【摘要】：蘇云金芽胞桿菌,為芽胞桿菌屬,是在世界范圍內(nèi)廣泛分布的一種革蘭氏陽性菌,作為一種環(huán)保型的殺蟲劑被廣泛用于害蟲的防治,其產(chǎn)生的蛋白可以對多種害蟲有高效的殺蟲活性。蘇云金芽胞桿菌被認(rèn)為是化學(xué)農(nóng)藥的一個有效的替代品。隨著轉(zhuǎn)基因技術(shù)的發(fā)展,蘇云金芽胞桿菌也成為轉(zhuǎn)基因作物抗蟲基因的主要來源。因此,尋找新的殺蟲活性基因?qū)οx的防治具有重要意義。由于鞘翅目葉甲科害蟲的危害日益嚴(yán)重,因此篩選出對其有高活性的菌株和基因具有重要意義。本研究的主要內(nèi)容如下:(1)本研究從海南地區(qū)的土壤中分離得到423株蘇云金芽胞桿菌,利用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)和聚丙烯酰胺凝膠電泳(SDS-PAGE)方法鑒定其菌株的基因型和殺蟲晶體蛋白的成分。經(jīng)PCR擴增結(jié)果顯示其中23株菌中含有cry7Ab基因,片段大小為3417 bp。對含有cry7Ab基因的BJH705(GenBank登錄號為KX010669)菌株進行了進一步的蛋白表達及生物活性測定。SDS-PAGE結(jié)果顯示該菌株可以表達分子量約為130 k Da的殺蟲晶體蛋白。對大猿葉甲進行生物活性測定,復(fù)篩結(jié)果顯示,該蛋白對大猿葉甲有較高的殺蟲活性其LC50為53.386μg/m L。(2)對實驗室保存的菌株3-1-a進行基因型鑒定,發(fā)現(xiàn)其中含有cry8基因,進一步用全長引物進行擴增,測序結(jié)果顯示該菌株同時含有cry8Ax和cry8Ea基因。與p EB表達載體連接,并在大腸桿菌BL21中表達,SDS-PAGE結(jié)果顯示,cry8Ax和cry8Ea基因可以在大腸桿菌中分別表達分子量為80 kDa、130 kDa的殺蟲蛋白。序列已提交GenBank注冊,登錄號分別為KU986725,KU955850。以大猿葉甲二齡幼蟲為供試?yán)ハx對兩種蛋白進行生物活性初步測定,校正死亡率分別為56.89%,77.87%,利用80 k Da的蛋白進行復(fù)篩,其LC50值為83.329μg/m L。本研究針對我國海南地區(qū)和實驗室保存菌株開展了菌株篩選與殺蟲基因克隆的研究,獲得有效新菌株BJH705和3-1-a,克隆有效殺蟲基因3個,研究結(jié)果對對大猿葉甲有殺蟲活性的新基因克隆及鞘翅目害蟲的可持續(xù)性防治具有重要意義。
[Abstract]:Bacillus thuringiensis, a genus of Bacillus thuringiensis, is a Gram-positive bacterium widely distributed worldwide. As an environmentally friendly insecticide, Bacillus thuringiensis is widely used in pest control. The protein produced by the protein can be highly insecticidal to a variety of pests. Bacillus thuringiensis is considered to be an effective substitute for chemical pesticides. With the development of transgenic technology, Bacillus thuringiensis has become the main source of insect resistance genes in transgenic crops. Therefore, it is of great significance to search for new insecticidal genes for pest control. It is of great significance to screen out the strains and genes which have high activity because of the serious damage caused by the insect pests of the family Elaeagnus. The main contents of this study are as follows: (1) 423 strains of Bacillus thuringiensis were isolated from the soil of Hainan area. Polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the genotype and the composition of insecticidal crystal protein. The results of PCR amplification showed that 23 of them contained cry7Ab gene, and the size of the fragment was 3417 bp.. The protein expression and biological activity of the strain BJH705 (GenBank accession number KX010669) containing cry7Ab gene were further studied. The results of SDS-PAGE showed that the strain could express the insecticidal crystal protein with molecular weight of 130k Da. The results of double screening showed that the protein had high insecticidal activity (LC50 was 53.386ug / m L. (2), and the genotypic analysis of the preserved strain was carried out in the laboratory, and the results showed that the protein had a high insecticidal activity to P. sibirica, and the bioactivity of the protein was 53,866 渭 g / m L. (2. The cry8 gene was found and amplified by full-length primers. Sequencing results showed that the strain contained both cry8Ax and cry8Ea genes. It was ligated with p-EB expression vector and expressed in E. coli BL21. The results of SDS-PAGE showed that cry8Ax and cry8Ea genes could express 80 kDa,130 kDa insecticidal protein in E. coli respectively. Sequence submitted for GenBank registration with login numbers of KU986725,KU955850. The biological activity of the two proteins was determined with the second instar larva of the great apes as the test insect. The corrected mortality rates were 56.89% and 77.87%, respectively. The LC _ (50) value of the two proteins was 83.329 渭 g / mL using the protein of 80 k Da for rescreening. In this study, strain screening and cloning of insecticidal genes were carried out for the preserved strains in Hainan province and laboratory in China. The effective new strains BJH705 and three effective insecticidal genes were obtained, and three effective insecticidal genes were cloned. The results of this study are of great significance for the cloning of new genes with insecticidal activity and the sustainable control of coleoptera pests.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S476

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