四川蒙顶山苏云金芽胞杆菌cry基因鉴定及其新型模式cry基因的克隆与表达
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摘要
苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)作为一种对人畜安全、无环境污染的微生物杀虫剂,在农、林及卫生害虫的防治中发挥了重要的作用,成为国内外开发应用最成功的一种微生物农药。因此深入发掘我国丰富的Bt资源,筛选具有高效广谱杀虫活性的Bt菌株,并对其基因型进行分析,克隆新型杀虫基因,这对微生物杀虫剂的研制、构建高效广谱工程微生物和培育转基因抗虫植物都具有重要的理论及实践意义。本研究对四川雅安蒙顶山土壤中Bt资源进行了较为系统地调查研究,从中克隆到一个新的cry2型模式基因,并对其进行了初步的表达研究,研究摘要如下：
1.根据海拔高度和生态环境的不同,从蒙顶山采集了285份土壤样本,采用醋酸钠-抗生素法对土样中Bt菌株进行分离,获得78株Bt菌株。土样中Bt菌的平均分离率为19%。显微镜观察显示,分离出的Bt菌株产生形态各异的伴胞晶体,有长菱形、短菱形、球形、方形、不定形等,充分显示了蒙顶山生态区Bt资源多样性的特点。利用PCR-RFLP鉴定体系鉴定了78株Bt菌的杀虫晶体蛋白基因类型：其中68株含有cry1型基因,28株含有cry2型基因,2株含有cry3型基因,2株含有cry9型基因,3株含有cry4/10型基因,3株含有cry30型基因。用SDS-PAGE对这些菌株的杀虫晶体蛋白进行分析,结果表明：这些菌株主要表达4种大小的晶体蛋白,分子量在40-130kDa之间。对于没有扩增产物的Bt菌株,其SDS-PAGE分析却检测到晶体蛋白,由此可推测,这些菌株中可能含有新型的杀虫基因。
2.用cry2型基因的通用引物鉴定出28株含有cry2型基因的Bt菌株,再用DdeI内切酶分别对它们的扩增产物进行基因分型,结果发现,菌株JF19-2的扩增片段的酶切产物大小与已知的cry2型基因的酶切片段大小存在差异,说明JF19-2中可能含有一个新的cry2型模式基因。
3.根据cry2型基因的通用引物对菌株JF19-2的扩增序列,在序列的上游和下游分别设计特异引物SP1和SP2,采用Tail-PCR技术获得两侧的未知序列,将扩增序列克隆并测序,测序结果与已知序列进行拼接,得到了3,285 bp的核苷酸序列,其中含有一个长1,905bp的开放阅读框(ORF),编码一个由635个氨基酸组成的蛋白,分子量为70.8kDa,等电点为9.18,该蛋白为弱碱性蛋白,其中异亮氨酸(Ile)、天冬酰氨(Asn)、色氨酸(Ser)和苏氨酸(Thr)四种氨基酸含量最高,分别为11.2%、10.57%、9.94%和8.36%。氨基酸同源性比较发现,其与Cry2Ab1蛋白同源性最高,为92%。该新基因已在GenBank中注册,其Accession number为ACH91610,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Ag1。
4.根据cry2Ag1基因ORF两端序列,设计一对特异引物P1和P2。PCR扩增获得cry2Ag1完整ORF。将获得的片段经NcoⅠ和XhoⅠ双酶切,与构建的大肠杆菌表达载体pET-22b (+)连接,构建了重组表达质粒pET-2Ag1。将该质粒导入E. coli BL21 (DE3) pLysS,经IPTG诱导能正常表达,SDS-PAGE分析表明：cry2Ag1基因在大肠杆菌中表达一个约60kDa的蛋白,比该基因推导的Cry2Ag1蛋白理论分子量小(70kDa)。生物活性测定表明表达的包涵体蛋白对鳞翅目的小菜蛾和棉铃虫,双翅目的伊蚊均具有杀虫活性,LC50分别为23.47μg/mL、9.74μg/mL、2.54μg/mL。
Bacillus thuringiensis (Bt), as a safeties to human and animals, pollution-free microbial insecticides, plays a very important role in pest prevention and cure of agriculture, forestry and sanitary insect pest. It becomes a most successful development and apply of microbial pesticide at abroad and home. So to futher dig abundant resource of Bt in our country, screen high effective and broad-spectrum insecticidal activity of Bt strain, analyse their cry gene types, isolate and clone novel pesticidal genes will have important meanings in theories and practices for developping microbial insecticides, constructing high toxicity and broadspectrum engineering strains and breeding insect resistant transgenic plants. This study described a systematic study of Bt resources from the soil in MengDing mountain of Yaan city in Sichuan province. A novel pesticidal protein genes was cloned and expressed. The concrete results are as follows:
1. According to different altitude and environment, we collected 285 soil samples and isolated 78 Bt strains from these soil samples by using sodium acetate-antibiotics method. The average, rate was 19%. Observed by electron microscope, These Bt strains produced different patterns of parasporal crystals, such as long bipyramid, short bipyramid, cuboidal, round and abnormity, which showed the diversity of Bt resources in Mengding mountain. The cry gene-types of 78 Bt isolates were identified by using PCR-RFLP system.68 isolates harbored cry1 genes,28 isolates harbored cry2 genes,2 isolates harbored cry3 genes,2 isolates harbored cry9 genes,3 isolates harbored cry4/10 genes and 3 isolates harbored cry3 genes. Using SDS-PAGE method to analyse insecticidal crystal proteins of these Bt strains, the result showed that four different kinds of insecticidal crystal proteins were expressed, whose molecular weight were between 40 kDa and 130 kDa. Some Bt strains didn't produce any PCR products in PCR-RFLP analysis. But SDS-PAGE assay indicated that these isolates produced crystal proteins, which suggested that they may contain potentially novel Cry toxin genes.
2.28 Bt strains which contained cry2 genes were identified by using general primers of cry2 gene. Then restriction enzyme DdeⅠwas used to digest these PCR productions for identification of cry gene-types. The results showed that the size of of the Bt strain JF19-2 amplified fragment was different from enzyme cut pieces of known cry2 genes, which indicated that JF19-2 contained a novel cry 2 holetype gene.
3. According to amplified fragment of JF19-2 by using general primers of cry2 genes, a pair of specific primers SP1 and SP2 was designed, in upstream and downstream of amplified fragment. Unknown sequence of the novel cry2 gene was amplified by Tail-PCR method. Then amplified sequences was cloned and sequenced. Sequencing result was matched with known sequence of the novel cry2 gene. A 3,285 bp nucleotide sequence was obtained. There was a 1,905 bp ORF in the obtained sequence, which encoded a protein of 635 amino acids with a predicted molecular mass of 70.8 kDa and isoelectric point of 4.952.This protein was an alkalescent protein, which mainly contained four kinds of Amino acids:Ile, Asn, Ser and Thr, accounted for 11.2%,10.57%,9.94% and 8.36%, respectively. Compared with other known Cry2 proteins, Cry2Ag1 and Cry2Ab1 had shown as high as 92% Amino acids sequence homology. This, novel gene was registered in the GeneBank databases under accession number ACH91610. This gene was designed as cry2Agl in the Bacillus thuringiensis Toxin Nomenclature Committee.
4. The full open reading frame sequence of the cry2Ag1 gene was amplified with a pair of PCR primers P1/P2 designed according to cry2Agl gene sequence, and inserted into the NcoⅠ/XhoⅠsite of E. coli expression vector pET-22b(+) to obtain the recombinant plasmid pET-2Ag1. The pET-2Agl was transduced into E. coli BL21 (DE3) pLysS. The analysis of SDS-PAGE showed that the cry2Agl gene produced 60kDa protein in E.coli by IPTG induction, which smaller than calculated Cry2Ag1 protein (70kDa) according to cry2Agl gene. Bioassay of the expressed product of the cry2Agl gene showed that Cry2Agl was highly toxic to both Lepidoptera P.xylostella, H.armigera and Dipteral A.aegypti with LC50 as 23.47μg/mL,9.74μg/mL and 2.54μg/mL, respectively.
1.根据海拔高度和生态环境的不同,从蒙顶山采集了285份土壤样本,采用醋酸钠-抗生素法对土样中Bt菌株进行分离,获得78株Bt菌株。土样中Bt菌的平均分离率为19%。显微镜观察显示,分离出的Bt菌株产生形态各异的伴胞晶体,有长菱形、短菱形、球形、方形、不定形等,充分显示了蒙顶山生态区Bt资源多样性的特点。利用PCR-RFLP鉴定体系鉴定了78株Bt菌的杀虫晶体蛋白基因类型：其中68株含有cry1型基因,28株含有cry2型基因,2株含有cry3型基因,2株含有cry9型基因,3株含有cry4/10型基因,3株含有cry30型基因。用SDS-PAGE对这些菌株的杀虫晶体蛋白进行分析,结果表明：这些菌株主要表达4种大小的晶体蛋白,分子量在40-130kDa之间。对于没有扩增产物的Bt菌株,其SDS-PAGE分析却检测到晶体蛋白,由此可推测,这些菌株中可能含有新型的杀虫基因。
2.用cry2型基因的通用引物鉴定出28株含有cry2型基因的Bt菌株,再用DdeI内切酶分别对它们的扩增产物进行基因分型,结果发现,菌株JF19-2的扩增片段的酶切产物大小与已知的cry2型基因的酶切片段大小存在差异,说明JF19-2中可能含有一个新的cry2型模式基因。
3.根据cry2型基因的通用引物对菌株JF19-2的扩增序列,在序列的上游和下游分别设计特异引物SP1和SP2,采用Tail-PCR技术获得两侧的未知序列,将扩增序列克隆并测序,测序结果与已知序列进行拼接,得到了3,285 bp的核苷酸序列,其中含有一个长1,905bp的开放阅读框(ORF),编码一个由635个氨基酸组成的蛋白,分子量为70.8kDa,等电点为9.18,该蛋白为弱碱性蛋白,其中异亮氨酸(Ile)、天冬酰氨(Asn)、色氨酸(Ser)和苏氨酸(Thr)四种氨基酸含量最高,分别为11.2%、10.57%、9.94%和8.36%。氨基酸同源性比较发现,其与Cry2Ab1蛋白同源性最高,为92%。该新基因已在GenBank中注册,其Accession number为ACH91610,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Ag1。
4.根据cry2Ag1基因ORF两端序列,设计一对特异引物P1和P2。PCR扩增获得cry2Ag1完整ORF。将获得的片段经NcoⅠ和XhoⅠ双酶切,与构建的大肠杆菌表达载体pET-22b (+)连接,构建了重组表达质粒pET-2Ag1。将该质粒导入E. coli BL21 (DE3) pLysS,经IPTG诱导能正常表达,SDS-PAGE分析表明：cry2Ag1基因在大肠杆菌中表达一个约60kDa的蛋白,比该基因推导的Cry2Ag1蛋白理论分子量小(70kDa)。生物活性测定表明表达的包涵体蛋白对鳞翅目的小菜蛾和棉铃虫,双翅目的伊蚊均具有杀虫活性,LC50分别为23.47μg/mL、9.74μg/mL、2.54μg/mL。
Bacillus thuringiensis (Bt), as a safeties to human and animals, pollution-free microbial insecticides, plays a very important role in pest prevention and cure of agriculture, forestry and sanitary insect pest. It becomes a most successful development and apply of microbial pesticide at abroad and home. So to futher dig abundant resource of Bt in our country, screen high effective and broad-spectrum insecticidal activity of Bt strain, analyse their cry gene types, isolate and clone novel pesticidal genes will have important meanings in theories and practices for developping microbial insecticides, constructing high toxicity and broadspectrum engineering strains and breeding insect resistant transgenic plants. This study described a systematic study of Bt resources from the soil in MengDing mountain of Yaan city in Sichuan province. A novel pesticidal protein genes was cloned and expressed. The concrete results are as follows:
1. According to different altitude and environment, we collected 285 soil samples and isolated 78 Bt strains from these soil samples by using sodium acetate-antibiotics method. The average, rate was 19%. Observed by electron microscope, These Bt strains produced different patterns of parasporal crystals, such as long bipyramid, short bipyramid, cuboidal, round and abnormity, which showed the diversity of Bt resources in Mengding mountain. The cry gene-types of 78 Bt isolates were identified by using PCR-RFLP system.68 isolates harbored cry1 genes,28 isolates harbored cry2 genes,2 isolates harbored cry3 genes,2 isolates harbored cry9 genes,3 isolates harbored cry4/10 genes and 3 isolates harbored cry3 genes. Using SDS-PAGE method to analyse insecticidal crystal proteins of these Bt strains, the result showed that four different kinds of insecticidal crystal proteins were expressed, whose molecular weight were between 40 kDa and 130 kDa. Some Bt strains didn't produce any PCR products in PCR-RFLP analysis. But SDS-PAGE assay indicated that these isolates produced crystal proteins, which suggested that they may contain potentially novel Cry toxin genes.
2.28 Bt strains which contained cry2 genes were identified by using general primers of cry2 gene. Then restriction enzyme DdeⅠwas used to digest these PCR productions for identification of cry gene-types. The results showed that the size of of the Bt strain JF19-2 amplified fragment was different from enzyme cut pieces of known cry2 genes, which indicated that JF19-2 contained a novel cry 2 holetype gene.
3. According to amplified fragment of JF19-2 by using general primers of cry2 genes, a pair of specific primers SP1 and SP2 was designed, in upstream and downstream of amplified fragment. Unknown sequence of the novel cry2 gene was amplified by Tail-PCR method. Then amplified sequences was cloned and sequenced. Sequencing result was matched with known sequence of the novel cry2 gene. A 3,285 bp nucleotide sequence was obtained. There was a 1,905 bp ORF in the obtained sequence, which encoded a protein of 635 amino acids with a predicted molecular mass of 70.8 kDa and isoelectric point of 4.952.This protein was an alkalescent protein, which mainly contained four kinds of Amino acids:Ile, Asn, Ser and Thr, accounted for 11.2%,10.57%,9.94% and 8.36%, respectively. Compared with other known Cry2 proteins, Cry2Ag1 and Cry2Ab1 had shown as high as 92% Amino acids sequence homology. This, novel gene was registered in the GeneBank databases under accession number ACH91610. This gene was designed as cry2Agl in the Bacillus thuringiensis Toxin Nomenclature Committee.
4. The full open reading frame sequence of the cry2Ag1 gene was amplified with a pair of PCR primers P1/P2 designed according to cry2Agl gene sequence, and inserted into the NcoⅠ/XhoⅠsite of E. coli expression vector pET-22b(+) to obtain the recombinant plasmid pET-2Ag1. The pET-2Agl was transduced into E. coli BL21 (DE3) pLysS. The analysis of SDS-PAGE showed that the cry2Agl gene produced 60kDa protein in E.coli by IPTG induction, which smaller than calculated Cry2Ag1 protein (70kDa) according to cry2Agl gene. Bioassay of the expressed product of the cry2Agl gene showed that Cry2Agl was highly toxic to both Lepidoptera P.xylostella, H.armigera and Dipteral A.aegypti with LC50 as 23.47μg/mL,9.74μg/mL and 2.54μg/mL, respectively.
引文
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