番茄GRAS转录因子家族的鉴定及SlGRAS24基因的功能研究

发布时间：2017-12-30 21:16

本文关键词：番茄GRAS转录因子家族的鉴定及SlGRAS24基因的功能研究　出处：《重庆大学》2016年博士论文　论文类型：学位论文

【摘要】：GRAS蛋白是一类植物特有的转录因子,广泛参与了植株发育、分生组织维持、根辐射形态建成、光信号转导、植物激素信号转导和胁迫响应等生理过程。目前,对该基因家族成员的鉴定工作已相继在拟南芥、水稻、杨树、大白菜、松树、果梅和葡萄等几个物种完成。然而大部分GRAS蛋白的功能尚未阐明。基于此,本论文主要利用重要果实模式作物番茄为材料,全面鉴定和分析了该物种GRAS家族成员并对其中一个受小RNA(miR171)调控的基因SlGRAS24的功能进行了深入研究,主要完成了以下几方面的工作:(1)在番茄中共鉴定出53个GRAS基因家族成员,其中19个已经在NCBI中存在命名信息(SlGRAS1-SlGRAS17,SlDELLA,SlLS),另34个成员根据其基因序列在番茄染色体的位置信息分别命名为SlGRAS18-SlGRAS51;多序列比对显示大多数SlGRAS蛋白C端具有典型的GRAS结构域。通过和拟南芥、水稻、杨树、梅中GRAS成员一起的系统发育树分析表明该家族一共分为13个亚家族;5’-RACE实验表明其中两个成员为miR171的靶基因:即SlGRAS24和SlGRAS40;运用qRT-PCR技术,分析了各成员在番茄中各组织器官中组织表达模式,同时分析了各成员在各激素处理和非生物胁迫条件下的响应情况;(2)SlGRAS24超表达转基因番茄植株表现出植株矮化,叶片变小,开花延迟,腋芽异位,根抑制,座果率降低等多重表型;qRT-PCR和SlGRAS24启动子GUS融合表达转基因番茄(pSlGRAS24::GUS)的组织化学染色分析结果均显示出SlGRAS24在番茄营养器官和生殖器官中的特异性表达;(3)启动子序列分析显示SlGRAS24启动子区含有赤霉素和生长素响应原件,外源GA3或IAA处理野生型番茄和pSlGRAS24::GUS转基因番茄幼苗均表明SlGRAS24的明显响应;SlGRAS24超表达幼苗中大多数赤霉素和生长素相关基因均下调,且这些基因对GA3或IAA的响应情况具有不同程度的改变;HPLC-MS/MS结果表明SlGRAS24超表达番茄籽苗中内源GA3和IAA呈显著增加;激素敏感性实验进一步表明SlGRAS24同时参与了赤霉素和生长素信号路径;(4)花粉互交实验、TCC花粉染色实验和花器官组织学切片分析表明SlGRAS24超表达植株中果实座果率低,果实和种子发育被抑制主要是由于SlGRAS24超表达造成花粉发育异常所引起;同时对野生型和转基因盛花RNA-seq测序分析发现数个花粉发育、激素信号转导相关基因和转录因子的表达变化;对果实初期发育不同时间果皮切片观察和相关基因的定量分析表明,SlGRAS24超表达植株果实细胞的分裂和伸长均受到抑制。综上,该论文首次全面分析了重要果实作物番茄中GRAS转录因子家族成员及其表达情况,这为我们深入了解GRAS家族基因的进化特征,鉴定GRAS家族基因的生理功能具有重要意义,也为以后利用基因工程手段改良番茄或者其他茄科作物的农业性状提供了基因资源。此外,通过转基因株系的表型观察和分析,发现该家族成员SlGRAS24参与了生长素-赤霉素两种植物激素的互作,且在番茄晚期雄蕊发育过程中起重要作用,加深了我们对这两种生理过程的分子机制的认识。
[Abstract]:GRAS protein is a kind of plant specific transcription factors, involved in a wide range of plant development, completion of root meristem maintenance, radiation patterns, signal transduction, signal transduction and response to stress hormones and other physiological processes. At present, the identification of the gene family members have been in Arabidopsis, rice, pine, poplar, Chinese cabbage. Mei fruit and grapes. Several species but most of the function of the GRAS protein has not been elucidated. Based on this, this paper mainly use the important fruit crop pattern of tomato, comprehensive identification and analysis of the species of GRAS family members and one of them by a small RNA (miR171) gene SlGRAS24 regulates the function of the depth the research, mainly completed the following work: (1) in tomato identified 53 members of GRAS gene family, 19 of them already exist in NCBI information (SlGRAS1-SlGRAS17, SlDELLA, named SlLS ), the other 34 members according to the gene sequence in tomato chromosome location information named SlGRAS18-SlGRAS51; multiple sequence alignment showed that most SlGRAS protein C end with a typical GRAS domain. The rice and Arabidopsis, poplar tree, GRAS, together with members of the phylogeny of plum in the analysis shows that the family is divided into 13 sub family; 5 "-RACE experiments show that the two members of miR171 target genes: SlGRAS24 and SlGRAS40; the use of qRT-PCR technology, analyzed the tissue expression pattern in different tissues and organs of each member in tomato, and analyzed the response of each member in the treatment of hormone and abiotic stress conditions; (2) SlGRAS24 over expression of transgenic tomato plants showed dwarfing, smaller leaves, delayed flowering bud root inhibition, decrease the rate of ectopic, multiple phenotypes; qRT-PCR and SlGRAS24 promoter GUS fusion gene expression Tomato (pSlGRAS24:: GUS) histochemical staining results showed specific expression of SlGRAS24 in tomato vegetative organs and reproductive organs; (3) promoter sequence analysis showed that the SlGRAS24 promoter region containing gibberellin and auxin response elements, exogenous GA3 or IAA and wild type tomato and pSlGRAS24:: GUS transgenic tomato seedlings SlGRAS24 showed obvious response; most of the over expression of SlGRAS24 gibberellin and auxin related genes in seedlings were reduced, and the response of these genes to GA3 or IAA with a different degree of change; HPLC-MS/MS results showed that SlGRAS24 over expression of endogenous GA3 and IAA in tomato seedlings were significantly increased; hormone sensitive experiments indicated that SlGRAS24 also participates in gibberellin and the auxin signal pathway; (4) pollen cross experiment, histology analysis showed that SlGRAS24 TCC pollen staining experiments and floral organ The expression of plant fruit low fruit, fruit and seed development was inhibited mainly due to the over expression of SlGRAS24 which caused by abnormal pollen development; flowers of wild type and transgenic Sheng and RNA-seq sequencing analysis found that the number of pollen development, expression of hormone signal transduction related genes and transcription factors; quantitative on fruit early development in different time slices of fruit observation and analysis of genes related to SlGRAS24 showed that over expression of cell division and elongation of plants were inhibited. In conclusion, this is the first comprehensive analysis of the GRAS transcription factor family member of important fruit crops in tomato and its expression, which for us to deeply understand the evolutionary characteristics of GRAS gene family, plays an important role in the physiological function of GRAS family identification genes, also for agricultural traits after using the genetic engineering methods to improve tomato or other solanaceous crops provide Gene resources. In addition, through the observation and analysis of the phenotype of transgenic lines, found that the interaction of the family members involved in the auxin SlGRAS24 gibberellin two kinds of plant hormones, which play an important role in the process of development of tomato late stamen, has deepened our understanding of the molecular mechanisms of the two kinds of physiological processes.

【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

【参考文献】