番茄花青素缺失基因Hoffman’s Anthocyaninless的图位克隆及功能分析

发布时间：2018-05-15 00:19

本文选题：番茄 + 花青素　；参考：《中国农业科学院》2016年博士论文

【摘要】：花青素是植物体内一类重要的次生代谢物质,不仅能吸引昆虫传粉和协助种子传播,还能提高植物对生物和非生物胁迫的抵抗能力。近年来,在拟南芥、玉米和矮牵牛中已克隆了大量的参与花青素合成调控的转录因子,但番茄中研究相对滞后,且都集中在R2R3-MYB型转录因子。为此,本研究通过图位克隆的方法在番茄中克隆了第一个参与花青素合成调控的b HLH型转录因子AH,并结合低温诱导、低温冷害试验以及转录组试验研究了AH基因的功能及其对提高番茄幼苗耐低温的作用,旨在为番茄花青素合成调控机理和番茄耐低温育种研究提供参考依据。主要结果如下:1.FMTT271中花青素缺失表型受一个单隐性基因控制,且与AH基因为等位基因,因此,将FMTT271中花青素缺失基因命名为ah。应用图位克隆的方法,我们将ah基因定位于番茄9号染色体长臂CAPS标记CAPS2和CAPS4之间,其物理距离约为130kb,根据番茄参考基因组注释信息(ITAG2.4),该区间共有5个ORFs。2.根据同源分析结果,结合基因表达分析,推测ORF5(Solyc09g065100)为AH基因。c DNA全长测序结果表明,ah突变体中Solyc09g065100基因的第6个外显子上一个碱基(G到T)的突变,使Gly突变成终止密码子,导致Solyc09g065100基因翻译提前终止。将Solyc09g065100基因在ah突变体FMTT271中过量表达,转基因番茄T1均能够互补FMTT271的表型,且叶片和果实中均大量积累花青素。以上试验结果表明Solyc09g065100即为AH。结合q RT-PCR分析结果及转录激活试验,我们推测AH可能通过上调花青素合成途径中晚期结构基因的表达促进花青素的合成。3.AH基因受发育调控,进而下调花青素合成途径中结构基因的表达,抑制花青素的合成。此外,低温可能通过激活AH的表达进而促进番茄叶片中花青素的积累。二氨基联苯胺(DAB)和台盼蓝染色试验表明,AH基因能够提高番茄幼苗对低温的抵抗能力。4.RNA-seq分析结果发现,NIL-PH和NIL-GH下胚轴中共含有551差异表达基因(DEGs)。相比于NIL-GH,在NIL-PH中285个表现为上调,266个表现为下调。GO terms分析表明PH/GH上调基因显著的富集于“响应非生物胁迫刺激”、“响应激素刺激”和“类黄酮物质合成”,而下调基因显著的富集于“响应非生物胁迫刺激”、“氧化还原反应”和“节律过程”。此外,根据低温诱导番茄叶片RNA-seq分析结果发现,39个基因的表达最有可能受AH的调控,其中10个在16-PL中表现为上调,包括花青素合成基因F3’5’H和DFR。GO term分析表明上调基因主要富集于“苯丙烷生物合成”、“细胞内氨基酸衍生物物质合成”和“芳香类物质合成”,而下调基因主要富集于“响应碳水化合物的刺激”。5.基因表达模式分析结果表明,在6个RNA-seq样品中,Sl AN2和Sl ANT1-like的表达模式与AH相一致(相关系数分别为0.938和0.818),推测AH可能与Sl AN2和Sl ANT1-like相互作用,共同调控花青素的合成。根据转基因试验表达分析以及RNA-seq分析结果,我们推测AH可能直接作用于F3’5’H和DFR。顺式作用元件分析结果表明,在F3’5’H和DFR两个基因的转录位点上游(-2000bp和-1500bp)均含有大量的b HLH结合位点E-box(CANNTG)。
[Abstract]:Anthocyanins are an important secondary metabolite in plants, which not only attract insect pollination and assist seed transmission, but also enhance resistance to biological and abiotic stresses. In recent years, a large number of transcription factors involved in the synthesis and regulation of anthocyanins have been cloned in Arabidopsis, corn and Petunia, but in tomato research phase In this study, we cloned the first B HLH type transcription factor AH, which was involved in the regulation of anthocyanin synthesis in tomato, and combined with low temperature induction, low temperature cold injury test and transcriptional test to study the function of AH gene and to improve the low temperature tolerance of tomato seedlings. The main results are as follows: the anthocyanin deletion phenotype in 1.FMTT271 is controlled by a single recessive gene, and the AH gene is the allele. Therefore, the anthocyanin deletion gene in FMTT271 is named as the ah. application map cloning method, We locate the ah gene between the CAPS marker CAPS2 and CAPS4 of the long arm of the tomato chromosome 9. The physical distance is about 130kb. According to the reference genomic information of tomato (ITAG2.4), there are 5 ORFs.2. according to the results of homologous analysis and the analysis of gene expression. It is concluded that ORF5 (Solyc09g065100) is the.C DNA whole sequence of AH gene. The mutation of one base (G to T) of the sixth exons of the Solyc09g065100 gene in the mutant of the H mutant causes Gly to turn into a terminating codon and lead to the early termination of the Solyc09g065100 gene translation. The Solyc09g065100 gene is overexpressed in the ah mutant FMTT271, and the transgenic tomato T1 can both complement the FMTT271 phenotype and have a large number of leaves and fruits. The above results show that Solyc09g065100 is AH. binding Q RT-PCR analysis and transcriptional activation test. We speculate that AH may promote the development regulation of anthocyanin synthesis.3.AH gene by up regulation of the expression of late structure gene in the anthocyanin synthesis pathway, and then down down the table of structural genes in the anthocyanin synthesis pathway. Two amino diphenyl amine (DAB) and trypan blue staining test showed that the AH gene could improve the resistance of tomato seedlings to low temperature by.4.RNA-seq analysis. The results showed that the NIL-PH and NIL-GH hypocotyls were 551 poor in the NIL-PH and NIL-GH hypocotyls. Different expression genes (DEGs). Compared with NIL-GH, 285 up regulation in NIL-PH and 266 down regulated.GO terms analysis showed that PH/GH up regulation genes were significantly enriched in "response to abiotic stress stimulation", "response to hormone stimulation" and "flavonoid synthesis", while the down regulated genes were significantly enriched in "response to abiotic stress spines." In addition, according to the RNA-seq analysis of tomato leaves induced by low temperature, the expression of 39 genes was most likely to be regulated by AH, and 10 of them were up-regulated in 16-PL, including the anthocyanin synthesis gene F3 '5' H and DFR.GO term analysis indicating that the up-regulated genes were mainly enriched in benzene. Propane biosynthesis, "synthesis of amino acid derivatives in cells" and "synthesis of aromatic substances", and the down regulated genes are mainly enriched in the "response to carbohydrate stimulus".5. gene expression pattern analysis results show that in 6 RNA-seq samples, the expression patterns of Sl AN2 and Sl ANT1-like are in accordance with AH (correlation coefficient, respectively) For 0.938 and 0.818), it is presumed that AH may interact with Sl AN2 and Sl ANT1-like to jointly regulate the synthesis of anthocyanins. According to the expression analysis of the transgenic test and the results of RNA-seq analysis, we speculate that AH may directly act on F3 '5' H and DFR. cis acting elements, indicating that the F3 '5' H and the transcriptional sites of two genes are on the F3 '5. Both -2000bp and -1500bp contain a large number of B HLH binding sites E-box (CANNTG).

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S641.2

【参考文献】