本文關(guān)鍵詞：番茄GRAS轉(zhuǎn)錄因子家族的鑒定及SlGRAS24基因的功能研究　出處：《重慶大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 番茄 GRAS轉(zhuǎn)錄因子 表達(dá)模式 SlGRAS24 激素互作 雄蕊發(fā)育

【摘要】：GRAS蛋白是一類植物特有的轉(zhuǎn)錄因子,廣泛參與了植株發(fā)育、分生組織維持、根輻射形態(tài)建成、光信號(hào)轉(zhuǎn)導(dǎo)、植物激素信號(hào)轉(zhuǎn)導(dǎo)和脅迫響應(yīng)等生理過程。目前,對(duì)該基因家族成員的鑒定工作已相繼在擬南芥、水稻、楊樹、大白菜、松樹、果梅和葡萄等幾個(gè)物種完成。然而大部分GRAS蛋白的功能尚未闡明�；诖�,本論文主要利用重要果實(shí)模式作物番茄為材料,全面鑒定和分析了該物種GRAS家族成員并對(duì)其中一個(gè)受小RNA(miR171)調(diào)控的基因SlGRAS24的功能進(jìn)行了深入研究,主要完成了以下幾方面的工作:(1)在番茄中共鑒定出53個(gè)GRAS基因家族成員,其中19個(gè)已經(jīng)在NCBI中存在命名信息(SlGRAS1-SlGRAS17,SlDELLA,SlLS),另34個(gè)成員根據(jù)其基因序列在番茄染色體的位置信息分別命名為SlGRAS18-SlGRAS51;多序列比對(duì)顯示大多數(shù)SlGRAS蛋白C端具有典型的GRAS結(jié)構(gòu)域。通過和擬南芥、水稻、楊樹、梅中GRAS成員一起的系統(tǒng)發(fā)育樹分析表明該家族一共分為13個(gè)亞家族;5’-RACE實(shí)驗(yàn)表明其中兩個(gè)成員為miR171的靶基因:即SlGRAS24和SlGRAS40;運(yùn)用qRT-PCR技術(shù),分析了各成員在番茄中各組織器官中組織表達(dá)模式,同時(shí)分析了各成員在各激素處理和非生物脅迫條件下的響應(yīng)情況;(2)SlGRAS24超表達(dá)轉(zhuǎn)基因番茄植株表現(xiàn)出植株矮化,葉片變小,開花延遲,腋芽異位,根抑制,座果率降低等多重表型;qRT-PCR和SlGRAS24啟動(dòng)子GUS融合表達(dá)轉(zhuǎn)基因番茄(pSlGRAS24::GUS)的組織化學(xué)染色分析結(jié)果均顯示出SlGRAS24在番茄營養(yǎng)器官和生殖器官中的特異性表達(dá);(3)啟動(dòng)子序列分析顯示SlGRAS24啟動(dòng)子區(qū)含有赤霉素和生長(zhǎng)素響應(yīng)原件,外源GA3或IAA處理野生型番茄和pSlGRAS24::GUS轉(zhuǎn)基因番茄幼苗均表明SlGRAS24的明顯響應(yīng);SlGRAS24超表達(dá)幼苗中大多數(shù)赤霉素和生長(zhǎng)素相關(guān)基因均下調(diào),且這些基因?qū)A3或IAA的響應(yīng)情況具有不同程度的改變;HPLC-MS/MS結(jié)果表明SlGRAS24超表達(dá)番茄籽苗中內(nèi)源GA3和IAA呈顯著增加;激素敏感性實(shí)驗(yàn)進(jìn)一步表明SlGRAS24同時(shí)參與了赤霉素和生長(zhǎng)素信號(hào)路徑;(4)花粉互交實(shí)驗(yàn)、TCC花粉染色實(shí)驗(yàn)和花器官組織學(xué)切片分析表明SlGRAS24超表達(dá)植株中果實(shí)座果率低,果實(shí)和種子發(fā)育被抑制主要是由于SlGRAS24超表達(dá)造成花粉發(fā)育異常所引起;同時(shí)對(duì)野生型和轉(zhuǎn)基因盛花RNA-seq測(cè)序分析發(fā)現(xiàn)數(shù)個(gè)花粉發(fā)育、激素信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因和轉(zhuǎn)錄因子的表達(dá)變化;對(duì)果實(shí)初期發(fā)育不同時(shí)間果皮切片觀察和相關(guān)基因的定量分析表明,SlGRAS24超表達(dá)植株果實(shí)細(xì)胞的分裂和伸長(zhǎng)均受到抑制。綜上,該論文首次全面分析了重要果實(shí)作物番茄中GRAS轉(zhuǎn)錄因子家族成員及其表達(dá)情況,這為我們深入了解GRAS家族基因的進(jìn)化特征,鑒定GRAS家族基因的生理功能具有重要意義,也為以后利用基因工程手段改良番茄或者其他茄科作物的農(nóng)業(yè)性狀提供了基因資源。此外,通過轉(zhuǎn)基因株系的表型觀察和分析,發(fā)現(xiàn)該家族成員SlGRAS24參與了生長(zhǎng)素-赤霉素兩種植物激素的互作,且在番茄晚期雄蕊發(fā)育過程中起重要作用,加深了我們對(duì)這兩種生理過程的分子機(jī)制的認(rèn)識(shí)。
[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.

【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 李桂英;田玉富;楊成君;;植物GRAS家族轉(zhuǎn)錄因子的研究現(xiàn)狀[J];安徽農(nóng)業(yè)科學(xué);2014年14期

2 孫欣;王晨;房經(jīng)貴;慕茜;王西成;;葡萄GRAS基因家族生物信息學(xué)分析[J];江西農(nóng)業(yè)學(xué)報(bào);2011年07期

3 ;Proteomics Study of COI1-regulated Proteins in Arabidopsis Flower[J];Journal of Integrative Plant Biology;2010年04期

4 李潔;植物轉(zhuǎn)錄因子與基因調(diào)控[J];生物學(xué)通報(bào);2004年03期

5 劉強(qiáng),張貴友,陳受宜;植物轉(zhuǎn)錄因子的結(jié)構(gòu)與調(diào)控作用[J];科學(xué)通報(bào);2000年14期

，

本文編號(hào)：1356543