【作者】 郑银英；

【导师】 王国平；

【作者基本信息】 华中农业大学 ， 果树学， 2005， 博士

【摘要】 苹果茎沟病毒(Apple stem grooving virus,ASGV)和苹果褪绿叶斑病毒(Apple cholortotic leaf spot virus,ACLSV)是危害梨和苹果及其它一些果树的重要病毒,对果树的生长、产量、果品质量等造成严重的影响。本研究以ASGV和ACLSV为对象研究其生物学、血清学特性和分子变异。目的就是要通过对不同来源分离物的分子变异与生物学和血清学特性的比较分析,从分子水平上探索ASGV和ACLSV生物学多样性和血清学差异与分子变异的关系,结果如下: 1.通过昆诺藜(Chenopodium.quinoa)接种鉴定和ELISA检测,从梨和苹果上获得ASGV 23个分离物。采用TC-RT-PCR对这些分离物进行扩增,均获得特异的扩增片段,PCR产物经5%PAGE电泳,出现大小约500、530和600bp的三种迁移率不同的泳动带型。根据PCR产物电泳迁移率的差异,选取3个来源于梨的分离物P-L4、P-6-1-17和P-3-2-67的PCR产物进行克隆与序列测定。经BLAST搜索,三个分离物的扩增片段与苹果分离物P-209的CP基因3′端核苷酸序列同源性分别为92.2%、90.4%和88.4%。3个分离物间的核苷酸序列也有较大差异,P-L4/P-6-1-17为95.5%、P-L4/P-3-2-67为90.4%、P-6-1.17/P-3-2-67为88.6%。 2.选取生物学和血清学特性上存在较大差异的ASGV梨分离物P-6-1-17、P-4-1-69、P-L2和P-D-21,采用RT-PCR法对3′端进行扩增,所获扩增片段经序列测定,全长分别为1089nt(P-6-1.17、P-L2)和1069nt(P-4-1-69、P-D-21)。4个分离物的CP基因均由714nt组成,其核苷酸和推导编码蛋白氨基酸序列同源性分别为89.8%～96.4%和94.9%～97.9%。将我国4个分离物的CP基因与已报道ASGV分离物及分子变种进行序列同源性比较和系统进化树分析,结果表明我国ASGV分离物至少可分为2个组群,其中生物学表现明显不同于其它分离物的P-L2,其CP基因核苷酸序列显示较大的变异。 3.通过昆诺藜接种鉴定和ELISA检测,从梨、苹果和桃上获得ACLSV 13个分离物。用PAS-ELISA和DAS-ELISA对这13个分离物进行检测,结果PAS-ELISA只检出来源于桃的HBP分离物,而DAS-ELISA检出来源于桃的HBP、PE-T-5和PE-5 3个分离物。然而采用RT-PCR和TC-RT-PCR对这些分离物进行扩增,均能获得特异的358bp目标片段。 4.在昆诺藜和苋色藜上均产生系统褪绿症状,且血清学上相关的来源于桃和苹果的HBP、ACLSV-C 2个分离物,通过RT-PCR法分别获得约1.8kb的目标片段。所获扩增片段经序列测定,全长分别为1768nt(GenBank登录号AY728180)和1751nt。2个分离物的CP基因均由582nt组成,其核苷酸和推导编码氨基酸序列同源性分别为87.8%和95.9%。将这2个分离物的CP基因与已报道ACLSV分离物进行序列同源性比较和系统进化树分析结果表明,HBP与从苹果和樱桃更多还原

【Abstract】 The apple stem grooving virus(ASGV) and apple cholortotic leaf spot virus (ACLSV) are important viruses in apple and pear trees which can cause sereious effect on the growth of fruit trees and the quantity, quality of fruit. The biology, serology and molecular variability of ASGV and ACLSV were studied in this reaserch. The aim of our studies was by analyze to study the relations in biological diversity, serological difference and molecular variability on the molecular level the relations in molecular variability, biological and serological characters among different isolates.The results are described as following:1. Twenty-three isolates of apple stem grooving virus were obtained from pear and apple by mechanically inoculating on Chenopodium quinoa and ELISA confirmation. Specific fragments were amplified from all these samples by tube capture reverse transcription-polymerase chain reaction (TC-RT-PCR). These PCR products showed three type bands with different migration rate (500bp, 530bp and 600bp) on 5%PAGE. The PCR products of three isolates P-L4, P-6-1-17 and P-3-2-67 from pear with different migration rates were cloned and sequenced. A BLAST search result revealed that these three isolates had 92.2%, 90.4% and 88.4% nucleotide sequence similarity with the 3’ ends of P-209, an isolate from apple. And the nucleotide sequences among these three isolates also showed some difference. The similarities of P-L4 and P-3-2-67, P-L4 and P-6-1-17, P-6-1-17 and P-3-2-67 were 90.4%, 95.5% and 88.6%, respectively.2. Four isolates of Apple stem grooving virus, P-6-1-17, P-4-1-69, P-L2 and P-D-21 from pear, which displayed different reactions in biological and serological tests, were studied for its molecular characteristics. RT-PCR products of 3 ’ end of four isolates were cloned and sequenced. The sizes of cloned fragments are 1089nt from P-6-1-17 and P-L2, and 1069nt from P-4-1-69 and P-D-21, respectively, which include 714nt CP coding region. The similarities of nucleotide and deduced amino acid sequences of CP genes among these isolates are 89.8% to 96.4% and 94.9% to 97.9%, respectively. The sequence data obtained in the study were compared with previously reported sequences of ASGV isolates and sequence variant species from different sources. Results suggested that isolates P-6-1-17, P-4-1-69, P-L2 and P-D-21 should be divided into two different subgroups. P-L2 showed different reactions in biological test and its CP gene also had a relatively high variability.3. Thirteen isolates of apple cholortotic leaf spot virus were obtained from pear, apple and peach by mechanically inoculating on Chenopodium quinoa and ELISA.PAS-ELISA and DAS-ELISA were used to detect these 13 isolates, in the former method one peach isolates (HBP) was positive reaction and in the latter method three peach isolates were positive reactions (HBP PE-T-5 和 PE-5) . Otherwise, when RT-PCR and TC-RT-PCR were used to detect these 13 isolates, specific products of 358bp fragment were obtained in all these isolates.4. Two isolates from peach and apple, HBP and ACLSV-C displayed systemic chlorotic symptoms on Chenopodium quinoa and Ch.amaranticolor had relations in serological tests. RT-PCR products of two isolates were cloned and sequenced. The sizes of cloned fragments are 1768nt from HBP(GenBank accession numer: AY728180) and 1751nt from ACLSV-C, respectively, which include 714nt CP coding region. The similarities of nucleotide and deduced amino acid sequences of CP genes among these isolates are 87.8% and 95.9% respectively. The sequence data obtained in the study were compared with previously reported sequences of ACLSV isolates from different sources. The similarities between HBP and SX/2 were high but the latter did not react (or reacted weakly) with some moloclonal and polyclonal antibodies prepared against ACLSV isolates from apple and cherry.5. A fragment 485nt corresponding to 83.3% of the 3’end coat protein gene of P-4-1-69 isolates of apple cholortotic leaf spot virus from pear was obtained. The similarities of nucleotide and deduced amino acid sequences of 3’end CP genes among P-4-1-69, HBP and ACLSV-C isolates are 86.2% to 99.8% and 96.2% to 100.0%, respectively. The sequence data obtained in the study were compared with previously reported sequences of ACLSV isolates from different sources. Results suggested that isolates P-4-l-69 HBP ACLSV-C Kuerle and MP-CI should be divided into two different subgroups.6. The result of SDS-PAGE of prokaryotic expression of the coat protein of HBP demonstranted that this gene was expressed in E.coli, the molecular weight of the fusion protein was 47.6kDa. Western-blot examination identified the gene was expressed rightly, and the fusion protein was immunogenic.更多还原