番茄LeCOI1基因的克隆与沉默载体的构建 摘 要：COI1(coronatine-insensitive 1)蛋白是茉莉酸信号途径的信号开关，在茉莉酸信号途径中起关键的调控作用。本研究以LeCOI1(Accession number: AY423549)序列为模板设计上、下游引物，采用反转录RT-PCR技术克隆MM番茄的LeCOI1序列。通过构建pMD18-T-LeCOI1重组载体，并转化大肠杆菌进行质粒验证，最后经序列测定，证明所克隆基因为目的基因；之后再构建烟草脆裂病毒(TRV)的pYY13-LeCOI1病毒诱导基因沉默的载体，本研究为后期深入研究番茄LeCOI1基因的功能验证提供了必要基础。
关键词：LeCOI1；反转录RT-PCR；番茄；防御应答
Cloning of LeCOI1 Gene and Vector Construction of Gene Silencing from Tomato
Abstract: COI1 (coronatine-insensitive 1) protein was a key factor in jasmonic acid(JA) signal transduction system, played the on-off switch role in JA signaling pathway. This study used the known sequence of tomato (Accession number: AY423549) as template, designed the upstream primer and downstream primer, then used the reverse transcription RT-PCR technology to clone LeCOI1 sequence from it. LeCOI1 was constructed to pMD18-T vector, and the recombinant pMD18-T-LeCOI1 vectors we constructed were successfully identified by PCR and sequencing, which proved that the cloned gene was the purpose gene. On the basis of VIGS technology, the target gene LeCOI1 was amplified by PCR as well, and then constructed the recombinant vector pYY13-LeCOI1 with tobacco rattle virus(TRV) vector pYY13. After that, the constructed vector was transformed into DH5α. Finally, the recombinant vectors pYY13-LeCOI1 were identified by PCR and sequencing, the results illustrated that the recombinant vectors were constructed successfully. The research laid foundation for deeply revealed gene functions of LeCOI1 in tomato.
Key words: LeCOI1; Reverse transcription RT-PCR; Tomato; Defense response
目 录
摘 要 1
Abstract 1
引言 2
1.材料与方法 2
1.1实验材料、试剂与仪器 2
1.1.1实验材料 2
1.1.2主要试剂 3
1.1.3主要仪器 3
1.2实验方法 3
1.2.1番茄RNA的提取、cDNA合成和LeCOI1的扩增 3
1.2.2重组pMD18-T-LeCOI1载体的构建 4
1.2.3重组载体鉴定 4
1.2.4 pYY13质粒单酶切及纯化 4
1.2.5 pYY13-LeCOI1重组载体的构建 4
1.2.6 pYY13-LeCOI1重组质粒的鉴定 5
2.结果与分析 5
2.1番茄RNA的提取、cDNA的制备和LeCOI1的扩增结果 5
2.2重组载体pMD18-T-LeCOI1的鉴定 6
2.3 pYY13单酶切电泳结果 6
2.4 pYY13-LeCOI1重组质粒的验证 7
3.讨论 7
参考文献 10
致谢 13，3331