【作者】 宋艳苏；

【导师】 王国平；

【作者基本信息】 华中农业大学 ， 植物病理学， 2011， 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 chlorotic leaf spot virus, ACLSV)是苹果和梨树上发生普遍的两种潜隐性病毒(Latent viruses),常与苹果茎痘病毒(Apple stem pitting virus, ASPV)复合侵染引起树势衰弱、减产和品质变劣。砂梨(Pyrus pyrifolia)在我国广泛种植,80%的梨树被ASGV和ACLSV侵染。为了建立砂梨上ASGV和ACLSV血清学检测技术体系以及研究ACLSV的血清学多样性,本研究构建了来源于砂梨的ASGV和ACLSV分离物外壳蛋白基因(Coat protein genes, cp genes)的原核表达载体,用诱导表达的重组外壳蛋白(Recombinant coat proteins, rCPs)免疫大耳白兔,制备了多克隆抗体；比较了来源于砂梨的ACLSV不同分离物的生物学特性、cp基因的分子变异和血清学多样性,分析了cp基因分子变异与血清学多样性的关系,为进一步了解ACLSV的群体结构提供实验依据,以及为梨ACLSV血清学检测提供技术支撑。取得的研究结果如下：1.将分别来源于砂梨和白梨(P. bretschneideri)的ASGV分离物P-4-1-69和P-L2cp基因(大小714 bp,编码237 aa,GenBank登录号分别为FJ608985和FJ608986)插入原核表达载体pET-28a(+),得到重组载体pET-P-4-1-69和pET-P-L2,转化大肠杆菌BL21(DE3),在1 mmol/L IPTG下诱导表达。SDS-PAGE和Western blot分析结果表明,两个ASGV分离物P-4-1-69和P-L2的cp基因在大肠杆菌中成功地进行了高效表达,重组外壳蛋白的大小均为约31 kDa,并具有抗原性。分别用含分离物P-4-1-69和P-L2的重组外壳蛋白的胶条经乳化处理后免疫大耳白兔,制备了抗ASGV重组外壳蛋白的抗血清,间接ELISA效价分别为1：512000倍和1：32000或1：64000。为了降低抗血清的非特异性反应,抗体被纯化。Western blot分析和组织免疫印迹检测(Tissue blotting immunoassay, TBIA)结果表明,纯化的抗体具有较强的特异性,可有效检测梨样品中的ASGV。2.克隆了来源于巴东桐子梨离体植株的ACLSV分离物ACLSV-BD的cp基因,其大小为582 bp,编码193aa (GenBank登录号分别为EJ608984)。将其cp基因插入原核表达载体pET-28a(+)得到重组载体pET-ACLSV-BD,在大肠杆菌BL21(DE3)中经1 mmol/L IPTG诱导表达。表达产物用SDS-PAGE和Western blot分析表明,分离物ACLSV-BD的cp基因在大肠杆菌中成功地进行了高效表达,重组外壳蛋白的大小为约26 kDa,并具有抗原性。用含分离物ACLSV-BD重组外壳蛋白的胶条经乳化处理后免疫大耳白兔,制备了抗ACLSV重组外壳蛋白的抗血清,间接ELISA效价为1：32 000或1：128 000。为了降低抗血清的非特异性反应,抗体被纯化。Western blot分析和TBIA结果表明,纯化的抗体具有较强的特异性,可有效检测梨样品中的ACLSV。3.从湖北省农科院果树茶叶研究所国家果树种质武昌砂梨圃、湖北省恩施市和云南省昆明市共采集了65份砂梨样品。采用TC-RT-PCR(试管捕捉RT-PCR, Tube capture RT-PCR)检测ACLSV结果表明,52份砂梨样品带有ACLSV,检出率为80%。将阳性样品接种昆诺藜(Chenopodium quinoa)和西方烟(Nicotiana occidentalis),在昆诺藜上表现为系统的褪绿斑、斑驳和叶片反卷,在西方烟上表现为系统的褪绿斑、坏死斑和叶脉黄化,这些症状是ACLSV和ASGV复合侵染引起的。4.从20个砂梨样品(不包括巴东桐子梨离体植株)、两个李样品(湖北省武汉市)和一个桃样品(陕西省西安市)中扩增了ACLSV分离物的cp基因,克隆后提取质粒。对这些分离物的克隆载体用EcoRⅠ和SacⅠ进行限制性片段长度多态性分析(Restricted fragment length polymorphisms, RFLP),结果表明cp基因的酶切产物可产生4种带型：Ⅰ、Ⅱ、Ⅲ和Ⅳ,其中Ⅰ和Ⅱ是主要类型,且同一个样品的ACLSVcp基因可产生多个类型,即ACLSV存在分子变种。同源性比较表明砂梨分离物PPl5的两个分子变种cp基因的核苷酸同源性较低(96.6%),被认为是两个分离物(即PP15-2和PP15-4),其它分离物分子变种间的同源性均高于99%。5.将获得的22个来源于砂梨的ACLSV分离物(包括分离物ACLSV-BD)和3个来源于核果类分离物(PL1、PL2和PE)的cp基因(GenBank登录号EJ608984、GU327981-GU328004)进行序列比对,结果表明ACLSV砂梨分离物的cp基因具有序列多样性,核苷酸序列和推导编码的氨基酸序列的同源性分别为87.3～100%和92.7～100%。将得到的25个分离物和45个已报道分离物的cp基因进行核苷酸和氨基酸系统进化分析,结果表明,这些分离物可以分成多个簇,所有的砂梨分离物都聚集在Ⅰ簇,并分成了两个亚簇(A和B)。19个砂梨分离物与5个核果类分离物(PE、PL1、PL2、HBP和SX/2)聚集于A亚簇,3个砂梨分离物(PP39、PP54和PP56)与我国的苹果分离物ACLSV-C、印度的西洋梨分离物Pear以及日本和印度的其它分离物聚集在B亚簇,而来源于我国新疆库尔勒香梨(P. sinkiangensis Yu)的分离物Kuerle与砂梨分离物的同源性较低,与来源于日本和印度的一些苹果和核果类分离物聚集在Ⅱ簇。外壳蛋白(Coat protein, CP)氨基酸序列的多重比对表明,本研究所获得的25个分离物在位点40、59、75、130和184均为S40-L59-Y75-T130-L184,为B6型分离物。B亚簇中的4个中国分离物(PP39、PP54、PP56和ACLSV-C)的CP在氨基酸位点70具有一个特异的氨基酸——谷氨酸(Glutamic acid, E)。6.根据系统进化分析,挑选A亚簇5个分离物(PP13、PP15-2、PP24、PP43和PE)和B亚簇3个分离物(PP54、PP56和ACLSV-C),构建其cp基因的原核表达载体,对表达的重组外壳蛋白进行电泳迁移率和血清学反应分析。重组外壳蛋白的SDS-PAGE、Western blot和PAS-ELISA分析表明,这8个分离物的重组外壳蛋白具有两种不同的电泳迁移率和血清学反应性。A亚簇的5个分离物的重组外壳蛋白与抗分离物ACLSV-BD(A亚簇)重组外壳蛋白的抗体和抗日本苹果分离物P-205(Ⅱ簇)病毒粒子的抗体的反应信号比与抗中国苹果分离物ACLSV-C (B亚簇)病毒粒子的抗体的反应信号强,而B亚簇的3个分离物的重组外壳蛋白与ACLSV-C的抗血清的反应较强。对CP的抗原决定簇进行预测表明,预测的9个抗原决定簇中有6个在A亚簇和B亚簇的分离物之间发生了特异变化。更多还原

【Abstract】 Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are two important latent viruses in commercially cultivated apple and pear trees. Two viruses are usually mixed infection with Apple stem pitting virus (ASPV) and cause greatly decreasing the growth and productivity of infected trees. Sand pear (Pyrus pyrifolia) is widely grown in China. Over 80% of pear trees in China were infected by ASGV and ACLSV. In this paper, in order to establish the foundation for high-through and fast detection techniques of ASGV and ACLSV in China and study the serological diversity of ACLSV, the prokaryotic expression vectors of coat protein genes (cp genes) of ASGV and ACLSV from sand pear were constructed. The antisera against recombinant coat proteins (rCPs) expressed in Escherichia coli (E. coli) were prepared. Furthermore, the biological, molecular and serological characteristics of ACLSV isolates from sand pear were compared with other isolates from different fruit trees and the molecular and serological diversity in ACLSV was analyzed. The obtained results contributed to increased understanding of the molecular population structure of ACLSV in different hosts and its implication in serological detection. The obtained results are as followings:1. The cp genes of two ASGV isolates (714 bp,237 aa, GenBank accession numbers FJ608985 and FJ608986), P-4-1-69 and P-L2 from sand pear and white pear (P. bretschneideri), respectively, were inserted into prokaryotic expression vector pET-28a(+). The recombinant plasmids were denoted as pET-P-4-1-69 and pET-P-L2. E. coli strain BL21 (DE3) was transformed with two expression constructs. Protein productions were induced by 1 mM isopropyl-β-D-thiogalactoside (IPTG). SDS-PAGE and Western blot analyses indicated that cp genes of two ASGV isolates were expressed effectively under the induction of 1 mmol/L IPTG and molecular weights of their rCPs were approximately 31 kDa. These rCPs were used to raise antisera in rabbits. The titers of antisera were 1:512 000 for isolate P-4-1-69 and 1:32 000 or 1:64 000 for isolate P-4-1-69 in indirect-ELISA, respectively. In order to reduce the nonspecific reaction, the raised antisera were purified. These purified antibodies showed to possess high specificity to their rCPs in Western blot and native virus coat proteins in infected pear shoot in-vitro by tissue blotting immunoassay (TBIA).2. The cp gene of an isolate ACLSV (ACLSV-BD) from "Badongtongzili" pear shoot in-vitro was cloned and sequenced (GenBank accession number EJ608984). The cloned fragment consisted of 582 bp and encoded 193 aa. The cp gene was inserted into prokaryotic expression vector pET-28a(+). The recombinant plasmid pET-ACLSV-BD containing cp gene was obtained and transformed into BL21 (DE3). Protein productions were induced by 1 mM IPTG. SDS-PAGE and Western blot analyses indicated that cp gene of isolate ACLSV-BD was expressed effectively under the induction of 1 mmol/L IPTG and molecular weight of the rCP was approximately 26 kDa. The rCP was used to raise antiserum in rabbits. The titer was 1:32 000 or 1:128 000 in indirect-ELISA. The purified antibody showed to possess high specificity to the rCP in Western blot and native virus coat protein in infected pear shoot in-vitro by TBIA.3. Sixty-five sand pear samples were collected from the National Germplasm Conservation Center of P. pyrifolia (Wuhan, Hubei), western Hubei province (Enshi) and Yunnan province (Kunming). TC-RT-PCR (Tube capture RT-PCR) test indicated that 80% (52/65) sand pear samples were infected by ACLSV. Chenopodium quinoa and Nicotiana occidentalis were inoculated with extracts from sand pear leaves infected by ACLSV. The symptoms of systemic chloroctic, mottle and leaf roll on C. quinoa and systemic chloroctic, necrotic and yellow vein on N. occidentalis, were observed and caused by the complex infection of ACLSV and ASGV.4. The cp genes of ACLSV from 20 sand pear samples, two plum samples (Wuhan, Hubei) and a peach sample (Xian, Shanxi) were cloned. The plasmids containing cp genes were digested with the restriction enzymes EcoR I and Sac I. The result showed that digested-products of cp genes contained four patterns, I, II, III and IV, and I and II were predominant patterns. The digested-products of ACLSV cp genes from the same sample showed different patterns, indicating that ACLSV isolate was composed of a population of variants. The cp genes sequence comparison revealed that two variants (PP15-2 and PP15-4) from isolate PP15 showed lower identity (96.6%) and were designed two isolates. The identities were more than 99% among variants from other isolates.5. The cp genes of 22 ACLSV isolates (including isolate ACLSV-BD) from sand pear, two isolates from plum and an isolate from peach were compared (GenBank accession numbers EJ608984, GU327981-GU328004). The sequences of cp genes from 22 sand pear isolates showed a high divergence, with 87.3～100% identities at the nucleotide (nt) level and 92.7～100% identities at the amino acid (aa) level. The phylogenetic trees generated from the nucleotide and deduced amino acid sequences of cp genes, including 25 isolates from this study and 45 isolates from GenBank, showed that the analyzed ACLSV isolates fell into different clusters and all isolates from sand pear were grouped into a larger cluster (Ⅰ) which was then divided into two subclusters (A and B). Nineteen out of 22 ACLSV sand pear isolates were grouped into the subcluster A with five isolates from stone fruit tree (PE, PL1, PL2, HBP and SX/2), whereas other three sand pear isolates (PP39, PP54 and PP56) together with a pear isolate (Pear) from India, an apple isolate (ACLSV-C) from China and other isolates from Japan and India were grouped into the subcluster B. The isolate (Kuerle) from kuerle pear (P. sinkiangensis Yu) specially grown in Xinjiang province showed low identities with 22 sand pear isolates and was grouped into a cluster (Ⅱ) together with some apple and stone fruit isolates from Japan and India. The multiple alignment of coat proteins (CPs) revealed that the five amino acids combination at positions 40,59,75,130 and 184 (S40-L59-Y75-T130-L184) was presented in 25 ACLSV isolates analyzed in this study, suggesting that these isolates were belonged to B6 type. An aa residue Glutamic acid (E) at position 70 was conserved in three sand pear isolates (PP39, PP54 and PP56) and isolate ACLSV-C grouped into the subcluster B.6. Based on the divergence deduced from phylogenetic analysis, the cp genes of five ACLSV isolates (PP13, PP15-2, PP24, PP43 and PE) grouped into the subcluster A and three (PP54, PP56 and ACLSV-C) grouped into the subcluster B were selected for producing recombinant proteins for further analyses of electrophoretic mobility and serological reactivity. SDS-PAGE, Western blot and PAS-ELISA analyses demonstrated that rCPs of eight ACLSV isolates had different mobility rates and serological reactivity. The rCPs of five isolates grouped into the subcluster A showed stronger reactivity with antibodies against rCPs of a sand pear isolate ACLSV-BD (subcluster A) and virions of a Japanese apple isolate P-205 (clusterⅡ) than that with the antibody against a Chinese apple isolate ACLSV-C (subcluster B). Three isolates grouped into the subgroup B showed stronger reactivity with the antibody against ACLSV-C. The antigenic determinants of CPs from eight isolates and isolates ACLSV-BD and P-205 predicted nine epitope regions in their CPs. Six predicted epitopes showed some variations among different isolates.更多还原