【作者】 谭芙蓉；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 苏云金芽胞杆菌（Bacillus thuringiensis,简称Bt）是目前研究最深入、使用最广泛的杀虫微生物之一。深入发掘我国丰富的苏云金芽胞杆菌资源,筛选高毒力及特异性菌株,对其基因型进行分析,分离克隆新型杀虫基因,其结果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。本研究对四川盆地不同生态区的Bt资源进行了较为系统的调查研究,从中分离克隆了数个新型杀虫基因,并进行了初步的表达研究,具体结果如下:1.采集四川不同生态区的土壤样品2650份,利用醋酸钠-抗生素法分离Bt菌791株,平均分离率为13.2%。分离出的菌株产生的伴胞晶体形态各异,有长菱形、短菱形、大菱形、小菱形、方形、球形、不定形等,充分显示了四川生态区Bt菌株资源多样性的特点。利用PCR-RFLP鉴定体系鉴定了791株Bt菌的杀虫晶体蛋白基因类型:其中522株含有cry1型基因,312株含有cry2型基因,20株含有cry3型基因,33株含有cry9型基因,28株含有cry4/10型基因,33株含有cry30型基因,3株含有cry40型基因。有80株菌未鉴定出基因型,对这些菌株的伴胞晶体SDS-PAGE分析结果表明:有的菌株表达130kDa和60kDa的蛋白;有的菌株表达90kD和40kDa的蛋白;有的菌株表达60kDa的蛋白;有的则表达90kDa左右的蛋白,可以推测,这些菌株极有可能含有新型的杀虫基因。2.系统地研究了四川生态条件下分离的Bt菌株Rpp02和Rpp39的生物学特性。根据形态特征、培养特征、细胞壁化学组分和生理生化分析,Rpp02菌株被定名为苏云金芽孢杆菌莫里逊亚种（Bacillus thuringiensis subsp.morrisoni）,Rpp39被定名为苏云金芽孢杆菌杀虫变种（Bacillus thuringensis var.entomocidus）。Rpp02生长3h进入对数生长期,生长速率略快于Rpp39（4h）,Rpp39与已知标准菌株HD-1生长速率相当。Rpp02产生大菱形、小菱形、圆形、不定形伴胞晶体,Rpp39产生菱形、方形和圆形伴胞晶体。PCR-RFLP鉴定结果表明Rpp02含有cry1Ab、cry1Ac、cry1Ca和cry1Ia四种基因,Rpp39含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五种基因。SDS-PAGE电泳分析Rpp02主要产生130kDa和40kDa大小的蛋白,Rpp39主要产生130kDa和60kDa的蛋白。3.设计了一对cry1Ac基因的特异引物,对Rpp02菌株基因组DNA进行PCR扩增,得到大约4kb的产物。测序结果表明,克隆到的片段含有一个较大的ORF框,该基因编码区为3534bp,编码1177个氨基酸,分子量为133.144kDa;等电点pI=4.825,为弱酸性蛋白质;亮氨酸（Leu）、丝氨酸（Ser）、谷氨酸（Glu）3种氨基酸含量最高,分别为7.98%、7.81%、7.73%。与已报道的cry1Ac序列同源性达到99%,存在着几个核苷酸以及编码氨基酸的差别。该基因序列的Accessionnumber为DQ285666,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry1Ac20。该基因在大肠杆菌中得到了表达,表达产物具有较强的杀虫效果。4.以Rpp39为出发菌株,分离克隆了cry2Aa类杀虫晶体蛋白全长基因。序列分析显示该基因的开放阅读框（ORF）为1902 bp,编码由634个氨基酸组成的蛋白质。其氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12。根据cry2Aa12基因ORF两端序列,设计1对特异引物P3/P4,PCR扩增获得cry2Aa12完整ORF。将获得的片段与大肠杆菌表达载体pET-30a连接,构建了重组表达质粒pET-2Aa。将该质粒导入E.coli BL21（DE3）,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65kDa表达蛋白。生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC₅₀分别为5.4μg/mL和22.3μg/mL。5.采用PCR-RFLP鉴定法,从菌株BtMC28中挖掘出一个新型杀虫模式基因,其部分序列与已知模式基因cry4Aa和cry10Aa的同源性分别为57%和60.5%。进一步采用Tail-PCR和Son-PCR技术获得了基因的全长序列。该基因的编码区为2022bp,编码673个氨基酸组成的蛋白,与Cry10Aa蛋白所氨基酸序列同源性最高为38%;其分子量为76.32kDa:异亮氨酸（Ile）、亮氨酸（Leu）、天冬酰胺（Asn）含量最高,分别为9.65%、9.36%、8.91%;它的等电点为7.535,属弱碱性蛋白。该新基因已在GenBank中注册,Accession number为EU339367。根据cry基因国际命名原则（即新发现的基因编码的氨基酸序列与已知蛋白同源性在45%以下为第一分类等级,用阿拉伯数字表示,如cry1、cry2、cry3…）,该基因被国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry54Aa1基因。根据cry54Aa1基因ORF两端序列,设计1对特异引物经扩增获得了cry54Aa1完整ORF。随后与大肠杆菌表达载体pET-30a连接,构建了重组表达质粒pET-54Aa。将该质粒导入E.coliBL21（DE3）,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有76kDa表达蛋白。生物活性测定表明表达的包涵体蛋白对甜菜夜蛾的LC₅₀为5.8μg/mL,对伊蚊的LC₅₀为6.02μg/mL。更多还原

【Abstract】 Bacillus thuringiensis（Bt） is currently one of the most extensive studied and widely used pesticidal microorganism.To futher dig abundant resource of Bt in our country,screen high toxicity and specific strains,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 in different ecological regions in Sichuan.Several novel pesticidal protein genes were cloned and expressed.The concrete results are as follows:1.In totle,791 B.thuringiensis isolates have been screened from 2650 soil samples with an average rate as 13.2%,which were collected from different ecological regions in Sichuan.Observed by electron microscope,these B.thuringiensis parasporal crystal shapes were long bipyramid,short bipyramid,big bipyramid,small bipyramid, cuboidal,round and abnormity,which showed the diversity of Bt resources in Sichuan ecology.The cry gene-types of 791 B.thuringiensis isolates were identified by use of PCR-RFLP.522 isolates harbored cry1 genes,312 isolates harbored cry2 genes,20 isolates harbored cry3 genes,33 isolates harbored cry9 genes,28 isolates harbored cry4/10 genes,33 isolates harbored cry3 genes and 3 isolates harbored cry40 genes.In addition,80 isolates did not produce any PCR products when assayed with the primers. However,SDS-PAGE assay indicated that these isolates produced crystal inclusions, suggesting that they may contain potentially novel Cry toxins.2.This paper systematically investigated the biological characteristics of Bt strains Rpp02 and Rpp39 isloated from Sichuan ecology.Based on their morphologic characteristics,culture characteristics,biochemical reactions and cell wall compounds analysis,the strain Rpp02 was approved to be a kind of Bacillus thuringiensis subsp. morrisoni while Rpp39 was Bacillus thuringiensis var entomocidus.After growing three hours,the strain Rpp02 entered logarithmic growth phase.The growth velocity of Rpp02 was quicker slightly than Rpp39（four hours） whose growth velocity was the same as that of the standard strain HD-1.The strain Rpp02 produced big bipyramid, small bipyramid,round and abnormity parasporal crystal while Rpp39 produced diamond,cuboidal and round parasporal crystal.The strain Rpp02 contained cry1Ab, cry1Ac,cry1Ca and cry1Ia genes by use of PCR-RFLP method and Rpp39 contained cry1Aa,cry1Ab,cry1Ac,cry1Ia and cry2Aa genes.SDS-PAGE analysis showed that two kinds of molecular mass of insecticidal crystal proteins,one was about 130kDa and other 40kDa,were expressed in Rpp02 while 130kDa and 60kDa proteins in Rpp39.3.According to the whole length of cry1Ac gene published on GenBank,a pair of primers was designed to amplify the genomic DNA of Rpp02 and a fragment of about 3.7kb was obtained.The sequence analysis showed that the novel gene cry1Ac20, named by B.thuringiensis Pesticidal Crystal Protein Nomenclature Committee, contained an open reading frame of 3534 nucleotides encoding a protein of 1177 amino acids with a predicted molecular mass of 133.144 kDa and isoelectric point of 4.952. Compared with other known cry1Ac genes,cry1Ac20 has shown as high as 99% nucleotide sequence homology.Bioassay showed that the toxic protein appeared high insecticidal activity against Pieris rapae L.with LC₅₀ as 9.01μg / mL4.One cry2Aa-type gene of Rpp39 was cloned and designated as cry2Aa12 by Bt Insecticidal Crystal Proteins Nomenclature Committee.Sequence analysis revealed this gene contained an open reading frame of 1902 nucleotides encoding a protein of 634 amino acids.Compared with Cry2Aa1 protein,Cry2Aa12 protein has shown as high as 99.7%amino acid homology.The full open reading frame sequence of the cry2Aa12 gene was amplified with a pair of PCR primers P3/P4 designed according to its DNA sequence,and inserted into the NdeⅠ/ BamHⅠsite of E.coli expression vector pET-30a to obtain the recombinant plasmid pET-2Aa.The result of SDS-PAGE proved that Cry2Aa12 could be expressed as 65kDa protein in E.coli BL21（DE3） strain induced by IPTG Bioassay of the expressed product of the cry2Aa12 gene showed that Cry2Aa12 was highly toxic to the larvae of Plutella xylostella and Chilo supperssalis, with LC₅₀ as 5.4μg / mL and 22.3μg / mL,respectively.5.One novel holetype genes was found from the strain BtMC28 by the method PCR-RFLP.The sequence analysis revealed that the partial sequence of one had 57% and 60.5%identical to cry4Aa and cry10Aa,respectively.Furthermore,the full-length sequence of the novel gene was obtained by Tail-PCR and Son-PCR.The sequence analysis showed that the novel 8ene cry54Aa1,named by B.thuringiensis Pesticidal Crystal Protein Nomenclature Committee,contained an open reading frame of 2022 nucleotides encoding a protein of 673 amino acids with a predicted molecular mass of 76.32 kDa and isoelectric point of 7.535.Compared with other known proteins, Cry54Aa1 has shown 38%amino acids homology.The full open reading frame sequence of the cry54Aa1 gene was amplified with a pair of PCR primers designed according to its DNA sequence,and inserted into the NdeⅠ/ EcoRⅠsite of E.coli expression vector pET-30a to obtain the recombinant plasmid pET-54Aa.The result of SDS-PAGE proved that Cry54Aa1 could be expressed as 76 kDa protein in E.coli BL21（DE3） strain induced by IPTG.Bioassay of the expressed product of the cry54Aa1 8ene showed that cry54Aa1 was highly toxic to the larvae of Laphygma exigua and Aedes aegypti with LC₅₀as 5.08μg / mL and 6.02μg / mL,respectively.更多还原