對華北大黑鰓金龜幼蟲具有活性的蘇云金芽胞桿菌篩選與鑒定
發(fā)布時間：2018-08-25 14:47
【摘要】：華北大黑鰓金龜[Holotrichia oblita(Falderman)]是難以防治的地下害蟲,給農(nóng)作物造成嚴重的損失。蘇云金芽胞桿菌(Bacillus thuringiensis,Bt)是一種已被廣泛用于農(nóng)業(yè)害蟲防治的昆蟲病原微生物,其在芽胞形成期可產(chǎn)生主要由cry和cyt基因編碼的殺蟲晶體蛋白。發(fā)掘Bt菌株資源是實現(xiàn)此類害蟲綠色防控的重要手段,因此,為了獲得對華北大黑鰓金龜具有較高殺蟲活性的菌株,本研究以實驗室分離的500株Bt菌株材料為基礎(chǔ),通過殺蟲活性測定篩選對華北大黑鰓金龜幼蟲有活性的菌株,進一步對菌株多樣性分析、菌株晶體形態(tài)觀察和蛋白表達分析、基因鑒定和華北大黑鰓金龜幼蟲感染Bt后中腸組織病理切片等方面進行了一系列的研究,其主要結(jié)果如下:對華北大黑鰓金龜幼蟲有活性菌株的篩選:以實驗室分離的500株Bt菌株為材料,通過殺蟲活性測定篩選到了42株對華北大黑鰓金龜幼蟲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天校正死亡率就達到100%,其余菌株的14天校正死亡率在30%~60%之間。菌株多樣性分析:利用本實驗室建立的便捷的Bt菌株比較鑒別技術(shù),對這42株菌株進行多樣性分析,以42株菌株的基因組為模板,利用優(yōu)化后的簡并引物和PCR擴增體系進行擴增,并利用HinfI對PCR產(chǎn)物進行酶切,通過電泳分析獲得了33個菌株的RFLP圖譜,分析比較顯示它們屬于14個不同的菌株類型,從而有效的去除了冗余菌株,結(jié)合菌株對華北大黑鰓金龜?shù)臍⑾x活性數(shù)據(jù),選擇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菌株進行進一步鑒定。菌株晶體形態(tài)觀察和蛋白表達分析:通過對14株Bt菌株的晶體形態(tài)電子顯微鏡觀察,結(jié)果顯示,78-3、127-2和1198-1菌株能產(chǎn)生雙錐體型晶體;261-1、FCD114、P65-1、FTL84、78-2和1126-1菌株能產(chǎn)生球形晶體,而其它5個菌株沒有晶體存在。通過SDS-PAGE分析了14株代表菌株表達蛋白情況,結(jié)果顯示菌株P(guān)65-1、1126-1、127-2、78-3、261-1、FCD114、1198-1和78-2能表達典型的約130 kDa的Bt Cry蛋白,QDL47-1和B8E11表達了約97 kDa蛋白,其余菌株表達了少量60 kDa的蛋白。cry基因鑒定:利用cry3、cry8、cry18、cry23、cry37、cry43類通用引物對14株Bt菌株進行進一步基因鑒定,結(jié)果顯示這14個菌株中,只有P65-1、1126-1、FCD114和78-2四個菌株分別含有cry8Ma、cry8Ca、cry8Ab、cry8Ga、cry8Ea等5類cry8類新基因;而包括對華北大黑鰓金龜幼蟲活性最高的261-1菌株在內(nèi)的其它10個菌株均不含有cry3、cry8、cry18、cry23、cry37、cry43等對鞘翅目害蟲有效的殺蟲基因。質(zhì)譜鑒定結(jié)果也顯示了P65-1、1126-1、FCD114和78-2等菌株有Cry8類蛋白的表達。中腸組織病理學(xué)觀察:進一步通過對華北大黑鰓金龜幼蟲感染Bt后中腸組織病理切片的觀察,發(fā)現(xiàn)在感染菌株261-1、1198-1、FCD114、1126-1、QDL40-2后,幼蟲腸壁細胞組織有明顯的病理變化。本研究結(jié)果對防治大黑鰓金龜?shù)腂t菌株和殺蟲基因資源發(fā)掘均具有重要意義。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433.5;S476.1
本文編號：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433.5;S476.1
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