【作者】 宋萍；

【导师】 王勤英；

【作者基本信息】 河北农业大学 ， 农业昆虫与害虫防治， 2012， 博士

【摘要】 苏云金芽胞杆菌(Bt)是世界上研究最多应用范围最广的杀虫微生物,杀虫活性物质包括Cry和Cyt两大类蛋白,在害虫生物防治中发挥了重要作用,但是也暴露了一些弊端,如杀虫谱窄、昆虫易产生抗性等问题,所以进一步分离特异活性或者高活性的野生菌株,鉴定其基因型,为害虫防治提供更多的基因资源。本论文针对含有cry7类基因的苏云金芽胞杆菌野生菌株进行了鉴定和评价,从中筛选出了对马铃薯二十八星瓢虫特异活性的新菌株,针对这一新菌株生物学特性、蛋白型、基因型以及作用机理等方面进行了一系列的研究,主要内容和结果如下。1.利用醋酸钠筛选法并经基因型分析,从河北省土壤中分离筛选出了56株含有cry7基因的Bt菌株。在这些菌株中,10株菌同时含有cry7基因和其他cry基因,46株菌只含有单一cry7基因。大部分Bt菌株表达130kDa蛋白,仅有BQZ-12菌株产生80kDa和70kDa蛋白,BQZ-8菌株产生130kDa和80kDa蛋白。通过生物活性测定得到一株对马铃薯二十八星瓢虫幼虫具有特异杀虫活性野生菌Bt WZ-9。2. WZ-9菌株在胞晶分离期产生菱形伴胞晶体,蛋白分子量为130kDa,对马铃薯二十八星瓢虫2龄幼虫的LC50值为0.209 mg/mL。基因型鉴定结果表明该菌只含有单一的cry7基因,序列分析显示该基因与cry7Ab1同源性达到99%,被Bt命名委员会命名为cry7Ab3(登录号ABX 24522)。3.为了明确Cry7Ab3蛋白的杀虫活性,从Bt WZ-9菌株中克隆了cry7Ab3基因,该基因在大肠杆菌BL21和Bt无晶体突变株HD73 cry-中都能正确表达130kDa蛋白,其表达产物对马铃薯二十八星瓢虫2龄幼虫都具有较高毒力,LC50值分别为0.460 mg/mL和0.205 mg/mL,cry7Ab3基因在转Bt无晶体突变株HD73-Cry7Ab3中形成的晶体(0.86μm×1.23μm)比野生菌产生的晶体(0.73μm×1.00μm)略大一些。这些结果证实了cry7Ab3基因编码蛋白发挥杀虫作用。4.为明确Cry7Ab3蛋白最短活性区序列,利用特异性引物分别克隆表达不同长度的cry7Ab3基因片段,E. coli Cry7Ab3-1(Met1-Phe657)、E. coli Cry7Ab3-2( Met1-Phe626 )、E. coli Cry7Ab3-3 ( Asn30-Phe657 )和E. coli Cry7Ab3-4(Lys87-Phe657),并测定了表达产物对马铃薯二十八星瓢虫2龄幼虫的毒力。结果表明E. coli Cry7Ab3-1表达的75kDa蛋白毒力最高,LC50值为0.114mg/mL,可以确定Met1-Phe657为Cry7Ab3蛋白的最短活性区。5.分别利用胰蛋白酶和马铃薯二十八星瓢虫中肠蛋白酶液体外酶解130kDa的Cry7Ab3原毒素(质量比为100:1),均得到75kDa的酶解产物。与相同浓度的原毒素相比,75kDa的酶解产物对马铃薯二十八星瓢虫2龄幼虫具有较高杀虫毒力。对马铃薯二十八星瓢虫幼虫中肠组织病理学研究表明,3龄幼虫取食Cry7Ab3原毒素蛋白3d后,中肠细胞严重破坏,细胞间出现明显孔洞,幼虫表现出活动缓慢,取食量下降的现象。取食5d后,中肠组织完全被破坏,幼虫开始死亡。通过Ligand blotting技术,检测到马铃薯二十八星瓢虫中肠BBMV上存在分子量为220kDa的Cry7Ab3毒素结合受体,经质谱分析鉴定该受体蛋白为钙粘蛋白。本论文创新之处在于发现了对马铃薯二十八星瓢虫幼虫特异活性的Cry7Ab3蛋白,构建了转cry7Ab3基因的Bt工程菌,明确了Cry7Ab3蛋白最短活性区为75kDa的Met1-Phe657蛋白片段以及Cry7Ab3毒素的作用靶标是马铃薯二十八星瓢虫幼虫中肠组织上的BBMV,初步确定了其在中肠BBMV上结合蛋白可能是钙粘蛋白。这些研究结果为更好地应用Cry7Ab3蛋白防治马铃薯二十八星瓢虫提供了理论基础。更多还原

【Abstract】 Bacillus thuringiensis is a spore-forming entomopathogenic bacterium that produces parasporal crystals composed of proteins calledδ-endotoxins during sporulation. There are two known classes ofδ-endotoxins in B. thuringiensis: the insect-specific insecticidal crystal (Cry) proteins and the Diptera-specific cytolytic (Cyt) proteins. Currently, the Cry proteins have been shown to be highly active against a wide variety of insect larvae. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. Although any particular toxin has a desirably restricted host range, there is a large number of different toxins, each showing toxicity to one of many diverse pests. For these reasons there is currently great interest in isolating novel strains of B. thuringiensis with either unique host specificity or elevated toxicity. New B. thuringiensis strains with cry7 gene were isolated and identified in this paper. Furthermore, characterization of the new Bt strain with cry7 gene against Henosepilachna vigintioctomaculata was studied. The results were as the following.1. The detection of cry gene of these isolates was done by a method based on polymerase chain reaction (PCR). 56 Bt isolates were identified to contain cry7 gene in which 10 isolates possessed cry7 gene and other cry gene, 46 isolates had only cry7 gene. SDS–PAGE analysis indicated that most of 56 isolates produced 130kDa protein bands, but Bt BQZ-12 produced 80kDa and 70kDa proteins, Bt BQZ-8 produced 130kDa and 80kDa proteins. The results of bioassay showed that just only Bt WZ-9 strain was toxic to larvae of H. vigintioctomaculata.2. Bt WZ-9 strain produces a bipyramidal crystal consisting of a 130 kDa protein. A novel gene, cry7Ab3, encoding a polypeptide of 1138 amino acids with a deduced molecular mass of 129.6 kDa was identified from Bt WZ-9 strain and submitted to GenBank with accession numbers ABX 24522. The results of bioassay demonstrated that Bt WZ-9 crystal proteins had high toxicity to larvae of H. vigintioctomaculata (LC500.209 mg/mL).3. The cry7Ab3 gene was successfully expressed as 130kDa protein in Escherichia coli BL21 and Bt acrystalliferous strain HD73 cry-. The expressed Cry7Ab3 proteins all had significant activity against second instar larvae of H. vigintioctomaculata, the estimated 50% lethal concentrations (LC50) were 0.460mg/mL and 0.205 mg/mL,respectively. Bt recombinant HD73-Cry7Ab3 produced bipyramidal crystals which were larger than those from Bt WZ-9 strain. The crystal volume were 0.86×1.23μm and 0.73×1.00μm.4. To determine the minimal active fragment of Cry7Ab3 protein, the four different cry7Ab3 fragments gene were cloned and expressed in E. coli BL21 respectively. Bioassay results showed that the toxicity (LC50=0.114 mg/mL) of expressed protein of E. coli Cry7Ab3-1(Met1-Phe657) was one time higher than that from Bt WZ-9. The expressed protein from E. coli Cry7Ab3-3(Asn30-Phe657) and E. coli Cry7Ab3-4(Lys87-Phe657) were no toxic to H. vigintioctomaculata. The minimal activated Cry7Ab3 toxin fragment was located at N-terminal of Cry7Ab3 between position Met1 and Phe657.5. The trypsin and gut juice proteases from H. vigintioctomaculata larvae all could proteolyse 130kDa Cry7Ab3 protoxin into 75kDa active toxin. The toxicity of 75kDa processing products against H. vigintioctomaculata was the higher than that of Cry7Ab3 protoxin. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae caused acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. Ligand blotting experiment demonstrated that Cry7Ab3 toxin bounded a 220kDa BBMV protein from midgut of H. vigintioctomaculata larvae. This putative receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS).Highlights of this paper are finding of a novel Cry7Ab3 toxin specific to H. vigintioctomaculata larvae, identifying the minimal active fragment of Cry7Ab3 and putative receptor protein of Cry7Ab3 in BBMV of H. vigintioctomaculata larvae. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae and a better usage of Cry7Ab3 for controlling H. vigintioctomaculata larvae.更多还原