番茄mir172的抗病功能研究生物化学与分子生物学专业论文.docxVIP

研究生优秀毕业论文 大连理工大学硕士学位论文摘 大连理工大学硕士学位论文 摘 要 MicroRNA(miRNA)作为一类生物体内普遍存在的长度为21—25个核苷酸的内源 性非编码小分子RNA，在植物抵抗病原物过程中起着至关重要的调控作用。miRl72作 为植物体内一类保守的miRNA，其所发挥的抗病功能已在多个物种中被相继报道，而 在番茄中却相对较少，更多研究亟待深入。据此，本研究利用分子生物学的技术手段， 将番茄miRl72家族的两个成员即miRl72a和miRl72b的前体基因序列分别在番茄中过 量表达，探究了miRl72在番茄抗病过程中的功能。 首先，本研究利用生物信息学的方法分析了番茄miRl72a和miRl72b的序列特性， 并对二者共同的靶基因进行预测。结果表明：miRl72a和miRl72b的前体序列不同而成 熟体序列相同，共同靶向APETALA2(AP2)乙烯响应转录因子，且miRl72和miRl72b 的基因上游启动子区均含有与生物胁迫相关的顺式作用元件。 其次，本研究利用实时定量PCR技术分析了病原菌和激素处理下，番茄miRl72a 和miRl72b的成熟体、前体及靶基因的表达特性。结果表明Pre—MIRl72a和Pre—MIRl72b 均能对上述胁迫条件作出响应，且它们的成熟体即miRl72与靶基因的表达水平呈负相 关。 为了进一步研究miRl72的抗病功能，本研究将miRl72a和miRl72b的前体基因序 列分别导入番茄中，并对转基因番茄的抗病性进行检测。结果表明：过表达MIRl72a 和MIRl 72b可有效提高番茄对病原菌的抗性。 本研究从miRNA的角度探究了番茄病害可能的防治途径，并首次利用过表达技术 系统地分析了番茄miRl72的抗病功能，为今后全面解析番茄体内miRNA的功能奠定 理论基础。 关键词：番茄；miRl72；AP2；病原茵 万方数据 Functional Functional Analysis of Tomato miR1 72 in Disease Resistance Abstract MicroRNA(miRNA)iS a class of non—coding RNA molecules with 2 l一25 nucleotide in length，and play vital roles in the interaction between tomato and pathogen．MiR 1 72 is highly conserved plant miRNA family，its function on disease defense has been well studied in various plants，unfortunately，few research in tomato．Therefore，we over-expressed the precursor gene of miRl 72a and miRl 72b in tomato by using the molecular biology technologies in the study，and analyzed their function and acting mechanism in tomato disease defense． Firstly，the sequence characteristics of tomato miRl 72a and miR 1 72b were analyzed and target genes were predicted by using of the bioinformatics．The results showed that miRl 72a and miRl 72b had different precursor but same mature miRNA sequence．They targeted a class of AP2 ethylene-response transcription factor．In addition，analysis of upstream sequences indicated that there are a lot of cis-elements which related to biotic stress in their promoter regions． Secondly，qRT-PCR was performed to determine the expression pattem of individual miR 1 72 genes(precursor and mature miRNA