【作者】 关鹏；

【导师】 李平； 郑爱萍；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 苏云金芽胞杆菌Bacillus thuringiensis,Bt)MC28是从四川盆地沐川原始森林土壤中分离得到的,能在芽胞形成期产生大量球形伴胞晶体蛋白,对鳞翅目和双翅目昆虫具有明显的杀虫活性。前期研究采用PCR-RFLP (restricted fragment length polymorphisms, RFLP)鉴定方法从MC28中鉴定并克隆了5个杀虫晶体蛋白基因cry30Fa1、 cry53Ba1、cry54Aa1和cyt2Aa3),其中cry54Aa1编码蛋白对棉铃虫Helicoverpa armigera, Lepidoptera)、甜菜夜蛾(Laphygma exigua, Lepidoptera)和伊蚊(Aedes aegypti, Diptera)具有特异的杀虫活性；cry30Fa1编码蛋白对水稻褐飞虱(Nilaparvata lugens, Homoptera)具有特异的杀虫活性。为了研究MC28的基因组结构和进化特点,我们采用Illumina GA Ⅱ测序技术对MC28菌株进行全基因组测序。全基因组测序获得原始reads并过滤得到18,324,961个双末端reads,其对基因组的覆盖度达到451倍。采用SOAPdenovo组装软件将大约97.13%的reads组装成113个scaffolds。通过PCR、长片段PCR扩增以及构建Fosmid文库等策略对Scaffold内部和scaffold之间的gaps进行补洞,最终获得大小为6.68Mb的基因组完成图。利用Glimmer3.02对Bt MC28全基因组进行基因的预测,然后使用blast软件,将预测得到的蛋白序列与NR库进行比对,选择最好的比对结果作为相对应蛋白的注释,最终,MC28基因组总共注释得到6,555个蛋白。采用perl+SVG工具对Bt MC28和BtCT43及Bt YBT-020分别做共线性分析,结果表明：MC28与CT-43及YBT-020具有非常相似的基因组结构和共线性关系。采用MEGA和SplitsTree软件分别对Bt MC28和其它相关菌株构建系统发育树,两个结果均表明：Bt MC28菌株同其它Bt菌株在进化上距离较远,而是同Be Rock3-28、Bc Rock4-18、Bc Rock1-3和Be Rock3-29处在一个独立的分支上Bt MC28全基因组由8个环状复制子构成(1个染色体DNA和7个内生质粒),总长为6.68Mb。其中环状染色体DNA长度为5,414,461bp,编码5,279个预测的开放阅读框(ORFs),染色体DNA的G+C的百分含量为35.41%；7个环状内生质粒分别为pMC8、pMC54、pMC95、pMC183、pMC189、pMC319和pMC429,这些质粒共编码1,278个预测的开放阅读框(ORFs),其G+C的百分含量在32.11%和34.78%之间。Bt MC28基因组共编码74个tRNA和45个rRNA。通过Bt MC28全基因组测序,我们从中发现了3个编码杀虫晶体蛋白基因的质粒,而且从这些质粒上发现了6个新型的杀虫晶体蛋白基因分别为cry54Ab1、cry68Aa1、cry69Aa1、cry69Aa2、cry70Ba1和cyt1Da1,至此在Bt MC28菌株中共得到11个新型杀虫晶体蛋白基因。其中质粒pMC189上共携带了7个杀虫晶体蛋白基因分别为cry30Fa1、cry53Ab1、cry54Aa1、cry54Ab1、cry68Aa1、, cyt1Da1和cyt2Aa3；而质粒pMC95上共携带有3个杀虫晶体蛋白基因分别cry4Cc1、 cry69Aa1和cry70Ba1;质粒pMC183上仅含有一个与cry69Aa1同源性为96.2%的杀虫晶体蛋白基因cry69Aa2。cry68Aa1、cry70Ba1和cyt1Da1的长度分别为2,511bp、2,427bp和1,527bp。为了研究这三个基因编码蛋白的杀虫活性,我们通过PCR扩增将这三个基因分别插入到原核表达载体pET-28a中,再转入到原核表达感受态细胞BL21(DE3)pLysS中,通过IPTG诱导表达,cry68Aa1, cry70Ba1和cyt1Da1基因分别产生约95kDa、90kDa和60kDa的表达产物,这与其预测表达蛋白的大小一致。生物活性测定结果表明：Cry68Aa1和Cry70Ba1均只对鳞翅目的甜菜夜蛾幼虫具有杀虫活性,其LC50分别为20.16μg/ml(95%的置信区间为16.34-35.72μg/ml)和30.22μg/ml(95%的置信区间为27.15-36.53μg/ml)，而两者对鳞翅目的棉铃虫和双翅目的蚊虫无杀虫活性；Cyt1Da1对所测的昆虫均无杀虫活性。cry69Aa1基因全长为3,651bp,编码1,216个氨基酸,其预测编码蛋白大小约为137.1kDa,与已知的Cry4Ba蛋白的同源性最高,但仅为39%。比较结果表明：Cry69Aa1和已知大分子量的Cry蛋白均含有8个保守区域(见图5-1),因此,Cry69Aa1蛋白属于大分子量Cry蛋白中的一类新型Cry蛋白。为了研究cry69Aa1基因编码蛋白的杀虫活性,我们将扩增得到cry69Aa1全长基因插入到穿梭载体pSTK中,再转入到Bt无晶体突变株HD73-中。重组菌株的扫描电镜(SEM)观察结果表明：cry69Aa1在Bt无晶体突变株HD73-中表达时,能够形成大量的球形伴胞晶体。生测结果表明：重组菌株的芽胞晶体混合物对双翅目的致倦库蚊(Culex quinquefasciatus)幼虫具有明显杀虫活性,其LC5o为23.7μg/ml,95%的置信区间为20.5-26.4μg/ml。cry54Ab1操纵子是由两个相同编码方向的ORFs组成的,第一个ORF是与ory54A类似的cry基因,长度为2,232bp,其编码743个氨基酸,预测编码蛋白分子量84.5kDa,根据编码蛋白的同源性,这个cry基因被命名为cry54Ab1;第二个ORF位于cry54Ab1的下游,两者之间被一个长67bp的非编码区所间隔,这个ORF在本研究中暂命名为orf2基因,该基因长1,524bp,编码503个氨基酸,其预测蛋白分子量为58.2kDa。为了研究cry54Ab1操纵子的杀虫活性,我们将cry54Ab1基因、orf2基因以及完整的cry54Ab1操纵子通过PCR扩增后分别插入到穿梭载体pSTK上,再转入到Bt无晶体突变株HD73-中表达。重组菌株的扫描电镜观察表明：当完整的cryS4Ab1操纵子在Bt无晶体突变株HD73-中表达时,能够形成大量的球形伴胞晶体；当cry54Ab1基因或orf2基因单独在Bt无晶体突变株HD73-中表达时,均不能形成伴胞晶体。SDS-PAGE电泳分析结果表明：完整的cry54Ab1操纵子在Bt无晶体突变株HD73-中表达时,两个ORFs均能够大量表达,然而当cry54Ab1基因和orf2基因单独表达时,两个基因的表达量非常低,尤其是orf2基因几乎不表达。另外生测结果表明：完整的cry54Ab1操纵子和cty54Ab1基因的表达产物均对双翅目的致倦库蚊有杀虫活性,但是完整的cry54Ab1操纵子表达产物的杀虫活性是cry54Ab1基因单独表达时的杀虫活性的10倍之多。由以上结果可知,orf2基因的表达不仅能够使Cry54Ab1蛋白形成球形伴胞晶体,而且能够提高Cry54Ab1蛋白的杀虫活性。更多还原

【Abstract】 Bacillus thuringiensis (Bt) strain MC28was isolated from Mu Chuan virgin forest in Sichuan Province of China. It can form spherical parasporal crystals during the sporulation stage and exhibit a broad insecticidal activity against Dipteran and Lepidopteran pests. In previous study,5insecticidal crystal protein genes were identified and cloned using PCR-RFLP (restricted fragment length polymorphisms, RFLP) method. Among these genes, Cry54Aal protein has insecticidal activity against Helicoverpa armigera.(Lepidoptera), Laphygma exigua (Lepidoptera) and Aedes aegypti (Diptera); Cry30Fal protein was toxic to Nilaparvata lugens (Homoptera).For studying genomic structure and evolutionary relationship of Bt strain MC28, whole-genome sequencing of MC28was performed using Illumina GA II sequencing technology. A total of18,324,961filtered paired-end reads were obtained and451-fold coverage of the genome was achieved using an Illumina Solexa GA II. About97.13%of the reads were assembled into113scaffolds using the SOAPdenovo alignment tool. Gaps within and between the scaffolds were confirmed and closed using primer walks, long-distance PCR amplification, and the construction of a fosmid library using an ABI3730capillary sequencer. Finally, the6.68Mb whole-genome sequence of MC28was obtained.The gene prediction of the whole-genome of Bt MC28was performed using the Glimmer3.02software. Then, the blast software was used to compare the predicted protein with the Nr (non-redundant protein sequences) databases. The best alignment results were as the annotation of the corresponding proteins. Synteny of Bt MC28, Bt CT-43, and Bt YBT-020was performed using the perl+SVG tool. The results indicated that Bt MC28, Bt CT-43, and Bt YBT-020had similar genomic structure and significant genomic synteny. Both the MEGA and the Splits Tree softwares were used to structure phylogenetic tree of Bt MC28and other relatived strains. These results all showed that there was further evolutionary distance between Bt MC28and other Bt strains. But Bt MC28, Be Rock3-28, Be Rock4-18, Be Rock1-3, and Be Rock3-29were in a in a separate branch.The6.68Mb genome of MC28is found to contain8replicons:a circular chromosome (5,414,461bp) encoding5,279predicted open reading frames (ORFs), and7circular plasmids: pMC8, pMC54, pMC95, pMC183, pMC189, pMC319and pMC429, These plasmids contain a total of1,278predicted ORFs (Table1). The G+C content of the chromosome is35.41%, and those of the plasmids range from32.11%to34.78%(Table1). The MC28genome encodes74tRNA and45rRNA operons. Based on Bt MC28genome, we found three plasmids which all contain insecticidal crystal protein genes from Bt strain MC28, and other six novle insecticidal crystal protein genes (cry54Ab1, cry68Aa1, cry69Aa1, cry69Aa2, cry70Bal, and cytlDa1) also were found from these plasmids. So far, eleven insecticidal crystal protein genes have been found from Bt strain MC28. Plasmid pMC189is found to harbor seven insecticidal crystal genes:cry30Fal, cry53Ab1, cry54Aa1, cry54Ab1, cry68Aa1, cyt1Da1, and cyt2Aa3. Plasmid pMC95is found to harbor three cry genes:cry4Cc1, cry69Aa1, and cry70Ba1. Plasmid pMC183is found to contain only one cry gene, cry69Aa2whose encoding protein is96.2%identical with Cry69Aal protein.Length of cry68Aa1, cry70Ba1, and cytlDal gene is2,511bp,2,427bp and1,527bp, respectively. In order to study insecticidal activity of these genes, these genes were amplified by PCR and inserted into prokaryotic expression vector pET-28a, respectively. Then, recombinant plasmids were transferred into E. coli BL21(DE3) pLysS and expressed by induction of IPTG. The molecular weight of expressed products of cry68Aa1, cry70Ba1, and cytlDal were95kDa,90kDa, and60kDa, respectively. These were the same as deduced molecular mass of Cry68Aal, Cry70Ba1, and CytlDal, respectively. Results of insect toxicity assay indicated that both Cry68Aal and Cry70Bal were toxic to L. exigua (Lepidoptera) with LC50as20.16μg/ml (95%confidence limit16.34-35.72μ/ml) and30.22μg/ml (95%confidence limit27.15-36.53μg/ml), but were no toxic to H. armigera (Lepidoptera) and A. aegypti (Diptera). The CytlDal protein had no any insecticidal activity against these insects.The cry69Aa1gene (3,651bp) encoded1,216amino acids. It had a deduced molecular mass of137.1kDa and39%identity with Cry4Ba protein as indicated by amino acid homology. Sequence analysis indicated that Cry69Aal and other known large Cry proteins contain eight conserved blocks. So Cry69Aal is a novel member of large Cry protein. For studying insecticidal activity of Cry69Aa1protein, the full-length sequence of cry69Aa1gene was inserted into a shuttle vector, pSTK, and expressed in a crystalliferous mutant Bt subsp. kurstaki HD73-. The transformant was examined with scanning electron microscope (SEM). The result showed that when cry69Aa1gene was expressed in the crystalliferous mutant Bt HD73-, it could form a large number of spherical parasporal crystals. The bioassay results inicated that spore-crystal complex of the transformants was highly toxic to the larvae of Culex quinquefasciatus (Diptera)(LCso=23.7μug/ml,95%confidence:20.5-26.4μg/ml).The cry54Ab1operon is composed of two ORFs oriented in the same direction. The first ORF, a cry54A-like gene, encoded743amino acids and it had a deduced molecular mass of84.5kDa. Based on encoded amino acid sequence homology, this cry gene was classified as cry54Ab1. The second ORF, located downstream of cry54Ab1, was separated from the cry54Ab1gene by a67bp untranslated gap. It is here called orf2. The orf2gene corresponded to a polypeptide of503amino acids and it had a deduced molecular mass of58.2kDa. In order to study insecticidal activity of cry54Ab1operon, the cry54Ab1, orf2, and the whole cry54Ab1operon were used to construct recombinant plasmid using shuttle vector pSTK, respectively. These recombinant plasmids were then expressed in the crystalliferous mutant Bt HD73-. The transformant was examined with SEM. The result showed that when the whole cry54Ab1operon were expressed in the crystalliferous mutant Bt HD73-, it could form a large number of spherical parasporal crystals. However, expression of cry54Abl or orf2alone in the crystalliferous mutant Bt HD73-did not produce any parasporal crystals. SDS-PAGE analysis indicated that when the whole cry54Ab1operon were expressed in the crystalliferous mutant Bt HD73-, it could produce a protein of approximately80kDa Cry54Ab1and another protein of approximately60kDa ORF2. However, expression of cry54Ab1or orf2alone in the crystalliferous mutant Bt HD73-, less expressed products were observed. The orf2gene even was not expressed in the crystalliferous mutant Bt HD73-. The bioassay results showed that the transformants expressing either the whole cry54Ab1operon or the cry54Abl gene alone showed larvicidal activity against C. quinquefasciatus (Diptera). However, transformants contained recombinant plasmid pSTK-orf2showed no larvicidal activity against C. quinquefasciatus. Transformants expressing the cry54Ab1gene alone had less than one-tenth the insecticidal activity of transformants expressing the whole cry54Ab1operon. Thus, ORF2protein can not only contribute to formation of parasporal crystals of Cry54Abl protein but also enhance the insecticidal activity of Cry54Ab1.更多还原