番茄miR828靶基因验证及SlyMYB7-Like功能探究
发布时间：2018-12-14 01:19
【摘要】：MicroRNA是广泛存在于真核生物中的一类内源性非编码小RNA。植物miRNA王要通过剪切靶基因在转录后水平调控植物的生长发育、抗逆抗病和次生代谢等生命活动。靶基因的鉴定及功能分析是研究miRNA生物学功能的关键。miR828是新近发现的一种保守miRNA,目前的研究多集中在拟南芥中花青素的生物合成领域。本试验以番茄为材料,首先采用RLM-5'Race技术成功鉴定出SlyMYB7-Like是番茄miR828的靶基因。为了更好的诠释番茄miR828的功能,试验中运用生物信息学、qRT-PCR、拟南芥转基因和干旱胁迫等方法初步探究了SlyMYB7-Like的基因的表达模式及功能。生物信息学分析结果显示：SlyMYB7-Like基因编码R2R3型MYB类转类因子,与拟南芥MYB7,金鱼草MYB330,丹参PAP等具有较高的同源性。qRT-PCR分析结果显示：SlyMYB7-Like基因在果和芽中的表达量最高,茎中次之,花和叶中的含量较低,根中最低。过表达SlyMYB7-Like转基因拟南芥研究结果显示：和野生型拟南芥相比,转基因拟南芥植株颜色发紫,花青素含量及花青素合成途径中关键酶基因的表达量上升,暗示SlyMYB7-Like可能在花青素生物合成中起一定的作用。拟南芥干旱胁迫结果显示：转基因拟南芥中离体叶片失水率、丙二醛含量低于野生型,过氧化氢酶、过氧化物酶活性均高于野生型,表明SlyMYB7-Like过量表达可以增强转基因拟南芥对干旱胁迫的耐受性。靶基因的鉴定为后期研究番茄miR828的生物学功能奠定一定的基础。SlyMYB7-Like正调控花青素的功能为研究番茄花青素调控网络提供一定的理论基础,对提高番茄的营养与药用价值具有一定的实际意义。SlyMYB7-Like的抗旱特性对挖掘和利用番茄优异抗旱基因有一定的指导意义。
[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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S641.2;Q943.2
，
本文编号：2377626
[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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S641.2;Q943.2
，
本文编号：2377626
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