【作者】 宋锐；

【导师】 潘光堂；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2009， 硕士

【摘要】 microRNA(miRNA)是广泛存在于真核生物细胞内的一大类内源性的、18-25 bp的小分子非编码RNA(non-coding RNA,ncRNA),在多细胞生物的基因表达调节和控制中扮演着十分重要的角色,并参与植物对外界环境压力胁迫的应答和病害防御。纹枯病真菌性病害是西南玉米主产区生产上的主要病害之一。研究miRNA在玉米纹枯病抗性中的角色与机制对于培育抗纹枯病玉米品种具有重要的理论与实践研究意义。本研究通过拟南芥、水稻等植物中发现的miRNA与玉米EST和GSS数据库进行比对搜索,分析玉米中可能存在的同源miRNA,并利用miRU软件在线预测靶基因。对纹枯病抗性自交系R15和感病自交系掖478在人工接种的条件下,采用半定量RT-PCR和实时荧光定量PCR(QPCR)方法,对获选miRNA进行抗纹枯病组织特异性表达验证,探讨了发掘与鉴定玉米抗真菌病害调控相关miRNA策略与方法,获得了6个抗病相关的候选miRNA。主要研究结果如下:1.从miRBase数据库下载拟南芥、水稻等植物中已发现的miRNA,利用生物信息学手段共获得了31条物种间保守的玉米同源miRNA序列,并利用miRU预测其靶基因。2.通过半定量RT-PCR的方法检测31条miRNA与miR168的前体在病菌胁迫下R15(耐)和掖478(感)中的表达情况,其中10条miRNA的前体在R15和掖478中均能获得RT—PCR阳性条带,并通过PCR产物的回收、克隆及测序证明序列的真实性;其中5条miRNA前体序列在两材料间的组织表达存在一定的差异,分别为miR168-1、miR168-2、miR165、miR173和miR845,推测5条miRNA可能与玉米纹枯病胁迫表达调控有关。3.通过改进的加尾-引物延伸实时荧光定量PCR法,对在前体水平上在两材料中有明显差异表达的miRNA(miR165、miR168b、miR168b~*、miR173)以及现已发现的与玉米抗病相关的miRNA(miR171和miR319),设计并合成相关引物进行成熟miRNA的PCR扩增和表达验证。对上述6条miRNA成熟分子的表达定量结果发现:接菌处理下玉米耐病材料R15和感病材料掖478中miR165、miR168b、miR168b~*和miR173有一定的上调表达趋势,而miR171和miR319则下调表达。并且全部6条miRNA分子在接菌12h后耐感材料间叶鞘组织中的表达量存在显著差异,叶片中虽存有类似差异,但差异较小。4.通过植物miRNA靶基因预测软件miRU分析结果表明,miR165、miR173、miR168b、miR168b~*、miR171和miR319等6个耐感材料组织差异表达的miRNA分子的靶基因多与植物的抗逆性有关。具体包括:病原菌毒素相关的多聚半乳糖醛酸酶(PG);植物细胞壁相关的纤维素合成酶、富含羟脯氨酸糖蛋白(HRGP);植物抗病反应信号传导相关的泛素酶、植物激素调控因子载体蛋白、丝氨酸/苏氨酸磷酸酶、丝氨酸/苏氨酸激酶、PP-2C类蛋白磷酸酶等;以及谷胱甘肽-S-转移酶、压迫诱导蛋白和创伤诱导蛋白等抗逆相关蛋白。5.综合本研究与本课题前期研究结果,推测miRNA在玉米抗纹枯病过程中可能存在以下调控机制:(1)当玉米受到真菌病原菌入侵时,miR168b~*,miR173可能通过上调表达参与降解病原菌分泌的毒素及酶类,从而抑制病原菌的侵害;(2)miR165,miR168,miR173通过上调表达从而负调控靶基因丝氨酸/苏氨酸磷酸酶,PP-2C类磷酸酶等蛋白磷酸酶下调表达;miR171和miR319则下调表达使丝氨酸/苏氨酸磷酸酶等蛋白激酶得到释放,从而共同打破蛋白激酶/蛋白磷酸酶的平衡,使得蛋白激酶大量表达,导致蛋白磷酸化级联放大,启动与上调防卫基因表达,抑制病原菌的侵害和生长。(3)miR171和miR319还可能通过下调表达,使谷胱甘肽-S-转移酶、压迫诱导蛋白和创伤诱导蛋白等抗逆相关蛋白大量表达,使植物对病原菌入侵进行抗病反应。更多还原

【Abstract】 microRNAs(miRNAs) is widely present in eukaryotic cells of a large class of endogenous,18-25 bp length of the non-coding small RNA(non-coding RNA,ncRNA).It plays an important role in multicellular organisms to regulate and control gene expression,and work in response to the outside plant environmental stress and disease stress defense.Sheath blight and other fungal diseases of the hazard are one of the major diseases of the main maize growing areas southwest of the production.It is an important study of the meaning of theory and practice for sheath blight of maize resistance varieties to cultivate by researching miRNAs in the role of sheath blight resistance and mechanisms.Downloaded from the miRBase database of Arabidopsis,rice and other plants have been discovered miRNAs,miRNAs were predicted in the maize by bioinformatics homologous blast Methods,their target genes were predicted using miRU online prediction software.In conditions of artificial inoculation of inbred lines R15(R) and Ye 478(S),using semi-quantitative RT-PCR and real-time fluorescence quantitative PCR(QPCR) method,it is analysised the expression of miRNA under the conditions of resistant to sheath blight.Excavation and identification of anti-fungal diseases of maize related to miRNAs regulation and control strategies and methods,access to a number of maize candidate miRNAs.The main findings are as follows:1.Downloaded from the miRBase database of Arabidopsis,rice and other plants have been discovered miRNAs,a total of 31 miRNAs were predicted in the maize by bioinformatics homologous blast Methods,their target genes were predicted using miRU online prediction software.2.Through semi-quantitative RT-PCR method to detect the expression of precursor of 31 of miRNAs and the miR168 in R15(resistant disease) and Ye 478(susceptible) under bacteria stress:the 10 miRNA precursors can be obtained RT-PCR-positive bands in Ye 478 and R15,through the recovery of PCR products,cloning and sequencing to prove the authenticity of sequence;the expression of the 5 miRNAs precursor sequences(miR168-1, miR168-2,miR165,miR173 and miR845) is a certain difference between the two tissue materials,it may be related to maize self-expression and regulation under sheath blight bacteria stress.3.By improving the tail-primer-extension real-time fluorescence quantitative PCR method,Level of precursor materials in two significant differences in the expression of miRNA molecule(miR165,miR168b,miR168b(~*),miR173),and has been found associated with maize miRNAs resistance elements(miR171 and miR319),Designed and synthesized the relevant primers for PCR of mature miRNAs molecular amplification and expression of authentication.The expression of 6 mature miRNAs and quantitative:In disease resistant maize material R15 and susceptible materials Ye 478,miR165,miR168b,miR168b(~*) and miR173 were an Up-regulated expression,miR171 and miR319 were down-regulated expression.The expression of all 6 miRNAs was a significant difference in 12h tissue sheath treatment between esistant/susceptible materials,although the leaves have a similar difference, but the difference was smaller.4.Through the plant miRNA target gene prediction software miRU,it is found that 6 miRNAs’ targets are related to the resistance of the plant:Multi-pathogen-associated polygalacturonase enzyme(PG);plant cell wall-related cellulose synthase,hydroxyproline rich glycoprotein(HRGP);plant disease resistance signal transduction related ubiquitin-enzymes,Auxin effiux carrier protein,serine/threonine phosphatase,serine/ threonine kinase,PP-2C-like protein phosphatase;and glutathione-S-transferase,Stress inducible protein and Wound-induced basic protein.5.According to a large number of our group research and the results of this study,that miRNAs in maize may control the following resistance mechanisms:(1) When the maize by fungal pathogens invasion,Up-regulated expression of miR168b~*,miR173 may be involved in the degradation of pathogenic bacteria secrete toxins and enzymes to inhibite against pathogens;(2) up-regulated expression of miR165,miR168,miR173 through negative regulation of target gene serine/threonine phosphatase,PP-2C-type phosphatase enzymes reduced the expression of protein phosphatase;miR171 and miR319 were down-regulated expression so that serine/threonine protein kinase phosphatase enzymes to be released, breaking the protein kinase/phosphatase balance,making a large number of protein kinase expression,leading to protein phosphorylation cascade,start with the increase of defense gene expression to inhibit the growth of pathogenic bacteria;(3) Down-regulated expression of miR171 and miR319 may be induce the expression of glutathione-S-transferase,Stress inducible protein and Wound-induced basic protein,so that the whole plant defense against pathogen invasion.更多还原