【作者】 张计育；

【导师】 章镇；

【作者基本信息】 南京农业大学 ， 果树学， 2011， 博士

【摘要】 湖北海棠(Malus hupehensis (Pamp.) Rehd)是原产于我国的抗性较强的苹果砧木之一,是研究木本植物抗逆性机制非常重要的植物材料。本文以‘湖北海棠’为试材,构建了水杨酸处理后的湖北海棠全长cDNA文库,从中分离了MhNPRl、MhTGA2转录因子以及病程相关蛋白基因(Mhchititl、MhGlu、MhPR1、和MhPR5)序列,对这些基因表达特性和功能进行了分析,建立了利用口蹄疫病毒2A序列构建植物三价融合表达载体的技术体系,构建了三价融合表达载体MhTNC (MhGGA2-2A-MhNPR1-2A-Mhchitl),并对其功能进行了初步分析,主要结果如下：1.以湖北海棠为材料,经水杨酸处理后,通过改良的CTAB法提取总RNA,纯化后构建全长cDNA文库。结果表明：提取的总RNA无降解,无污染。mRNA弥散带主要集中在500～2000 bp左右,没有rRNA残留。dscDNA弥散带主要分布于300-2000bp之间,PCR验证后片段大小分布于200-2000bp之间,说明合成dscDNA质量较好,成功地构建了全长cDNA文库。2.湖北海棠MhNPRl基因的全编码区cDNA序列为1761bp,命名为MhNPR1 GenBank序列登录号FJ5981431。基因组全编码区序列长为2259bp,GenBank序列登录号GU183100。序列比对结果表明,该基因编码区与拟南芥AtNPR1,水稻OsNPRl基因类似,含有3个内含子和4个外显子。MhNPRl基因组序列的长度和拟南芥相近,其内含子相对位置也同于拟南芥。该蛋白包括9个保守的半胱氨基酸残基、BTB和ANK_REP_REGION两个结构域。分离了1238bp的上游启动子序列,序列分析表明,该基因的5’UTR区含有一个601bp的内含子,该启动子区域含有1个水杨酸作用元件、2个乙烯响应元件、2个茉莉酸甲酯响应元件、3个赤霉素响应元件、2个热胁迫响应元件、1个厌氧响应元件,1个加强转录元件。另外,还包括一些光响应元件。3.利用实时荧光定量RT-PCR(qRT-PCR)研究了植物激素SA、MeJA、ACC和生物胁迫苹果轮纹病病原菌和苹果蚜虫处理后的MhNPR1基因的表达情况。结果表明：MhNPRl在湖北海棠叶片中的表达量最大；SA在叶片、茎、根中均可以诱导该基因的表达,MeJA、ACC仅在根中诱导该基因的表达；苹果轮纹病病原菌在转录水平上未能诱导该基因的表达；苹果蚜虫在叶片和茎中可以诱导该基因的表达。4.构建了湖北海棠MhNPR1基因的植物双元表达载体,通过农杆菌介导法将其转化模式植物烟草,通过PCR和RT_PCR对其抗性株系进行检测,MhNPR1基因已经成功地插入烟草基因组中并得到了表达。转基因烟草株系中NtPR1、NtPR3和NtPR5基因的表达量得到了上调,T1代植株在苗期表现出较强的抗灰霉病能力。转基因烟草株系中抗渗透胁迫NtSPS、NtSAM1、TOBLTP、ERD10A、ERD10B、ERD10C、ERD10D和抗氧化基因NtCA、NtSOD、NtRbohD的表达量得到了上调。转基因烟草株系T1植株在种子发芽、苗期都表现出较强的抗盐性,To代植株、T1代植株在种子发芽、苗期表现出较强的抗渗透胁迫能力。总之,湖北海棠MNPR1基因在植物的防御反应中具有多重抗性。5.通过RACE技术结合电子克隆的方法克隆了湖北海棠一个bZIP类转录因子,利用生物信息学的方法对其进行序列分析。实验结果表明,该基因全长1541bp,最大开放阅读框为999bp,编码333个氨基酸,5’UTR区有191bp,3’UTR区有351bp。氨基酸序列含有典型的bZIP结构域,亮氨酸结构域,在氨基酸的C端含有YX2RL[RQ]ALSS[LS]W结构,属于典型的bZIP-D结构。系统进化树分析表明：湖北海棠MhTGA2与菜豆TGA2.1、菜豆TGA2.2、葡萄TGA2、杨树TGA2.1亲缘关系最近,聚为一类,说明我们克隆的bZIP类转录因子属于TGA2类转录因子,命名为MhTGA2, Gen Bank上的登录号为FJ598138。亚细胞定位实验表明MhTGA2蛋白定位于细胞核中。MhTGA2在叶片中的表达量最大；SA、MeJA、ACC可以诱导MhTGA2基因的表达；苹果轮纹病病原菌未能诱导该基因的表达。转MhTGA2基因烟草中NtPRs (NtPR1、NtPR2、NtPR3)以及抗逆相关基因NtSOD、NtPPO、NtPAL、NtAPX的表达量得到了提高。6.克隆了湖北海棠第1类几丁质酶基因,命名为Mhchitl。亚细胞定位研究表明该基因位于细胞膜和细胞壁。植物激素SA.MeJA.ACC可以诱导湖北海棠叶片、茎、根中Mhchitl基因的表达。苹果轮纹病病原菌可以诱导Mhchitl基因的表达,3h后表达量增加,6h后表达量达到最大,随后表达量降低。在湖北海棠叶片、茎中,苹果蚜虫可以诱导该基因的表达。将该基因构建植物表达载体,通过农杆菌介导法转化烟草。与非转基因烟草相比,转基因烟草中抗逆相关基因NtSOD.NtAPX.NtPPO,和NtPAL的表达量增加。转基因烟草株系抗灰霉病能力增加,抗PEG6000能力增强。说明Mhchot1基因不仅参与SA介导的抗病性,而且参与JA/ET介导的抗病性,具有多种抗逆功能。7.克隆了湖北海棠β-1,3-葡聚糖酶基因的全长cDNA序列和基因组DNA序列,该基因编码区含有一个内含子。该基因与桃、李和葡萄的核苷酸序列同源性为84、83、和77%,氨基酸序列同源性分别为84、74、和76%。通过基因组步移法克隆了该基因上游的启动子序列,启动子序列含有SA、MeJA和ET作用元件。利用荧光定量RT-PCR分析表明SA、MeJA和ACC均可以诱导湖北海棠叶片、茎、和根中的MhGlu基因的表达。在苹果轮纹病病原菌处理的96h内,湖北海棠MhGlu基因在24h时表达量开始上调,48h达到最大,随后降低。在湖北海棠叶片和茎中,苹果蚜虫可以诱导MhGlu基因的表达。总之,MhGlu基因是湖北海棠中抗生物胁迫的基因。8.分离了病程相关蛋白基因MhPR1、MhPR5的全编码区的cDNA和基因组DNA序列,MhPR1、MhPR5的最大开放阅读框分别为492bp、741 bp,分别编码162、246个氨基酸。MhPR5在基因编码区含有一个内含子,MhPR1没有内含子。这两个基因与苹果、梨的同源性较高,亲缘关系较近。通过与苹果基因组序列分析比较结果表明：MhPR1、MhPR5在苹果基因组中有多个拷贝,分别有4、3个拷贝。这两个基因在N端均含有一个信号肽,并且MhPR1、MhPR5分别含有6、10个保守的半胱氨基酸残基。荧光定量PCR分析结果表明MhPR1、MhPR5在湖北海棠各种组织中的表达存在差异。SA、MeJA、ACC在湖北海棠的叶、茎、和根中均可以诱导MhPR1和MhPR5基因的表达,分析表明MhPR1和MhPR5是湖北海棠SAR中的标记基因。9.以pMD19-T为中间载体,首先对其进行了改造,然后将MhTGA2、MhNPRl、和Mhchit1基因依次连接在中间载体上,命名为T2AN2AC,然后将其一并酶切,连接于植物表达载体上,成功地构建了植物三价融合表达载体,命名为MhTNC。通过农杆菌介导法转化烟草,获得转基因烟草株系。通过PCR和RT-PCR实验表明,MhTGA2、MhNPR1、和Mhchit1三个基因已经成功地插入到烟草的基因组中,并且得到了转录。与野生型株系相比,转基因烟草株系表现出早花现象,矮化、节间数显著较少,节间显著增长。并且具有抗灰霉病和抗PEG6000的能力。更多还原

【Abstract】 Malus hupehensis, originated in China, has shown strong resistance to various apple pathogens and is an important materials to study woody plant resistance mechanisms. In this study, we constructed the complete cDNA library of Malus hupehensis treated by SA. Sequences of MhNPR1, MhTGA2, and pathogens related proteins (Mhchitl, MhGlu, MhPR1, and MhPR5) were isolated from this library. Eexpressions of these genes were detected by real-time quantitative RT-PCR (qRT-PCR), and their functions were analyzed through overexpressing in transgenic tobacco plants. In additional, we established a technology system of plant trivalent fusion expression vector utilizing foot and mouth disease virus 2A sequence, and constructed the trivalent fusion expression vector MhTNC (MhTGA2-2A-MhNPR1-2A-Mhchitl)We preliminarily analysised its function. The main results are as follows:1. In this experiment, total RNA was extracted from leaves of Malus hupehensis treated with SA by improved CTAB method and mRNA was purified. The full-length cDNA library was constructed using the SMARTTM PCR cDNA Synthesis Kit. The results showed that the total RNA was non-degradable, non-polluting. mRNA dispersion were mainly concentrated in the 500～2000 bp, and there was no rRNA residues. Dispersion of ds cDNA was mainly distributed between the 300 and 2000bp. PCR fragment size was between 200 and 2000bp, indicating that the quality of ds cDNA synthesized was better and we successfully constructed a full-length cDNA library.2. The cDNA sequence of M.hupehensis NPR1 has an entire coding region of 1761 bp, and was named MhNPRl (GenBank sequence accession number FJ5981431). Its corresponding genomic DNA sequence was 2259 bp, and GenBank sequence accession number was GUI83100. Sequence comparison results showed that the gene contains three introns and four exons, the same with the Arabidopsis AtNPR1 and rice OsNPRl. The amino acids included 9 conserved cysteine residues, BTB and ANK_REP_REGION structure domains.1238 bp of upstream promoter sequence was isolated, and sequence analysis showed that the gene contains an intron of 601bp in 5’UTR. Promoter region of this gene contains a salicylic acid component, two ethylene response elements, two methyl jasmonate response elements, three gibberellin response elements, two heat stress response elements, an anaerobic response element, an enhanced transcription component, and a number of light response elements.3. Expression pattern of MhNPRl gene in M. hupehensis treated by plant hormones (SA, MeJA, and ACC), biotic stress (apple ring rot pathogen (Botryosphaeria berengeriana) and apple aphid(Aphis citricota)) were analyzed through qRT-PCR. The results showed that the expression level in leaf was higher than that in stem and root. S A could induce the expression of MhNPR1 gene in leaves, stems, and roots in M. hupehensis. And MeJA and ACC only induce the expression of this gene in roots. However, apple ring spot pathogens could not induce the expression at the transcription level. Apple aphid could induce the expression of this gene in leaves and stem of M. hupehensis.4. A plant binary expression vector of MhNPR1 was constructed. The vector was transformed into tobacco through the method of Agrobacterium-mediated. PCR and RT-PCR results showed that MhNPR1 gene was inserted into the genomic DNA of tobacco, and was expressed successfully in transgenic tobacco plants. Compared to wide type tobacco plants, the expression of PR1, PR3 and PR5 gene were upregulated in transgenic tobacco plants. T1 generation of transgenic tobacco plants showed strong resistance to Botrytis cinerea at the seedling stage. The expression level of osmotic stress related genes, such as NtSPS, NtSAMl, NtTOBLTP, NtERD10A, NtERD10B, NtERD10C, and NtERD10D, and antioxidant related gene such as NtCA, NtSOD, and NtRbohD were up-regulated in transgenic tobacco lines. T1 line of transgenic tobacco plants showed strong tolerance to salt at the stage of seed germination, seedling. To generation of transgenic tobacco plants, T1 generation of transgenic tobacco plants in seed germination, seedling showed strong resistance to osmotic stress. In short, MhNPR1 gene has multiple resistances to defense response in M. hupehensis.5. A bZIP transcription factor gene was cloned by RACE technology combined with in silico cloning. The full-length cDNA sequence of this gene is 1541bp, which includes 191bp of the 5’UTR, an ORF of 999bp, and 351bp of 3’UTR. The deduced amino acid sequence of MhTGA2 is 333 amino acids. MhTGA2 contain a typical bZIP domain, leucine domain, YX2RL [RQ] ALSS [LS] W structure domain at the C terminal of the protein. Phylogenetic analysis showed that MhTGA2 close with the bean TGA2.1, beans TGA2.2, grapes TGA2, and poplar TGA2.1, indicating that we isolated a bZIP transcription factor transcription factor, named MhTGA2, and GenBank accession number is FJ598138. MhTGA2 localize in the nucleus. The expression of MhTGA2 gene was higher in leaf than that in stem and root. And accumulation of MhTGA2 gene was enhanced in M. hupehensis after treatment with plant hormones SA, MeJA, and ACC. however, apple ring rot pathogen failed to induce the gene expression. PRs (NtPR1, NtPR2, and NtPR3) and stress related gene (NtSOD, NtPPO, NtPAL, NtAPX) were upregulated in tobacco plants of overexpression of MhTGA2 gene, comparing with the WT plants.6. Mhchitl, a class I chitinase gene from Malus hupehensis, was cloned, and its expression and function in seedlings were observed. Treatment with SA, MeJAand ACC resulted in the elevation of Mhchitl transcript levels in leaves, stems and roots. Infection with B. berengeriana caused an accumulation of Mhchitl transcripts, with maximum levels at 6 h post-inoculation. Mhchitl expression was also induced by the A. citricota. Transgenic tobacco plants that over-expressed Mhchitl showed enhanced resistance to B. cinerea, relative to wild type control plants, and were not susceptible to PEG. In addition, transcript levels for NtSOD, NtAPX, NtPPO and NtPAL were up-regulated in the transgenic plants. These results suggest that Mhchitl is not only involved in the SA-signal pathway, but also with the JA/ET-signal pathway. Our data support the role of Mhchitl in M. hupehensis as an important part of the plant’s defense strategy, through promotion of resistance to a number of stress abiotic and biotic factors.7. MhGlu, aβ-1,3-glucanase cDNA, was cloned from Malus hupehensis by in silico cloning and validated with RT-PCR. MhGlu has an intron and possess 84,83, and 77% nucleotide identity and 84,74, and 76% amino acid identity with Prunus persica, Prunus avium, and Vitis riparia, respectively. MhGlu genomic DNA sequence and promoter sequence including the SA motif, MeJA responsive, and ET responsive elements were isolated. MhGlu expression was detected in M.hupehensis seedlings treated with SA, MeJA and ACC. qRT-PCR revealed constitutive expression of MhGlu in leaf but not in the stem and root where it was silent and induced by SA, MeJA, and ET. This result suggests that MhGlu might be involved in the SA-and the JA/ET-signaling pathways in M. hupehensis. The expression of the gene monitored in a 96 h course after inoculation with B. berengeriana. Inoculation with B. berengeriana, up-regulated MhGlu 24 h post inoculation (PI), the expression reached to maximum at 48 h, and then decline. Moreover, A. citricota could enhance MhGlu expression in the leaf and stem compared to healthy control plants. It can be concluded from the results that MhGlu is involved in resistance to biotic stress in M. hupehensis.8. Full coding region of cDNA and genomic DNA sequence of MhPRl and MhPR5 were isolated from M. hupehensis. There were 492 bp and 741 bp of ORF and 162 and 246 amino acids for MhPRl and MhPR5, respectively. MhPRl gene has not intron, but MhPR5 contains an intron. Sequence comparing with the Apple genome showed that MhPR1, MhPR5 have 4, and 3 copies, respectively. These two genes have highly homologous and closer relationship with apple and pear. Both genes contain an N terminal signal peptide, and MhPR1, MhPR5 containing 6 and 10 conservative cysteine residues. QPCR analysis results showed that the expression of MhPRl and MhPR5 in different tissues of M hupehensis were different. The accumulation of MhPRl and MhPR5 were up regulated in leaves, stem, and roots after treatment with SA, MeJA, and ACC, suggesting that MhPRl and MhPR5 are the marker genes of SAR in M. hupehensis.9. Transformation of pMD19-T vector was carried out in this paper; then, MhTGA2, MhNPRl, and Mhchitl gene were inserted into this vector in turn, named T2AN2AC. Trivalent fusion plant expression vector, named MhTNC, was constructed successfully through digested and inserted combo of three genes into the plant expression vector. Transgenic MhTNC tobacco plants were obtained through the method of Agrobacterium-mediated. Compared with wild-type strains, transgenic tobacco lines showed early flowering, dwarfing, significantly less internode number, significantly longer. And transgenic tobacco plants were higher resistance to Botrytis cinerea and PEG6000.更多还原