玉米蚜和麦二叉蚜内共生菌groEL基因的克隆与原核表达
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摘要
蚜虫是传播植物病毒的最重要的昆虫介体，由其传播的病毒病害造成多种农作物、蔬菜及经济作物的严重损失。阐明蚜虫传毒的分子机制可望为蚜传病毒病的防治找到新的思路。从病毒方面已对多种参与蚜虫传毒的因子进行了鉴定和功能分析，但对参与传毒的蚜虫介体因子报道很少。蚜虫Buchnera GroEL是一种与循回传播病毒密切相关的蛋白，因而受到广泛重视。本研究设计了特异性引物，利用PCR技术从玉米蚜(Rhopalosiphummaize)和麦二叉蚜(Schizaphis graminum)中克隆出蚜虫Buchnera groEL基因并进行原核表达研究，主要结果如下：
(1)从杨凌生物型玉米蚜和麦二叉蚜中在国内外首先扩增出Buchnera groEL基因，测定了全长基因序列，并对相关的同源基因核苷酸进行了比较。玉米蚜和麦二叉蚜杨凌生物型Buchnera groEL基因(GenBank登录号分别为AF387863和AF434719)全长均为1647bp；推导GroEL蛋白序列结果表明其编码548个氨基酸。通过比较核苷酸和推导的氨基酸序列并绘制进化树，发现Buchnera spp.的亲缘关系不但与寄主的亲缘关系有关，还与不同的地理种群相关。
(2)将克隆的玉米蚜和麦二叉蚜Buchnera groEL基因连接到原核表达载体pBV221、pET30a和pTrcHisA上，构建了含2个目的基因的6个表达原核表达载体：pBVRM、pBVSG，pETRM、pETSG和pTrcRM、pTrcSG。转化大肠杆菌进行诱导表达，SDS-PAGE检查表达结果：载体pBVRM和pBVSG在42℃诱导2h-6h，2个载体均可表达出63KD的非融合蛋白；pETPdVl和pETSG以及pTrcRM和pTrcSG经IPTG诱导2h-6h，4个载体均可表达出分子量为69KD的融合蛋白。在这6个原核表达产物中，pETRM和pETSG表达量最高，其次是pBVRM和pBVSG，pTrcRM和pTrcSG表达量最低。
(3)纯化融合蛋白pETSGGroEL，免疫家兔，制备了GroEL蛋白的特异抗血清。
(4)通过Western blot检测确证了原核表达的非融合蛋白与融合蛋白的同源性。
蚜虫内共生菌Buchnera groEL基因的获得、初步的原核表达研究以及GroEL蛋白抗血清的制备，为进一步研究GroEL蛋白在蚜虫传毒中的作用方式和位点以及在基因工程途径的利用打下了基础。
Aphid is the most important insect vector transmitting plant virus.Many virus
diseases of crop, vegetable and economic crop transmitted by aphid cause severe economic
loss. Control on aphid-borne virus diseases may be wishful and have new methods if we
fully understand the molecular mechanism of aphid transmission. Previous work about aphid transmission have identified and analyzed a lot of molecular determinants from plant virus. However, the aphid vector factors involved in aphid transmission only have a few reports. Buchnera GroEL of aphid is a protein involved in virus circulative transmission and paid much attention to.Our study designed two pairs of specific primers and cloned two molecular weight of 63ku from Schizaphis graminum, and Rhopalosiphum maize utilizing PCR technique and have done the prokaryotic expression, the main results are as follows:
(1)Amplified the Buchnera groEL of Schizaphis graminum, and Rhopalosiphum maize Yangling Biotype at first time all over the world ,and sequenced the full lenghth gene.Furthermore,we compared the related nucleotide of ten homogenous genes.The two Buchnera groEL gene of Schizaphis graminum and Rhopalosiphum maize are all 1647bp.Their GenBank accession numbers are AF3 87863 and AF434719.The deduced amino acid sequences indicated the Buchnera groEL encode 548 amino acids.By compared the nucleotides and the deduced amino acid we protract their evolution tree and found the relative of Buchnera spp is related not only to the relative of the vector,but also to the vectors'biotype.
(2) The cloned Buchnera groEL genes of Schizaphis graminum and Rhopalosiphum maize were ligated into pBV221,pET30a and pTrcHisA expression vector for construction of pBVRM,pBVSG, pETRM,pETSGPR,pTrcRM and pTrcSG. They were transformated into E. coli BL21(DE3),DH5a and JM109 for studying their expression. The SDS-PAGE electrophoresis results suggested that pBVRM and pBVSG containing aim gene can express 63 ku aim proteins, but have lower expression quantities;pETRM and pETSG
fusion expression vectors can promote aim gene expression in high levels when induced by IPTG.The fusion proteins expressed have a molecular weight of 69 ku, expression quantities were higher and highest when induced by IPTG for 4h. Vector pTrcHisA could express aim fusion proteins whose MW is 69ku,too,but the product of pTrcSG and pTrcRM is the lowest in the three prokaryotic expression vectors by different receptor cell,sometimes even they can't be detected by SDS-PAGE.
(3)Purified the fusion protein pETSG GroEL and immuned the rabbit to prepare for the antibody of the GroEL.
(4)The Western blot indicated the prokaryotic expression is successful and the fusion
and non-fusion protein are homogenous.
We gained the Buchnera groEL gene of Schizaphis graminum, and Rhopalosiphum
maize biotype through this study , and have done a primary study of expression. Based on this study, we can further probe the role during aphid transmission of Buchnera GroEL and utilize them through the ways of genetic engineering.
(1)从杨凌生物型玉米蚜和麦二叉蚜中在国内外首先扩增出Buchnera groEL基因，测定了全长基因序列，并对相关的同源基因核苷酸进行了比较。玉米蚜和麦二叉蚜杨凌生物型Buchnera groEL基因(GenBank登录号分别为AF387863和AF434719)全长均为1647bp；推导GroEL蛋白序列结果表明其编码548个氨基酸。通过比较核苷酸和推导的氨基酸序列并绘制进化树，发现Buchnera spp.的亲缘关系不但与寄主的亲缘关系有关，还与不同的地理种群相关。
(2)将克隆的玉米蚜和麦二叉蚜Buchnera groEL基因连接到原核表达载体pBV221、pET30a和pTrcHisA上，构建了含2个目的基因的6个表达原核表达载体：pBVRM、pBVSG，pETRM、pETSG和pTrcRM、pTrcSG。转化大肠杆菌进行诱导表达，SDS-PAGE检查表达结果：载体pBVRM和pBVSG在42℃诱导2h-6h，2个载体均可表达出63KD的非融合蛋白；pETPdVl和pETSG以及pTrcRM和pTrcSG经IPTG诱导2h-6h，4个载体均可表达出分子量为69KD的融合蛋白。在这6个原核表达产物中，pETRM和pETSG表达量最高，其次是pBVRM和pBVSG，pTrcRM和pTrcSG表达量最低。
(3)纯化融合蛋白pETSGGroEL，免疫家兔，制备了GroEL蛋白的特异抗血清。
(4)通过Western blot检测确证了原核表达的非融合蛋白与融合蛋白的同源性。
蚜虫内共生菌Buchnera groEL基因的获得、初步的原核表达研究以及GroEL蛋白抗血清的制备，为进一步研究GroEL蛋白在蚜虫传毒中的作用方式和位点以及在基因工程途径的利用打下了基础。
Aphid is the most important insect vector transmitting plant virus.Many virus
diseases of crop, vegetable and economic crop transmitted by aphid cause severe economic
loss. Control on aphid-borne virus diseases may be wishful and have new methods if we
fully understand the molecular mechanism of aphid transmission. Previous work about aphid transmission have identified and analyzed a lot of molecular determinants from plant virus. However, the aphid vector factors involved in aphid transmission only have a few reports. Buchnera GroEL of aphid is a protein involved in virus circulative transmission and paid much attention to.Our study designed two pairs of specific primers and cloned two molecular weight of 63ku from Schizaphis graminum, and Rhopalosiphum maize utilizing PCR technique and have done the prokaryotic expression, the main results are as follows:
(1)Amplified the Buchnera groEL of Schizaphis graminum, and Rhopalosiphum maize Yangling Biotype at first time all over the world ,and sequenced the full lenghth gene.Furthermore,we compared the related nucleotide of ten homogenous genes.The two Buchnera groEL gene of Schizaphis graminum and Rhopalosiphum maize are all 1647bp.Their GenBank accession numbers are AF3 87863 and AF434719.The deduced amino acid sequences indicated the Buchnera groEL encode 548 amino acids.By compared the nucleotides and the deduced amino acid we protract their evolution tree and found the relative of Buchnera spp is related not only to the relative of the vector,but also to the vectors'biotype.
(2) The cloned Buchnera groEL genes of Schizaphis graminum and Rhopalosiphum maize were ligated into pBV221,pET30a and pTrcHisA expression vector for construction of pBVRM,pBVSG, pETRM,pETSGPR,pTrcRM and pTrcSG. They were transformated into E. coli BL21(DE3),DH5a and JM109 for studying their expression. The SDS-PAGE electrophoresis results suggested that pBVRM and pBVSG containing aim gene can express 63 ku aim proteins, but have lower expression quantities;pETRM and pETSG
fusion expression vectors can promote aim gene expression in high levels when induced by IPTG.The fusion proteins expressed have a molecular weight of 69 ku, expression quantities were higher and highest when induced by IPTG for 4h. Vector pTrcHisA could express aim fusion proteins whose MW is 69ku,too,but the product of pTrcSG and pTrcRM is the lowest in the three prokaryotic expression vectors by different receptor cell,sometimes even they can't be detected by SDS-PAGE.
(3)Purified the fusion protein pETSG GroEL and immuned the rabbit to prepare for the antibody of the GroEL.
(4)The Western blot indicated the prokaryotic expression is successful and the fusion
and non-fusion protein are homogenous.
We gained the Buchnera groEL gene of Schizaphis graminum, and Rhopalosiphum
maize biotype through this study , and have done a primary study of expression. Based on this study, we can further probe the role during aphid transmission of Buchnera GroEL and utilize them through the ways of genetic engineering.
引文
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