【摘要】：MicroRNA是廣泛存在于真核生物中的一類內(nèi)源性非編碼小RNA。植物miRNA王要通過剪切靶基因在轉(zhuǎn)錄后水平調(diào)控植物的生長發(fā)育、抗逆抗病和次生代謝等生命活動。靶基因的鑒定及功能分析是研究miRNA生物學(xué)功能的關(guān)鍵。miR828是新近發(fā)現(xiàn)的一種保守miRNA,目前的研究多集中在擬南芥中花青素的生物合成領(lǐng)域。本試驗以番茄為材料,首先采用RLM-5'Race技術(shù)成功鑒定出SlyMYB7-Like是番茄miR828的靶基因。為了更好的詮釋番茄miR828的功能,試驗中運(yùn)用生物信息學(xué)、qRT-PCR、擬南芥轉(zhuǎn)基因和干旱脅迫等方法初步探究了SlyMYB7-Like的基因的表達(dá)模式及功能。生物信息學(xué)分析結(jié)果顯示：SlyMYB7-Like基因編碼R2R3型MYB類轉(zhuǎn)類因子,與擬南芥MYB7,金魚草MYB330,丹參PAP等具有較高的同源性。qRT-PCR分析結(jié)果顯示：SlyMYB7-Like基因在果和芽中的表達(dá)量最高,莖中次之,花和葉中的含量較低,根中最低。過表達(dá)SlyMYB7-Like轉(zhuǎn)基因擬南芥研究結(jié)果顯示：和野生型擬南芥相比,轉(zhuǎn)基因擬南芥植株顏色發(fā)紫,花青素含量及花青素合成途徑中關(guān)鍵酶基因的表達(dá)量上升,暗示SlyMYB7-Like可能在花青素生物合成中起一定的作用。擬南芥干旱脅迫結(jié)果顯示：轉(zhuǎn)基因擬南芥中離體葉片失水率、丙二醛含量低于野生型,過氧化氫酶、過氧化物酶活性均高于野生型,表明SlyMYB7-Like過量表達(dá)可以增強(qiáng)轉(zhuǎn)基因擬南芥對干旱脅迫的耐受性。靶基因的鑒定為后期研究番茄miR828的生物學(xué)功能奠定一定的基礎(chǔ)。SlyMYB7-Like正調(diào)控花青素的功能為研究番茄花青素調(diào)控網(wǎng)絡(luò)提供一定的理論基礎(chǔ),對提高番茄的營養(yǎng)與藥用價值具有一定的實際意義。SlyMYB7-Like的抗旱特性對挖掘和利用番茄優(yōu)異抗旱基因有一定的指導(dǎo)意義。
[Abstract]:MicroRNA is a kind of endogenous non-coding small RNA. widely found in eukaryotes. MiRNA target genes are used to regulate plant growth and development, stress resistance and secondary metabolism at post-transcriptional level. The identification and functional analysis of target genes are the key to study the biological function of miRNA. MiR828 is a newly discovered conservative miRNA,. The current research focuses on the biosynthesis of anthocyanins in Arabidopsis thaliana. In this experiment, tomato was used as material. Firstly, SlyMYB7-Like was identified as the target gene of tomato miR828 by RLM-5'Race technique. In order to better explain the function of tomato miR828, bioinformatics, qRT-PCR, transgenic and drought stress were used to explore the expression pattern and function of SlyMYB7-Like gene. Bioinformatics analysis showed that the SlyMYB7-Like gene encodes R2R3 type MYB transcription-like factor, which is associated with MYB330, of MYB7, in Arabidopsis thaliana. The results of qRT-PCR analysis showed that the expression of SlyMYB7-Like gene was the highest in fruit and bud, followed by stem, lower in flower and leaf, and lowest in root. The results of overexpression of SlyMYB7-Like in transgenic Arabidopsis thaliana showed that compared with wild-type Arabidopsis thaliana, transgenic Arabidopsis plants were purple in color, anthocyanin content and expression of key enzyme genes in anthocyanin synthesis pathway were increased. It suggests that SlyMYB7-Like may play a role in anthocyanin biosynthesis. The results of drought stress in Arabidopsis thaliana showed that the rate of water loss and malondialdehyde content in leaves of transgenic Arabidopsis thaliana were lower than those of wild type, and the activities of catalase and peroxidase were higher than those of wild type. It was suggested that overexpression of SlyMYB7-Like could enhance the tolerance of transgenic Arabidopsis thaliana to drought stress. The identification of target gene lays a foundation for studying the biological function of tomato miR828. The function of SlyMYB7-Like regulating anthocyanins provides a theoretical basis for studying the regulatory network of tomato anthocyanin. It has certain practical significance to improve the nutrition and medicinal value of tomato, and the drought resistance characteristic of SlyMYB7-Like has certain guiding significance to excavate and utilize the excellent drought-resistant genes of tomato.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S641.2;Q943.2
，

本文編號：2377626