杨树miR393及其靶基因相互作用研究毕业论文

 2021-04-12 11:04

摘 要

miRNA不仅在植物生长发育、新陈代谢、组织分化、响应各种逆境胁迫等过程中起到调控作用。大多数情况下,植物miRNA作用的靶标主要是一些在植物生长发育过程中起作用的转录调控因子。

为了验证杨树中miR393a和其预测靶基因AFB2-3间是否也存在这一相互作用,采用PCR诱变的大引物法对AFB2-3进行多位点突变,利用GATEWAY技术构建表达载体p2GWF7.0- AFB2-3 和p2GWF7.0- AFB2-3M。然后共转化杨树原生质体。共转化24小时后,在荧光显微镜下,采用450~490nm蓝光波长激发GFP荧光,通过观察绿色荧光蛋白eGFP的表达情况,研究miRNA与其靶基因间的相互作用。

实验结果表示,通过对AFB2-3上作用靶标的多位点突变,可以有效地解除miR393a对于其靶基因AFB2-3的表达抑制,验证了对AFB2-3miR393a作用靶标的预测,故该方法对于靶标鉴定确实简单有效。

关键词:杨树;miRNA 393aAFB2-3; 靶基因鉴定

The research on the interaction between poplar miR393a and it's target gene AFB2-3

ABSTRACT

miRNA is involved in multiple biological functions of plant, such as plant development, hormones single transduction, and environmental stimuli. Many research showed that miRtargets transcription factor genes.

To confirm the interaction between miR393a and its predicted target gene, AFB2-3, in poplar, a Site-directed mutant sequence of AFB2-3 was cloned, named AFB2-3M with PCR-based megaprimer method for site-director mutagenesis. Using gateway technology, two plant expression vectors:p2GWF7.0- AFB2-3 and p2GWF7.0- AFB2-3M were constructed and co-transferred in poplar protoplasts to observe the florescence of eGFP 24h later under blue light between 450~490nm.

As a result, we confirmed the interaction between miR393a and AFB2-3 and confirmed the simple and intuitive method for miRNA target detection in plant cell.

key words: Poplar ; miRNA 393aAFB2-3; ; Target gene identification;

目 录

1.文献综述.....................................................................................................................................01

1.1 植物miRNA.......................................................................................................................01

1.2 植物miRNA的起源与进化..............................................................................................02

1.3 植物miRNA靶基因分析方法..........................................................................................02

1.4 植物miRNA与非生物胁迫响应......................................................................................02

1.5 本研究的目的意义与方法.................................................................................................03

2.实验材料………………………………………………………………………………………04

2.1植物材料………………………………………………………………………………….04

2.2菌株及载体……………………………………………………………………………….04

2.3实验试剂………………………………………………………………………………….04

2.4缓冲液及培养基………………………………………………………………………….05

2.5仪器设备………………………………………………………………………………….05

3.实验方法………………………………………………………………………………………06

3.1基因编码区克隆及载体构建…………………………………………………………...06

3.1.1 基因编码区克隆………………………………………………………………....07

3.1.2 目的片段连接入门载体…………………………………………………………07

3.1.3 载体构建…………………………………………………………………………08

3.2 靶基因AFB2-3的多位点突变…………………………………………………………09

3.3 杨树原生质体分离与转化……………………………………………………………..09

2.3.1 杨树叶肉原生质体分离…………………………………………………………10

4.结果与分析……………………………………………………………………………………11

4.1 靶基因的多位点突变…………………………………………………………………..12

4.2 基因表达载体的构建…………………………………………………………………..12

4.3 miR393a基因与靶基因AFB2-3的相互作用………………………………………….13

结 论……………………………………………………………………………………………..14

致 谢……………………………………………………………………………………………..15

参考文献…………………………………………………………………………………………16

1. 文献综述

1.1 植物miRNA

植物的发育、代谢和胁迫反应等一系列功能的正常发挥都依赖于基因表达的合理调控。除转录水平的基因调控之外,大量数据表明micro RNA ( 简称miRNA) 参与的转录后调控也不可缺少。miRNA 是一类小分子的非编码RNA,可通过对其靶基因mRNA 的降解或翻译抑制调控基因表达[1]

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