对华北大黑鳃金龟幼虫具有活性的苏云金芽胞杆菌筛选与鉴定
发布时间：2018-08-25 14:47
【摘要】：华北大黑鳃金龟[Holotrichia oblita(Falderman)]是难以防治的地下害虫,给农作物造成严重的损失。苏云金芽胞杆菌(Bacillus thuringiensis,Bt)是一种已被广泛用于农业害虫防治的昆虫病原微生物,其在芽胞形成期可产生主要由cry和cyt基因编码的杀虫晶体蛋白。发掘Bt菌株资源是实现此类害虫绿色防控的重要手段,因此,为了获得对华北大黑鳃金龟具有较高杀虫活性的菌株,本研究以实验室分离的500株Bt菌株材料为基础,通过杀虫活性测定筛选对华北大黑鳃金龟幼虫有活性的菌株,进一步对菌株多样性分析、菌株晶体形态观察和蛋白表达分析、基因鉴定和华北大黑鳃金龟幼虫感染Bt后中肠组织病理切片等方面进行了一系列的研究,其主要结果如下:对华北大黑鳃金龟幼虫有活性菌株的筛选:以实验室分离的500株Bt菌株为材料,通过杀虫活性测定筛选到了42株对华北大黑鳃金龟幼虫具有不同程度活性的菌株,其中有10株14天的校正死亡率大于60%,而261-1菌株7天校正死亡率就达到100%,其余菌株的14天校正死亡率在30%~60%之间。菌株多样性分析:利用本实验室建立的便捷的Bt菌株比较鉴别技术,对这42株菌株进行多样性分析,以42株菌株的基因组为模板,利用优化后的简并引物和PCR扩增体系进行扩增,并利用HinfI对PCR产物进行酶切,通过电泳分析获得了33个菌株的RFLP图谱,分析比较显示它们属于14个不同的菌株类型,从而有效的去除了冗余菌株,结合菌株对华北大黑鳃金龟的杀虫活性数据,选择1126-1、P65-1、QDL47-1、261-1、FTL84、78-2、127-2、78-3、1198-1、S10A2、B8E12、QDL40-2、FCD114、B8E11等14株Bt菌株进行进一步鉴定。菌株晶体形态观察和蛋白表达分析:通过对14株Bt菌株的晶体形态电子显微镜观察,结果显示,78-3、127-2和1198-1菌株能产生双锥体型晶体;261-1、FCD114、P65-1、FTL84、78-2和1126-1菌株能产生球形晶体,而其它5个菌株没有晶体存在。通过SDS-PAGE分析了14株代表菌株表达蛋白情况,结果显示菌株P65-1、1126-1、127-2、78-3、261-1、FCD114、1198-1和78-2能表达典型的约130 kDa的Bt Cry蛋白,QDL47-1和B8E11表达了约97 kDa蛋白,其余菌株表达了少量60 kDa的蛋白。cry基因鉴定:利用cry3、cry8、cry18、cry23、cry37、cry43类通用引物对14株Bt菌株进行进一步基因鉴定,结果显示这14个菌株中,只有P65-1、1126-1、FCD114和78-2四个菌株分别含有cry8Ma、cry8Ca、cry8Ab、cry8Ga、cry8Ea等5类cry8类新基因;而包括对华北大黑鳃金龟幼虫活性最高的261-1菌株在内的其它10个菌株均不含有cry3、cry8、cry18、cry23、cry37、cry43等对鞘翅目害虫有效的杀虫基因。质谱鉴定结果也显示了P65-1、1126-1、FCD114和78-2等菌株有Cry8类蛋白的表达。中肠组织病理学观察:进一步通过对华北大黑鳃金龟幼虫感染Bt后中肠组织病理切片的观察,发现在感染菌株261-1、1198-1、FCD114、1126-1、QDL40-2后,幼虫肠壁细胞组织有明显的病理变化。本研究结果对防治大黑鳃金龟的Bt菌株和杀虫基因资源发掘均具有重要意义。
[Abstract]:Holotrichia oblita (Falderman) is an underground pest that is difficult to prevent and cure, causing serious losses to crops. Bacillus thuringiensis (Bacillus thuringiensis,Bt) is a kind of insect pathogen which has been widely used in agricultural pest control. It can produce insecticidal crystal proteins mainly encoded by cry and cyt genes during the spores formation. Therefore, in order to obtain the strains with high insecticidal activity on the basis of 500 strains of Bt isolated in the laboratory, this study is based on the discovery of the Bt strain resources to achieve the green control of these pests. The strains that were active to the larva of the black Gill turtle of North China were screened by insecticidal activity test. The diversity of the strains, the crystal morphology and the protein expression of the strains were further analyzed. A series of studies were carried out on gene identification and histopathological sections of the midgut after Bt infection. The main results are as follows: screening of the active strains of the larvae of the North China Black Gill Tortoise: 500 strains of Bt isolated in the laboratory were used as materials. Through insecticidal activity test, 42 strains with different activity to the larva of Blackbranchella sinensis were screened. The corrected mortality of 10 strains in 14 days was greater than 60%, while the corrected mortality rate of 261-1 strain reached 100% in 7 days, and the corrected mortality rate of the other strains in 14 days was between 30% and 60%. Analysis of strain diversity: using the convenient Bt strain comparison and identification technique established in our laboratory, the diversity of 42 strains was analyzed, and the genome of 42 strains was used as template. The optimized degenerate primers and PCR amplification system were used to amplify the PCR products and the PCR products were digested by HinfI. The RFLP profiles of 33 strains were obtained by electrophoretic analysis. The results showed that they belonged to 14 different strains. In order to effectively remove redundant strains, combined with the data of insecticidal activity of the strain, 14 Bt strains, such as 1126-1P65-1P65-1QDL47-1261-1, FTL8O78-2127-2, 78-38-3198-1, S10A2OB8E12QD40-2FCD114B8E11, were selected for further identification. Crystal morphology observation and protein expression analysis of 14 strains of Bt. The results showed that the bipyramidal crystals were obtained by using electron microscope. The results showed that the bipyramidal crystals could be produced by FCD114P65-1 FTL84-78-2 and 1126-1 strains. The other five strains had no crystals. The expression of protein in 14 representative strains was analyzed by SDS-PAGE. The results showed that strain P65-1126-1127-2O78-3261-1FCD114FCD114FCD111-1198-1 and 78-2 could express the typical Bt Cry protein QDL47-1 of about 130 kDa and B8E11 expressed about 97 kDa protein. The remaining strains expressed a small amount of 60 kDa protein. Cry genes were identified. 14 strains of Bt strains were further identified by using cry3,cry8,cry18,cry23,cry37,cry43 generic primers. The results showed that among the 14 strains, Only four strains of P65-1O1126-1FCD114 and 78-2 contained five new cry8 genes, such as cry8Ma,cry8Ca,cry8Ab,cry8Ga,cry8Ea, respectively, while none of the other 10 strains, including 261-1 strain, which had the highest activity to the larvae of the North China blackgill tortoise, did not contain the insecticidal genes such as cry3,cry8,cry18,cry23,cry37,cry43, which were effective against Coleoptera pests. The results of mass spectrometry also showed that P65-1A1126-1FCD114 and 78-2 had Cry8 protein expression. Histopathological observation of midgut: the histopathological changes of intestinal parietal cells were observed after infection with Bt in the larvae of the North China Black Gill tortoise. It was found that the tissue of intestinal parietal cells of the larvae had obvious pathological changes after infection with strain 261-1198-1 FCD114-1126-1QDL40-2. The results of this study are of great significance for the prevention and control of Bt strains and the discovery of insecticidal gene resources.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.5;S476.1
本文编号：2203201
[Abstract]:Holotrichia oblita (Falderman) is an underground pest that is difficult to prevent and cure, causing serious losses to crops. Bacillus thuringiensis (Bacillus thuringiensis,Bt) is a kind of insect pathogen which has been widely used in agricultural pest control. It can produce insecticidal crystal proteins mainly encoded by cry and cyt genes during the spores formation. Therefore, in order to obtain the strains with high insecticidal activity on the basis of 500 strains of Bt isolated in the laboratory, this study is based on the discovery of the Bt strain resources to achieve the green control of these pests. The strains that were active to the larva of the black Gill turtle of North China were screened by insecticidal activity test. The diversity of the strains, the crystal morphology and the protein expression of the strains were further analyzed. A series of studies were carried out on gene identification and histopathological sections of the midgut after Bt infection. The main results are as follows: screening of the active strains of the larvae of the North China Black Gill Tortoise: 500 strains of Bt isolated in the laboratory were used as materials. Through insecticidal activity test, 42 strains with different activity to the larva of Blackbranchella sinensis were screened. The corrected mortality of 10 strains in 14 days was greater than 60%, while the corrected mortality rate of 261-1 strain reached 100% in 7 days, and the corrected mortality rate of the other strains in 14 days was between 30% and 60%. Analysis of strain diversity: using the convenient Bt strain comparison and identification technique established in our laboratory, the diversity of 42 strains was analyzed, and the genome of 42 strains was used as template. The optimized degenerate primers and PCR amplification system were used to amplify the PCR products and the PCR products were digested by HinfI. The RFLP profiles of 33 strains were obtained by electrophoretic analysis. The results showed that they belonged to 14 different strains. In order to effectively remove redundant strains, combined with the data of insecticidal activity of the strain, 14 Bt strains, such as 1126-1P65-1P65-1QDL47-1261-1, FTL8O78-2127-2, 78-38-3198-1, S10A2OB8E12QD40-2FCD114B8E11, were selected for further identification. Crystal morphology observation and protein expression analysis of 14 strains of Bt. The results showed that the bipyramidal crystals were obtained by using electron microscope. The results showed that the bipyramidal crystals could be produced by FCD114P65-1 FTL84-78-2 and 1126-1 strains. The other five strains had no crystals. The expression of protein in 14 representative strains was analyzed by SDS-PAGE. The results showed that strain P65-1126-1127-2O78-3261-1FCD114FCD114FCD111-1198-1 and 78-2 could express the typical Bt Cry protein QDL47-1 of about 130 kDa and B8E11 expressed about 97 kDa protein. The remaining strains expressed a small amount of 60 kDa protein. Cry genes were identified. 14 strains of Bt strains were further identified by using cry3,cry8,cry18,cry23,cry37,cry43 generic primers. The results showed that among the 14 strains, Only four strains of P65-1O1126-1FCD114 and 78-2 contained five new cry8 genes, such as cry8Ma,cry8Ca,cry8Ab,cry8Ga,cry8Ea, respectively, while none of the other 10 strains, including 261-1 strain, which had the highest activity to the larvae of the North China blackgill tortoise, did not contain the insecticidal genes such as cry3,cry8,cry18,cry23,cry37,cry43, which were effective against Coleoptera pests. The results of mass spectrometry also showed that P65-1A1126-1FCD114 and 78-2 had Cry8 protein expression. Histopathological observation of midgut: the histopathological changes of intestinal parietal cells were observed after infection with Bt in the larvae of the North China Black Gill tortoise. It was found that the tissue of intestinal parietal cells of the larvae had obvious pathological changes after infection with strain 261-1198-1 FCD114-1126-1QDL40-2. The results of this study are of great significance for the prevention and control of Bt strains and the discovery of insecticidal gene resources.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.5;S476.1
【参考文献】
相关期刊论文 前1条
1 陈桂花;束长龙;李颖;宋福平;郭媛媛;李国勋;张杰;;基于聚合酶链式反应-高通量测序(PCR-HTS)联用的cry2A基因鉴定方法[J];中国生物防治学报;2014年05期
相关博士学位论文 前1条
1 束长龙;苏云金芽胞杆菌资源多样性分析与杀虫基因发掘[D];东北农业大学;2013年
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