【作者】 向征；

【导师】 王贵学； 黄俊丽；

【作者基本信息】 重庆大学 ， 植物学， 2016， 硕士

【摘要】 转录因子通过参与调控真核基因转录水平上的或转录后水平上的目的基因的表达,从而实现它对植物生长发育以及生理代谢等过程的调控作用。现目前已经报道的,有许多种类的转录因子家族参与到水稻的生长发育过程中,而MYB转录因子家族是其中最大的种类之一。MYB转录因子在结构上有共同的特征,它们的N端大约有由51~52个氨基酸残基组成的高度保守的MYB结构域。植物中根据MYB结构域的重复个数将MYB大家族主要可以分为三个亚类,即:只含有一个MYB结构域的特异群体——MYB like蛋白,含有两个重复MYB结构域的R2R3-MYB和含有三个相邻MYB重复结构域的R1R2R3-MYB。花青素作为植物重要的类群代谢产物之一,广泛存在于植物中,在植物的生长发育及抗逆性方面具有重要作用。近几年的研究表明花青素在医学上还有降低心血管疾病,关节炎、糖尿病以及癌症发病率的功效。所以对花青素合成相关的调控因子进行研究有助于通过控制花青素来改造植物的抗性以及帮助人们获得更高的抗氧化性食物。目前,在水稻中关于参与花青素合成调控的MYB基因少有报道。因此研究MYB家族对水稻花青素合成调控的影响,对阐明花青素合成机理具有重要参考价值。本研究对水稻转录因子OsMYB112进行研究,该基因全长1048 bp,编码含有251个氨基酸。OsMYB112转录因子具有非常保守的R2R3MYB结构域。进化分析结果表明,OsMYB112与其他物种的R2R3MYB亚族MYB蛋白在进化上聚为一个大分支。亚细胞定位实验结果表明,OsMYB112蛋白定位在细胞核中。通过遗传转化实验获得转基因植株,对转基因植株进行表型观察和花青素含量测定,结果表明,OsMYB112参与调控水稻花青素合成。本研究主要获得以下结果:(1)多重序列比对结果显示OsMYB112含有一个R2R3MYB亚家族特有的保守序列,进化树分析结果显示,OsMYB112和其他物种的R2R3MYB亚族蛋白在进化上聚为一个大分支;(2)通过表达模式分析,OsMYB112在分蘖期的叶片中表达量最高,对高温和机械损伤有胁迫应答,且OsMYB112蛋白定位在细胞核中;(3)对OsMYB112转录活性分析,OsMYB112有转录活性,且转录激活区在N端;(4)构建pANDA-OsMYB112干扰载体,通过遗传转化获得多个水稻转基因株系;(5)通过干扰植株表型分析,OsMYB112有影响花青素合成的功能。从上述结果中,我们发现水稻OsMYB112基因是R2R3MYB亚家族中的一员,在细胞核表达,在水稻花青素合成过程中起到关键作用。更多还原

【Abstract】 The MYB family of proteins is large, functionally diverse and represented in all eukaryotes. In plants, most MYB proteins function as transcription factors in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses. However, R2R3-MYB subfamily is the largest and most diverse transcription factors in plants. Recently, An explosion of research have reported R2R3-MYB genes involved in many biological pathways such as cell cycle and morphogenesis, meristem formation and floral and seed development, light and hormone transduction, and responses to the stress.R2R3MYB transcription factors are known to play a wide role in regulating the phenylpropanoid pathway in plants. In this study, we report isolation, cloning and characterization of an R2R3 MYB transcription factor gene OsMYB112. OsMYB112 consists of 705 bp coding sequence corresponding to 235 amino acids. Sequence alignment revealed that the N-terminal(MYB) domain of the gene shares up to 95 % similarity with subgroup 4(Sg4) members of R2R3 Myb gene family functionally known to be lignin repressors. Highly divergent C-terminal region of the gene carried an ERF-associated amphiphilic repression(EAR) motif, another characteristic of the Sg4. The gene was phylogenetically grouped closest with ZmMYB31, a known repressor of anthocyanin biosynthetic pathway genes. In this study, forward genetic analysis was used to investigte the biological functions of OsMYB112 in rice, and the findings are:(1) Cloning of full-length cDNAs encoding OsMYB112. Total RNA of various plants and tissues was isolated by Trizol(TaKaRa) according to the manual provided and was used for reverse transcription by Moloney murine leukemia virus(M-MLV).(2) Database search and phylogenetic analysis.The deduced amino acid sequence contains two highly conserved motifs in its N-terminus, which are known to be MYB DNA-binding domains R2 and R3.(3) Vector construction and rice transformation. To construct the RNAi vector, a 214-bp cDNA fragment of OsMYB112 was amplified from the cDNA clone using the primer set RNAi-OsMYB112 F and RNAi-OsMYB112 R and was inserted into the Kpn I and Sal I sites. For overexpression, the full-length cDNA of Os MYB112 amplified with the primer set 35S-OsMYB112 F and 35S- OsMYB112 R was inserted into the KpnI and SalI sites of pU1301. To construct the translational OsMYB112-GFP fusion, the coding region of OsMYB112 was amplified using the primers GFP-OsMYB112 F and GFP-OsMYB112 R. The amplified fragment was inserted into the KpnI and Sal I sites of modified pCAMBIA1301-GFP to create 35S:: OsMYB112-GFP.(4) Subcellur localization. The resulting 35S:: OsMYB112-GFP fusion construct was used for transient expression in onion epidermal cells following Zhang[1]. The location of the introduced genes in the onion cells was observed using confocal laser scanning microscopy.(5) Anthocyanin measurement. The relative total content of rice anthocyanin was determinated. The results show that the relative total content of anthocyanin in R2 line and R6 line are 17% and 51% lower than that in wild type, respectively.更多还原