【作者】 张洪磊；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 园林植物与观赏园艺， 2009， 硕士

【摘要】 本研究从杜梨(Pyrus betulaefolia)、白梨(P. bretschneideri)、西洋梨(P. communis)、秋子梨(P. ussriensis)、沙梨(P. pyrifolia)、新疆梨(P. sinkiangensis)和杂交种(P. hybrid)等7个种内选取46个主要栽培品种为实验试材,利用根据已知抗病基因的NBS(nucleotide binding site)保守结构域设计的引物,通过PCR技术展开分离梨抗病基因同源类似物(resistance gene analogs, RGAs)的研究。对获得的阳性克隆进行序列测定,通过生物信息学分析,鉴定并筛选RGAs。在此基础上,利用RACE(rapid amplification of cDNA ends, RACE)技术获得部分RGAs的全长cDNA序列,并通过生物信息学分析、Southern blot分析、梨黑星病菌诱导的表达分析等,考察其结构特点、拷贝数以及在抗黑星病过程中的表现等。本研究取得的成果主要有:1、梨属植物RGAs的克隆及分析从39个梨树品种中克隆获得了102个RGAs,GenBank登录号分别为:FJ262736和EU939772～EU939872。在102个RGAs中,因RGAs Pb-Dshs-1(GenBank登录号:EU939772)和Pb-Zsm-2(GenBank登录号:EU939785)推导的氨基酸序列因不具连续的开放阅读框(open reading frame, ORF),所以没有进行进一步的分析。其余100个RGAs的均能推导为具有连续ORF的氨基酸序列,并具有抗病相关的NBS功能域,如P-loop、Kinase2、Kinase3和疏水结构(GLPLAL)等基序。同源性分析表明这100个RGAs核苷酸序列的同源性在2%～100%之间,氨基酸序列的同源性在20%～100%。利用Clustal X和MEGA 4构建的系统发育进化树表明,这100个RGAs聚类为两大类,其中有93个属于TIR(Toll/interleukin-1 receptor, TIR)类,其余7个RGAs属于non-TIR类。Blast分析表明,本研究得到的梨RGAs与其它植物中已克隆的RGAs具有较高的同源性,与已知NBS类抗病基因也存在一定的同源性。本研究在梨的7个种内各选择1～4个RGAs检测其在RNA水平上的表达情况,共选出15个RGAs,根据其DNA序列设计特异PCR引物,通过RT-PCR检测到只有白梨、杜梨、秋子梨和杂交梨等4个种的8个RGAs在RNA水平上表达,其GenBank登录号分别为:FJ173825～FJ173829和FJ377558～FJ377561,而在西洋梨、新疆梨和沙梨中选择的7个RGAs均未检测到表达信号。2、早酥梨3个RGAs基因全长序列的克隆与分析根据早酥梨RGAs的cDNA序列,利用RACE技术获得了3个RGAs的基因全长cDNA序列。根据早酥梨RGAs Pb-Zs1获得的cDNA序列全长3498bp,包含一个3162bp的开放阅读框架,编码1053个氨基酸残基,分子量为120.3 kD,将该基因命名为Prg1,GenBank登录号为FJ943460。早酥梨Prg2基因全长为2384bp,包含一个完整的2325bp的ORF,编码774个氨基酸残基,分子量为88.8 kDa,GenBank登录号为FJ943461。早酥梨Prg1和Prg2基因核酸序列的同源性达91.9%,推测其可能为同一家族的基因。早酥梨RGAs Pb-Zs3基因的全长cDNA序列大小为1200bp,包含一个完整的921bp的ORF,其中起始密码子ATG位于175-177bp处,终止密码子TGA位于1093-1095bp,共编码306个氨基酸。分子质量为34.6 kD,命名为Vnp1(GenBank登录号为FJ763188)。生物信息学分析表明,Prg1和Vnp1基因都含有完整的抗病基因的NBS保守结构域和所有的基序P-Loop、Kinase2、Kinase3及疏水结构(GLPLA);早酥梨Prg1和Prg2基因含有与抗病相关的TIR和LRR结构域。同源性比对显示,Vnp1基因与蓖麻中克隆得到的烟草抗花叶病毒基因N类似蛋白,毛果杨TIR-NBS-LRR类抗病蛋白等具有较高的同源性;Prg1与Prg2基因编码的蛋白与从山荆子、拟南芥等中分离得到的TIR-NBS-LRR类抗病蛋白具有较高的同源性。另外,Prg1、Prg2和Vnp1基因与已克隆的西红柿的抗落叶病蛋白I2C-1、亚麻抗锈病基因L6和烟草抗花叶病毒N等NBS-LRR类抗病基也具有一定的同源性。3、梨黑星病菌诱导的早酥梨Prg1、Prg2和Vnp1基因的表达分析应用半定量RT-PCR技术,对早酥梨Prg1、Prg2和Vnp1基因在接种黑星病菌后的早酥梨叶片中的表达水平变化情况进行分析。结果表明:早酥梨Prg1基因的表达水平在接种黑星病菌后呈先升后降的趋势。接种黑星病菌后该基因表达水平呈明显上升趋势,在24小时后表达量达到最高点,随后又呈逐渐下降趋势,到72小时又降至未接种黑星病菌时的表达水平;早酥梨Prg2基因的表达量在接种黑星病菌后呈先降后升趋势。接种病菌后该基因表达水平迅速下降,在接种后12小时,观测到该基因的表达水平降至最低点,随后又呈现上升趋势,在接种黑星病菌48小时超过接种前的表达水平,随接种时间的增长又呈现下降趋势,在接种病菌72小时后,恢复至原来的表达水平。早酥梨Vnp1基因的表达量在接种黑星病菌216小时的范围内一直呈现上升趋势。梨黑星病菌诱导的早酥梨Prg1、Prg2和Vnp1基因的表达分析说明在早酥梨抗黑星病的过程中,早酥梨Prg1、Prg2和Vnp1基因在转录水平即受到调节,参与抗病反应。4、早酥梨Prg1基因的Southern blot分析将早酥梨基因组DNA分别用限制性内切酶EcoRⅠ,HindⅢ和XbaⅠ进行单酶切,将杜梨基因组DNA以EcoRⅠ酶切,以早酥梨Prg1基因制备探针,进行Southern blot。在HindⅢ酶切的早酥梨基因组DNA中最多检测到4个杂交信号,另外,在EcoRⅠ酶切的杜梨基因组DNA中也检测到2个杂交信号。Southern blot分析表明Prg1基因是以多拷贝的形式在梨属植物中广泛存在的。更多还原

【Abstract】 46 cultivars which were chosen from 7 species of pear including Pyrus betulaefolia, P. bretschneideri, P. communis, P. ussriensis, P. pyrifolia, P. sinkiangensis and P. hybrid were used in this study to isolate disease resistance gene analogs (RGAs). A PCR strategy with the primers designed according to the conserved NBS (nucleotide binding site) domain of known resistance genes were used to isolate RGAs from pear. Positive clones were sequenced and RGAs were identified by bioinformatics analysis. Based on the RGAs isolated from pear, full-length cDNA sequences were acquired with the technique of RACE (rapid amplification of cDNA ends). Further more, bioinformatics analysis, southern blot analysis and semi-quantitative RT-PCR analysis were used to analyze their structure, copy numbers and expression pattern during the period of defending against Venturia nashicola. The main results are as follows:1. Cloning and characterization of RGAs from pear102 RGAs were isolated from genomic DNA of 39 pear cultivars, and submitted to GenBank with accession numbers FJ262736 and EU939772 to EU939872. Among these 102 RGAs, owning to containing stop codens when translated into amino acid sequences, two RGAs Pb-Dshs-1(GenBank Accession No. EU939772) and Pb-Zsm-2 (GenBank Accession No. EU939785) were not further analyzed. All the other 100 RGAs had uninterrupted open reading frames (ORF) and disease resistance related NBS domains, such as the motifs of P-loop, Kinase2, Kinase3 and hydrophobic domain. Homologous analysis indicated that the identity of nucleotide sequences of these RGAs ranged from 2%～100% and the amino acid sequences ranged from 20%～100%. Phylogenetic tree which constructed by Clustal X and MEGA 4 indicated these 100 RGAs could be classified into two major classes. 93 RGAs belonged to TIR (Toll/interleukin-1 receptor, TIR) type; the other 7 RGAs belonged to non-TIR type. Blast analysis showed high identity between these RGAs from pear and RGAs cloned from other plants, certain identity were also found between these pear RGAs and some known NBS type disease resistance genes.In this study, 1～4 RGAs which were chosen from each species of pear were used to examine transcript expression profiles. Totally, 15 RGAs were selected. Specific PCR primers which were designed according to the DNA sequences of 15 RGAs successfully amplified 8 cDNA sequences of RGAs from P. betulaefolia, P. bretschneideri, P. ussriensis and P. hybrid, with GenBank accession numbers FJ173825～FJ173829 and FJ377558～FJ377561. The other 7 RGAs isolated from P. pyrifolia, P. sinkiangensis and P. communis were failed to be detected at mRNA level.2. Full-length isolation and characterization of RGAs from Zaosu pearBased on the cDNA sequences of RGAs cloned from Zaosu pear, three genes with full-length cDNA sequences were acquired with the technique of RACE. The full-length cDNA sequences of Zaosu pear RGAs Pb-Zs1 is 3498 base pairs, and contains an open reading frame with 3162 base pairs which encodes 1053-residue protein with a calculated molecular mass of 120.3 kD. This gene is named Prg1 with GenBank accession numbers FJ943460. The cDNA sequences of Prg2 gene in Zaosu pear is 2384 base pairs with a 2325 bp open reading frame. Prg2 gene encodes 774-residue protein with a calculated molecular mass of 88.8 kD, and has been submitted to GenBank with accession number FJ943461. The nucleotide acid sequences of Prg1 and Prg2 genes are highly conserved with 91.9% identity, and they may from same gene family. The full-length cDNA sequences of Zaosu pear RGAs Pb-Zs3 is 1200 base pairs. The complete open reading frame is 921bp in length, start conden ATG lies in 175-177bp and stop conden TGA lies in 1093-1095bp, encoding 306-residue protein with a calculated molecular mass of 34. 6 kD. This gene is named Vnp1 (GenBank Accession No. FJ763188). Bioinformatics analysis showed that Prg1 and Vnp1 genes contained unabridged NBS domain of disease resistance gene with all the motifs P-loop, Kinase2, Kinase3 and hydrophobic domain. Besides this NBS domain, Prg1and Prg2 also contained other disease resistance related domains TIR and LRR. Homologous analysis showed Vnp1 gene showed high identity to TMV like gene N in Ricinus communis and TIR-NBS-LRR type resistance genes in Populus trichocarpa; Prg1 and Prg2 genes showed high identity to TIR-NBS-LRR type resistance genes isolated from Malus baccata and Arabidopsis thaliana. What’s more these 3 genes isolated from Zaosu pear showed a certain identity to the known NBS-LRR type disease resistance genes like I2C-1 in tomato, L6 in falx and N in tobacco.3. Expression analysis of Prg1, Prg2 and Vnp1 genes from Zaosu pear after inoculated with Venturia nashicolaSemi-Quantitative RT-PCR was used to analyze the expression pattern of Prg1, Prg2 and Vnp1 genes in the leaves of Zaosu pear. The result showed the expression level of Prg1 was presented as increased at first and followed by decreasing trend. The expression level of Prg1 increased significantly after inoculated with V. nashicola,and reach to the highest level at 24 h post-inoculation. In the flowing time the expression level was slightly decreased and reached to the normal level at 72 h post-inoculation; the expression level of Prg2 was drastically decreased and reach to the lowest level at 12 h post-inoculation. Then, the expression level was slightly increased, and reached to the highest point at 48h post-inoculation. When at 72 h post-inoculation the expression level returned to the normal level before-inoculation; the expression level of Vnp1 gene was significantly increased in 216 h after inoculation. These results showed that these three genes positively involved in resistance to pear scab.4. Southern blot analysis of Prg1 gene from Zaosu pearGenomic DNA of zaosu pear was digested with EcoR I, HindⅢand Xba I, and genomic DNA of Duli was digested with EcoR I, then hybridised with the DNA sequence of Prg1 as probe. Four hybridised bands were present in lane which digested with HindⅢ. What’s more, two hybridized bands were found in lane with genomic DNA of Duli which digested by EcoR I. This indicated that Prg1 gene was widely distributed in pear with multiple copies.更多还原