编号为CCTCC NO.M2018930。
本发明还提供了上述苏云金芽胞杆菌CS72-2在防治鳞翅目类害虫中的应用。
进一步地,鳞翅目类害虫为玉米螟或棉铃虫。
本发明还提供了一种杀虫蛋白cyt2-like,该杀虫蛋白的氨基酸序列如SEQ IDNO:6所示。
本发明还提供了一种杀虫基因cyt2-like,可编码权利要求4所述杀虫蛋白cyt2-like,其核苷酸序列如SEQ ID NO:1。
本发明还提供了一种杀虫剂,包括上述苏云金芽胞杆菌CS72-2的培养菌悬液、发酵液或发酵分离上清液。
本发明提供的苏云金芽胞杆菌及其在防治鳞翅目类害虫中的应用,具有以下有益效果:
(1)本发明提供的苏云金芽胞杆菌CS72-2,通过产生杀虫晶体蛋白对鳞翅目害虫具有较高的毒力,尤其能抑制玉米螟和棉铃虫的生长和侵染。
(2)本发明筛选分离克隆新的、高毒力的Bt杀虫基因,该基因表达的蛋白能够对鳞翅目产生毒力,尤其是玉米螟和棉铃虫有一定的毒杀作用,可以应用于植物的遗传改造,提高其对害虫的抗性,具有较好的生物防治作用。本发明丰富杀虫基因资源,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。
(3)本发明提供的杀虫剂,具有制备简单,成本低,易于工业化生产,防治效果良好,扩大了苏云金芽胞杆菌杀虫剂的使用范围,且不会造成环境污染,降低农药的使用量,具有重要的经济价值和应用前景。
附图说明
图1为CS72-2菌株的电镜扫描图。
图2为CS72-2菌株SDS-PAGE电泳分析结果图;其中,M为蛋白质Marker;1为CS72-2菌株表达的蛋白。
图3为CS72-2菌株中cyt基因型的PCR鉴定结果图;其中M为DNA Marker,2、3、4、5和6为cyt2类基因扩增产物。
图4为CS72-2菌株中cyt2-like基因的PCR扩增产物;其中,M为DNA marker,1为cyt2-like基因。
具体实施方式
实施例1苏云金芽胞杆菌CS72-2的分离筛选及鉴定
1、分离筛选
从贵州省高原地区采取土壤,采用醋酸钠-抗生素分离法进行分离,具体过程为:称取10g土样放入装有50ml醋酸钠培养基的摇瓶中,分别加入青霉素钠盐和硫酸庆大霉素各400μg/ml,摇床培养(200r/min,30℃)4h。培养结束后取土壤悬液10ml,加入无菌的离心管3000r/min离心15min,取上层混浊液2ml于65℃水浴15min,取热处理后的混浊液0.1ml涂平板,将平板置30℃培养箱中培养;培养48h后从平板上挑取类似Bt的菌株涂片,发现一株含有菱形伴胞晶体形态的Bt菌株,将其命名为CS72-2。
2、鉴定方法
用光学显微镜和电子显微镜观察该菌株CS72-2细胞两端钝圆,能形成芽胞,同时能形成菱形伴胞晶体,菌株大小为(1.2-1.5)μm×(3.5-4.4)μm,通常单个或两个存在,一个营养细胞为一个胞子囊,每个胞子囊内含有一个芽胞,次端生,另一端有一个伴胞晶体,胞子囊不膨大(图1)。
将菌株CS72-2表达的蛋白进行SDS-PAGE电泳,结果如图2所示,从图2中可以看出菌株CS72-2主要产生约130kDa、25kDa和15kDa左右的蛋白。
综上所述,CS72-2菌株在分类学上属于苏云金芽胞杆菌(Bacillusthuringiensis),命名为苏云金芽胞杆菌CS72-2,保藏于中国典型培养物保藏中心,保藏单位地址:中国.武汉.武汉大学,保藏日期为2018年12月27日,保藏编号为CCTCCNO.M2018930。
实施例2杀菌基因的初步鉴定
根据cyt2类基因的保守序列设计1对特异引物,来鉴定菌株CS72-2中cyt基因;其中设计的引物序列为:
F:5'-ATTTGAGTTTCTAAATTTGTAAA-3'(SEQ ID No:2);
R:5'-TTTACAAATTTAGAAACTCAAAT-3'(SEQ ID No:3)。
PCR扩增体系为:Taq酶0.2μL,10×Buffer 2.5μL,正、反向引物(10μM)各1μL,模板(DNA)5μL,dNTP(2.5mM)2μL,MgCl2(25mM)1.5μl,无菌ddH2O补足至25μL。
PCR反应程序为:95℃预变性2min;95℃变性20s,55℃退火20s,72℃延伸90s,34个循环;最后,72℃延伸7min。
将PCR产物通过1%琼脂糖凝胶电泳分析,结果如图3所示,由图3可知,利用上述特异性引物对模板进行扩增,分别得到了目标扩增产物,表明CS72-2中含有cyt2类基因。
实施例3杀菌基因cyt2-like的全长基因的克隆及序列分析
采用基因组DNA纯化试剂盒(购自赛百盛公司)提取菌株CS72-2的基因组DNA,并以基因组DNA为模板,以cyt2-like-F和cyt2-like-R为引物来扩增cyt2-like全长基因。
cyt2-like-F:5'-ATGGAGAATAAAAATCAACAC-3'(SEQ ID No:4);
cyt2-like-R:5'-CTATTCCTCCATAAGGAG-3'(SEQ ID No:5)。
PCR扩增体系为:Taq酶0.2μL,10×Buffer 2.5μL,正向引物(cyt2-like-F,10μM)1μL,反向引物(cyt2-like-R,10μM)1μL,模板(DNA)5μL,dNTP(2.5mM)2μL,MgCl2(25mM)1.5μl,无菌ddH2O补足至25μL。
PCR反应程序为:95℃预变性2min;95℃变性20s,55℃退火20s,72℃延伸90s,34个循环;最后,72℃延伸7min。
获得PCR产物通过1%琼脂糖凝胶电泳分析,结果如图4所示,得到长1137bp的基因。按胶回收试剂盒(9672,Takara)将1137bp的片段纯化后备用。
通过TA克隆载体将目的片段与pET28a载体连接,获得重组质粒pET28a-cyt2-like,将重组质粒转化大肠杆菌DH5a,从含有氨苄霉素(100mg/L)的LB培养板上挑取阳性克隆,并将其进行测序分析。
结果表明,cyt2-like全长基因序列如SEQ ID NO:1所示,含有1137bp的开放阅读框,GC含量为32%,进行测序,在GenBank上进行检索,结果表明所获得的基因为新基因,并且菌株CS72-2与已知的任何其他菌株不同,是一个新菌株,其氨基酸序列为SEQ ID NO:6所示,编码378个氨基酸组成的蛋白。经测定,其蛋白的氨基酸组成如表1所示。
表1 cyt2-like蛋白的氨基酸组成
实施例4 cyt2-like基因表达及其杀虫活性测定
将实施例3构建的重组质粒pET28a-cyt2-like转入感受态细胞E.coli.BL21(DE3)中,获得表达菌株。IPTG诱导表达后,纯化得到cyt2-like蛋白。
将cyt2-like蛋白配制成10g/mL、5g/mL、2.5g/mL、1.25g/mL、0.625g/mL和0.01g/mL等6个不同的浓度梯度,分别取3ml与饲料搅拌混匀,然后分别挑选活跃的玉米螟幼虫和棉铃虫幼虫接于饲料上,以E.coli.BL21(DE3)混合的饲料为阴性对照,清水为空白对照。每个处理重复3次,每个重复为20头相应的害虫。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,培养3d后统计棉铃虫幼虫的死亡情况,培养7d后统计玉米螟幼虫的死亡情况,用SPSS 10.0软件计算LC50,结果如表2所示。
表2菌株CS72-2生物活性测定结果
由表2可知,,E.coli.BL21(DE3)和空白对照对玉米螟和棉铃虫不具杀虫活性,而菌株CS72-2表达的cyt2-like蛋白对鳞翅目害虫具有显著的毒杀活性。
综上,本发明苏云金芽胞杆菌CS72-2中cyt2-like基因序列及其表达蛋白能够对鳞翅目产生毒力,尤其是玉米螟和棉铃虫有一定的毒杀作用,可以应用于植物的遗传改造,提高其对害虫的抗性,具有较好的生物防治作用,且不会造成环境污染。
序列表
<110> 长江师范学院
<120> 一种苏云金芽孢杆菌及其在防治鳞翅目类害虫中的应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1135
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgcatacac aaatggcttt ttcccaacag aactttaaag ttcatggggt taaatttttt 60
gatggactat taaacatcgt aaaactagaa aatgaaagaa aagagttacc atcttcagct 120
ttagctactt acatcttgct tcactttgaa tgcaatgata taggaatgct accacgtgaa 180
tttcaaataa tggatcttgc taagaaaagt gggattccct acacaacaat ttatacaggg 240
tttcaagttt gtttggagcg tagactcgta aaagaaacac ctgttggaaa tcgtactgta 300
tatgaaattg tagattatgc gttgtataac cacactactg cagaaactac aaagatgaca 360
ggcatatcgc tatcgtattt ccgtattcca tttcacctaa ttcaaacaac gataatgagt 420
agtctagtaa aggctcgtga taatagaggg attatattcc ttttagattt aatcaatact 480
ttttcaagaa aagtaggtat ggaacattat aaacataaaa ttgaggagtt tcagattaca 540
agaaagatgt cttccttaaa aaagagatta aatcgtaatg caaagaaagt aagacgctac 600
atagaaatta ttaaccccat cgtgcaattt actgcaatcg attctaaaga gaagaaatct 660
aatgaaacaa gaattacaag tgtaaggaaa caggtaaaac aaatcatcat tgagaagttc 720
aacgttgtat tttcttcaaa ctgcgtaatt gagaatgata aaaaagaatt acatagacca 780
gtagcgaagt gcagaaaaga agcggtgtct cgtttgaaac acatgggaca ggcgttacgt 840
aaaaaagata aagaaaatat aatgactgca tttagacaag aaattgttga tatcgcaatc 900
tatttaccga cgaaacaaaa acaaagtgaa ttattaattt acgcaatgac ttatgcatta 960
gatcaagttg aaagttgttt gaaagaacaa gaaatattct ctattccagc atttatgcgt 1020
atacagttta gagagtcctg gagaagtttt aaggaagaaa acttgtctac aggggaaaga 1080
cacgatgcaa tgataaatta tcataagaaa aatggtgttt atccggaatt tatttaa 1137
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atttgagttt ctaaatttgt aaa 23
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tttacaaatt tagaaactca aat 23
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggagaata aaaatcaaca c 21
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctattcctcc ataaggag 18
<210> 6
<211> 378
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met His Thr Gln Met Ala Phe Ser Gln Gln Asn Phe Lys Val His Gly
1 5 10 15
Val Lys Phe Phe Asp Gly Leu Leu Asn Ile Val Lys Leu Glu Asn Glu
20 25 30
Glu Lys Glu Leu Pro Ser Ser Ala Leu Ala Thr Tyr Ile Leu Leu His
35 40 45
Phe Glu Cys Asn Asp Ile Gly Met Leu Pro Arg Glu Phe Gln Ile Met
50 55 60
Asp Leu Ala Lys Lys Ser Gly Ile Pro Tyr Thr Thr Ile Tyr Thr Gly
65 70 75 80
Phe Gln Val Cys Leu Glu Arg Arg Leu Asp Lys Glu Thr Pro Val Gly
85 90 95
Asn Arg Thr Asp Tyr Glu Ile Val Asp Tyr Ala Leu Tyr Thr His Thr
100 105 110
Thr Ala Glu Thr Thr Lys Met Thr Gly Ile Ser Leu Ser Tyr Phe Arg
115 120 125
Ile Pro Phe His Leu Ile Gln Thr Thr Ile Met Ser Ser Leu Val Lys
130 135 140
Ala Arg Asp Asn Arg Gly Ile Ile Phe Leu Leu Asp Leu Ile Asn Thr
145 150 155 160
Phe Ser Arg Lys Val Gly Met Glu His Tyr Lys His Lys Ile Glu Glu
165 170 175
Phe Gln Ile Thr Arg Lys Met Ser Ser Leu Lys Lys Arg Leu Asn Arg
180 185 190
Asn Ala Lys Lys Val Arg Arg Tyr Ile Glu Ile Ile Asn Pro Ile Val
195 200 205
Gln Phe Thr Ala Ile Asp Ser Lys Glu Lys Lys Ser Asn Glu Thr Arg
210 215 220
Ile Thr Ser Val Arg Lys Gln Val Lys Gln Ile Ile Ile Glu Lys Phe
225 230 235 240
Asn Val Val Phe Ser Ser Asn Cys Val Ile Glu Asn Asp Lys Lys Glu
245 250 255
Leu His Arg Pro Val Ala Lys Cys Arg Lys Glu Ala Val Ser Arg Leu
260 265 270
Lys His Met Gly Gln Ala Leu Arg Lys Lys Asp Lys Glu Asn Ile Met
275 280 285
Thr Ala Phe Arg Gln Glu Ile Val Asp Ile Ala Ile Tyr Leu Pro Thr
290 295 300
Lys Gln Lys Gln Ser Glu Leu Leu Ile Tyr Ala Met Thr Tyr Ala Leu
305 310 315 320
Asp Gln Val Glu Ser Cys Leu Lys Glu Gln Glu Ile Phe Ser Ile Pro
325 330 335
Ala Phe Met Arg Ile Gln Phe Arg Glu Ser Trp Arg Ser Phe Lys Glu
340 345 350
Glu Asn Leu Ser Thr Gly Glu Arg His Asp Ala Met Ile Asn Tyr His
355 360 365
Lys Lys Asn Gly Val Tyr Pro Glu Phe Ile
370 375
Claims (2)
1.一种杀虫剂,其特征在于,有效成分为苏云金芽胞杆菌CS72-2和/或氨基酸序列如SEQ ID NO:6所示的杀虫蛋白cyt2-like;所述苏云金芽胞杆菌CS72-2保藏于中国典型培养物保藏中心,保藏日期为2018年12月27日,保藏编号为CCTCC NO.M2018930;所述杀虫剂可杀害玉米螟。
2.根据权利要求1所述的杀虫剂,其特征在于,所述苏云金芽胞杆菌CS72-2为该菌株的培养菌悬液、发酵液或发酵分离上清液。
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CN112314631B (zh) * | 2020-11-09 | 2021-07-30 | 中国计量大学 | 一种生物源杀虫剂及其制备方法 |
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