苏云金芽胞杆菌的筛选鉴定及新型cry基因克隆、表达的研究
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摘要
苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)作为一种高效、安全、无污染的昆虫病原微生物,在害虫的生物防治中得到了广泛的应用,已发展成为全球产量最大、应用面积最广的微生物杀虫剂。因此,对Bt的研究越来越受到世界各国的重视,对Bt资源的调查也在逐渐扩大。我国地域辽阔,生态环境复杂多样,深入发掘我国丰富的苏云金芽胞杆菌资源,筛选高毒力菌株、分离克隆新的高效广谱杀虫基因,对构建高效广谱工程菌及培育转基因抗虫植物具有深远意义。
本研究对黑龙江省不同类型土壤采集的土样及四川省5个地区采集的土样进行了苏云金芽胞杆菌的分离、鉴定,并进行了新型cry基因的克隆和表达研究。结果如下：
1.从黑龙江省不同类型土壤中采集372份土样,共分离出Bt菌株41株,出菌率为11.02%。黑土与暗棕壤Bt出菌率较高,分别为15.53%及13.95%,黄沙土出菌率最低仅为5.88%,在采集的17份盐碱土中未分离出Bt菌,说明Bt分布的丰度与土壤类型有很大关系。采用PCR-RFLP方法,对41株菌株进行了基因型鉴定。结果表明：32株含有cry1型基因(其中有两株菌株的酶切图谱中含有新的特异性条带,推测含有新的未知基因),13株含有cry2型基因,12株含有crylI型基因,另有9株菌株未鉴定出已知基因。
2.从四川省采集的207份土样中,共分离出26株Bt野生菌株,平均出菌率为12.56%。26株Bt分离株伴胞晶体的形态有球形、方形、菱形、及不规则形,且多数菌株都同时含有多种形态的伴胞晶体,充分体现了四川省Bt菌株资源的多样性。基因型的鉴定结果也可看出四川生态条件下Bt菌株多样性的特点,26株菌株共含有cryl、cry2、cry1I及cry9四种基因型,其中21株含有cry1型基因,8株含有crylI型基因,11株含有cry2型基因,13株含有cry9型基因,5株菌株未鉴定出已知基因。多数菌株都同时含有多种类型的基因组合,以crylAa、cry1Ac、crylIb、cry2Ab、cry9Ba基因型最丰富。
3.对两省分离的67株Bt菌的SDS-PAGE分析表明：多数菌株表达了130kD的蛋白,少数菌株表达了60、120和140kD的蛋白,在个别菌株中还发现有45kD、70kD和90kD的蛋白表达,可以推测,在这些菌株中可能含有新型的杀虫基因,另有2株菌株未发现有任何蛋白表达,其原因有待于进一步研究。
4.系统地研究了Bt菌株BF-4和NJ-1的生物学特性。BF-4的10项生理生化反应结果与标准菌株猝倒亚种反应一致,NJ-1与蜡螟亚种一致；BF-4主要形成菱形、方形、球形晶体,NJ-1则形成菱形、球形和不规则形晶体；两株菌都表达140kD的蛋白,BF-4还同时表达了60kD的蛋白；BF-4含有3个大小不同的质粒,含有crylAa、cry1Ac、cry1Ib、cry2Ab和cry9Ba5种基因；菌株NJ-1含有2个大小不同的质粒,含有crylAa、cry1Ia和cry9Ba共3种基因。两株菌株的基因均定位于大质粒上。
5.从菌株BF-4中克隆了cry2Ab和cryllb两种基因。基因的核苷酸序列已经在GenBank中注册,Accession number分别为HM037126和HM051227,并经国际Bt杀虫晶体蛋白基因命名委员会分别正式命名为cry2Ab15和cryllb4o cry2Abl5的编码区为1905bps,编码634个氨基酸的蛋白,蛋白分子量为70.8kDa; cryllb4的编码区为2160bps,编码的蛋白质由719个氨基酸组成,该蛋白质的分子量为81.2kDa,等电点为6.71,为弱酸性蛋白质。序列分析结果表明cry2Ab15和cryllb4与已发表的基因在氨基酸序列上均有不同程度的差异,属于新的cry基因。
6.从菌株NJ-1中克隆了cry9Ba2。该基因的编码框由3495bp组成,所编码的蛋白质由1164个氨基酸组成,分子量为131.6kDa,pI=4.92,为弱酸性蛋白质。该基因序列与已报道的cry9Bal同源性最高达98%,该基因已在GenBank中注册,Accession number为GU299522,并经国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry9Ba2。
7. cry2Abl5和cryllb4均在大肠杆菌中获得了高效表达,cry2Ab15的表达产物对小菜蛾和棉铃虫均有很高的杀虫活性,3d的校正死亡率分别为100%和90.8%；Cry1Ib4对小菜蛾具有较好的活性,对大猿叶虫的毒力一般,96h的校正死亡率分别为82.5%和46.6%。
Bacillus thuringiensis (Abbreviation, Bt) as an efficient, safe, pollution-free insect pathogenic microorganisms has been widely used in the biological control of pests. It has been produced largest as a microbial pesticides and widely applied in most area in global. So the study on Bacillus thuringiensis was paid more and more attention by the countries in the world, and the investigation of Bt resources are gradually expanding. Because of the extensive terrain and diversity of environment in our country, it is very significant and necessary to explore the rich Bt resources, screen Bt isolates with high toxicity and clone the novel insecticidal toxin genes for construction of genetically engineered bacteria and transgenic plants. In this study, we described a screening and identification of Bacillus thuringiensis from different sort of soils of Hei Long Jiang Province and Sichuan province, Several novel insecticidal crystal protein genes were cloned and expressed.The concrete results are as follows:
1. Three hundred and seventy two soils samples were collected from Hei Long Jiang Province, and 41 strains of native Bacillus thuringiensis were isolated from the samples. The percentage of Bt strains isolation is 11.02%. The most high percentage of Bt isolation from black soil is 15.53% and yellow sand's is lowest as 5.88%, however none Bt strains were isolated from the seventeen saline-alkali soil. So the abundance of Bt distribution have relationship with the Soil kinds. The genotypes of 41 Bt isolates were identified by PCR-RFLP. The results show that 32 isolates harbored cry1 genes(Among them, two strains' restriction map contained a new specific band, speculated that there were unknown genes with them),13 isolates harbored cry2 genes,12 isolates harbored cry1I genes, the known genes were not identified in another nine strains.
2. Two hundred and seven soils samples were collected from Sichuan Province, and 26 strains of native Bacillus thuringiensis were isolated from the samples. The percentage of Bt strains isolation is 12.56%. The parasporal crystal shapes of Bacillus thuringiensis were pyramid, cuboidal, round and abnormity, furthermore most of them there were a variety of forms parasporal crystal in one strain.which showed the diversity of Bt resources in Sichuan province.The Identification results of genotype can also display the diversity of Bt strains under the ecological conditions in Sichuan. 26 strains contain cryl, cry2, cry1I and cry9 four genotypes,21 isolates harbored cry1 genes, 8 isolates harbored crylI genes,11 isolates harbored cry2 genes,13 isolates harbored cry9 genes, the known genes were not identified in another five strains. most of them contain a variety of parasporal crystal in one strain. Most of them are crylAa, crylAc, cryllb, cry2Ab and cry9Ba.
3. The SDS-PAGE analysis of 67 strains showed that most strains expressed 130kDa protein, and part of them expressed 60,120 and 140kDa protein, a few of them expressed 45,70 and 90kDa protein. Suggesting that they may contain potential novel Cry toxins. There were no proteins expressed in another two strains, the reasons need to be further studied.
4. The biological characteristics of Bt strains BF-4 and NJ-1 were studied systematically in this paper. Biochemical reaction indicated that BF-4 was identical with Bt subsp.sotto,while NJ-1 was the same as Bt subsp.galleriae. Crystal shape of BF-4 was bipyramidal, cuboidal and round, but in NJ-1 bipyramidal, spherical and irregular shapes were observed.140kDa ICP was encoded in the two strains, but 60kD ICPs were encoded in BF-4. Three different plasmids were detected from BF-4, there were crylAa、cry1Ac、cryllb、cry2Ab and cry9Ba in the strain. Two different plasmids were detected from NJ-1, there were crylAa, cry1Ia and cry9Ba in the strain..and all genes of these two strains were located on its plasmids seperately.
5. Two cry genes cry2Ab and cryllb were coloned from BF-4 successively.They had been registered in GenBank, The accession number is HM037126 and HM051227 respectively, The gene had been named cry2Ab15 and cryllb4 by Btδ-endotoxin gene Nomenclature Committee respectively. Cry2Ab15,70.8kD protein, was composed of 634 amino acids deduced from 1905bps nucletide sequence.Cry1Ib4,81.2kD protein, was composed of 719 amino acids deduced from 2160bps DNA sequences. Isoelectric point of the protein is 6.71, so it is a weak acid protein.Sequence analysis showed that cry2Ab and cryllb are different from the genes published in GenBank.They should be new cry genes.
6. cry9Ba2 was coloned from NJ-1.The encoding sequence is made up of 3495 bps, Cry9Ba2,131.6kD protein, was composed of 1164 amino acids. The amino acid sequences deduced from its DNA fragment as 98% homologous with Cry9Ba1. The gene sequence had been registered in GenBank, The accession number is GU299522. The gene had been named cry9Ba2 by Bt 8-endotoxin gene Nomenclature Committee.
7. cry2Abl5 and cryllb4 were expressed efficiently in E.coli. Cry2Ab15was highly toxic to the larvae of Plutella xylostella and Helicoverpa armigera, LC50 is 5.7μg/ml and 4.3μg/g respectively. Cry1Ib4 has higer toxic to Plutell axylostella L, but not higher to Colaphellus bowringi L.The Corrected mortality is 82.5% and 46.6% respectively.
本研究对黑龙江省不同类型土壤采集的土样及四川省5个地区采集的土样进行了苏云金芽胞杆菌的分离、鉴定,并进行了新型cry基因的克隆和表达研究。结果如下：
1.从黑龙江省不同类型土壤中采集372份土样,共分离出Bt菌株41株,出菌率为11.02%。黑土与暗棕壤Bt出菌率较高,分别为15.53%及13.95%,黄沙土出菌率最低仅为5.88%,在采集的17份盐碱土中未分离出Bt菌,说明Bt分布的丰度与土壤类型有很大关系。采用PCR-RFLP方法,对41株菌株进行了基因型鉴定。结果表明：32株含有cry1型基因(其中有两株菌株的酶切图谱中含有新的特异性条带,推测含有新的未知基因),13株含有cry2型基因,12株含有crylI型基因,另有9株菌株未鉴定出已知基因。
2.从四川省采集的207份土样中,共分离出26株Bt野生菌株,平均出菌率为12.56%。26株Bt分离株伴胞晶体的形态有球形、方形、菱形、及不规则形,且多数菌株都同时含有多种形态的伴胞晶体,充分体现了四川省Bt菌株资源的多样性。基因型的鉴定结果也可看出四川生态条件下Bt菌株多样性的特点,26株菌株共含有cryl、cry2、cry1I及cry9四种基因型,其中21株含有cry1型基因,8株含有crylI型基因,11株含有cry2型基因,13株含有cry9型基因,5株菌株未鉴定出已知基因。多数菌株都同时含有多种类型的基因组合,以crylAa、cry1Ac、crylIb、cry2Ab、cry9Ba基因型最丰富。
3.对两省分离的67株Bt菌的SDS-PAGE分析表明：多数菌株表达了130kD的蛋白,少数菌株表达了60、120和140kD的蛋白,在个别菌株中还发现有45kD、70kD和90kD的蛋白表达,可以推测,在这些菌株中可能含有新型的杀虫基因,另有2株菌株未发现有任何蛋白表达,其原因有待于进一步研究。
4.系统地研究了Bt菌株BF-4和NJ-1的生物学特性。BF-4的10项生理生化反应结果与标准菌株猝倒亚种反应一致,NJ-1与蜡螟亚种一致；BF-4主要形成菱形、方形、球形晶体,NJ-1则形成菱形、球形和不规则形晶体；两株菌都表达140kD的蛋白,BF-4还同时表达了60kD的蛋白；BF-4含有3个大小不同的质粒,含有crylAa、cry1Ac、cry1Ib、cry2Ab和cry9Ba5种基因；菌株NJ-1含有2个大小不同的质粒,含有crylAa、cry1Ia和cry9Ba共3种基因。两株菌株的基因均定位于大质粒上。
5.从菌株BF-4中克隆了cry2Ab和cryllb两种基因。基因的核苷酸序列已经在GenBank中注册,Accession number分别为HM037126和HM051227,并经国际Bt杀虫晶体蛋白基因命名委员会分别正式命名为cry2Ab15和cryllb4o cry2Abl5的编码区为1905bps,编码634个氨基酸的蛋白,蛋白分子量为70.8kDa; cryllb4的编码区为2160bps,编码的蛋白质由719个氨基酸组成,该蛋白质的分子量为81.2kDa,等电点为6.71,为弱酸性蛋白质。序列分析结果表明cry2Ab15和cryllb4与已发表的基因在氨基酸序列上均有不同程度的差异,属于新的cry基因。
6.从菌株NJ-1中克隆了cry9Ba2。该基因的编码框由3495bp组成,所编码的蛋白质由1164个氨基酸组成,分子量为131.6kDa,pI=4.92,为弱酸性蛋白质。该基因序列与已报道的cry9Bal同源性最高达98%,该基因已在GenBank中注册,Accession number为GU299522,并经国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry9Ba2。
7. cry2Abl5和cryllb4均在大肠杆菌中获得了高效表达,cry2Ab15的表达产物对小菜蛾和棉铃虫均有很高的杀虫活性,3d的校正死亡率分别为100%和90.8%；Cry1Ib4对小菜蛾具有较好的活性,对大猿叶虫的毒力一般,96h的校正死亡率分别为82.5%和46.6%。
Bacillus thuringiensis (Abbreviation, Bt) as an efficient, safe, pollution-free insect pathogenic microorganisms has been widely used in the biological control of pests. It has been produced largest as a microbial pesticides and widely applied in most area in global. So the study on Bacillus thuringiensis was paid more and more attention by the countries in the world, and the investigation of Bt resources are gradually expanding. Because of the extensive terrain and diversity of environment in our country, it is very significant and necessary to explore the rich Bt resources, screen Bt isolates with high toxicity and clone the novel insecticidal toxin genes for construction of genetically engineered bacteria and transgenic plants. In this study, we described a screening and identification of Bacillus thuringiensis from different sort of soils of Hei Long Jiang Province and Sichuan province, Several novel insecticidal crystal protein genes were cloned and expressed.The concrete results are as follows:
1. Three hundred and seventy two soils samples were collected from Hei Long Jiang Province, and 41 strains of native Bacillus thuringiensis were isolated from the samples. The percentage of Bt strains isolation is 11.02%. The most high percentage of Bt isolation from black soil is 15.53% and yellow sand's is lowest as 5.88%, however none Bt strains were isolated from the seventeen saline-alkali soil. So the abundance of Bt distribution have relationship with the Soil kinds. The genotypes of 41 Bt isolates were identified by PCR-RFLP. The results show that 32 isolates harbored cry1 genes(Among them, two strains' restriction map contained a new specific band, speculated that there were unknown genes with them),13 isolates harbored cry2 genes,12 isolates harbored cry1I genes, the known genes were not identified in another nine strains.
2. Two hundred and seven soils samples were collected from Sichuan Province, and 26 strains of native Bacillus thuringiensis were isolated from the samples. The percentage of Bt strains isolation is 12.56%. The parasporal crystal shapes of Bacillus thuringiensis were pyramid, cuboidal, round and abnormity, furthermore most of them there were a variety of forms parasporal crystal in one strain.which showed the diversity of Bt resources in Sichuan province.The Identification results of genotype can also display the diversity of Bt strains under the ecological conditions in Sichuan. 26 strains contain cryl, cry2, cry1I and cry9 four genotypes,21 isolates harbored cry1 genes, 8 isolates harbored crylI genes,11 isolates harbored cry2 genes,13 isolates harbored cry9 genes, the known genes were not identified in another five strains. most of them contain a variety of parasporal crystal in one strain. Most of them are crylAa, crylAc, cryllb, cry2Ab and cry9Ba.
3. The SDS-PAGE analysis of 67 strains showed that most strains expressed 130kDa protein, and part of them expressed 60,120 and 140kDa protein, a few of them expressed 45,70 and 90kDa protein. Suggesting that they may contain potential novel Cry toxins. There were no proteins expressed in another two strains, the reasons need to be further studied.
4. The biological characteristics of Bt strains BF-4 and NJ-1 were studied systematically in this paper. Biochemical reaction indicated that BF-4 was identical with Bt subsp.sotto,while NJ-1 was the same as Bt subsp.galleriae. Crystal shape of BF-4 was bipyramidal, cuboidal and round, but in NJ-1 bipyramidal, spherical and irregular shapes were observed.140kDa ICP was encoded in the two strains, but 60kD ICPs were encoded in BF-4. Three different plasmids were detected from BF-4, there were crylAa、cry1Ac、cryllb、cry2Ab and cry9Ba in the strain. Two different plasmids were detected from NJ-1, there were crylAa, cry1Ia and cry9Ba in the strain..and all genes of these two strains were located on its plasmids seperately.
5. Two cry genes cry2Ab and cryllb were coloned from BF-4 successively.They had been registered in GenBank, The accession number is HM037126 and HM051227 respectively, The gene had been named cry2Ab15 and cryllb4 by Btδ-endotoxin gene Nomenclature Committee respectively. Cry2Ab15,70.8kD protein, was composed of 634 amino acids deduced from 1905bps nucletide sequence.Cry1Ib4,81.2kD protein, was composed of 719 amino acids deduced from 2160bps DNA sequences. Isoelectric point of the protein is 6.71, so it is a weak acid protein.Sequence analysis showed that cry2Ab and cryllb are different from the genes published in GenBank.They should be new cry genes.
6. cry9Ba2 was coloned from NJ-1.The encoding sequence is made up of 3495 bps, Cry9Ba2,131.6kD protein, was composed of 1164 amino acids. The amino acid sequences deduced from its DNA fragment as 98% homologous with Cry9Ba1. The gene sequence had been registered in GenBank, The accession number is GU299522. The gene had been named cry9Ba2 by Bt 8-endotoxin gene Nomenclature Committee.
7. cry2Abl5 and cryllb4 were expressed efficiently in E.coli. Cry2Ab15was highly toxic to the larvae of Plutella xylostella and Helicoverpa armigera, LC50 is 5.7μg/ml and 4.3μg/g respectively. Cry1Ib4 has higer toxic to Plutell axylostella L, but not higher to Colaphellus bowringi L.The Corrected mortality is 82.5% and 46.6% respectively.
引文
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