森林草莓黄果表型形成机制研究

发布时间：2018-06-25 08:23

本文选题：森林草莓 + 黄果　；参考：《沈阳农业大学》2017年博士论文

【摘要】：果色是影响草莓果实外观品质和商品价值的核心指标,草莓果色是花青苷积累的结果,目前对于草莓花青苷积累调控机制的研究还不充分。森林草莓(Fragaria vesca)是多年生草本植物,基因组小,是研究草莓属植物以及蔷薇科植物的重要模式材料。草莓的黄果性状由c位点基因控制。为了研究与森林草莓黄果表型相关的关键基因,我们首先利用黄果森林草莓'Yellow Wonder'验证FvF3H(报道称与c位点基因共分离)的基因功能,随后,结合转录组高通量测序、遗传图谱构建和比较基因组分析进一步筛选c位点可能的候选基因。主要结果如下:1.利用HPLC方法测定森林草莓'Yellow Wonder'(YW)和'Ruegen'(RG)果实中花青苷含量和组分,结果显示红果类型RG中花青苷含量随果实成熟而持续增加,而黄果类型YW果实成熟过程中花青苷含量变化很小。RG果实中花青苷组分以Cy 3-glucoside和Pg3-glucoside为主,还有Pg3-rutinoside和Pg3-malonylglucoside;YW中能够检测到少量的Cy 3-glucoside、Pg 3-glucoside 和 Pg 3-malonylglucoside 三类花青苷。2.开展FvF3H基因功能验证,发现在YW中过量表达红果的FvF3H基因没有引起果实花青苷含量的增加,也没有引起果色变红。3.采用Illumina测序技术对森林草莓YW和RG转熟期果实进行转录组高通量测序。经de novo装配得到75,426条Unigene,鉴定获得595个差异表达基因,其中224个在YW中上调表达,371个下调表达。4.分析转录组测序结果和qRT-PCR结果,发现6个花青苷合成催化酶的编码基因(C4H、CHS、CHI、F3H、DFR和ANS)在YW中同时下调表达;多个调控花青苷合成的转录因子也存在显著表达差异,其中MYB1、MADS等转录因子在YW中下调表达,MYB1R在YW中上调表达。对花青苷合成相关结构基因和转录因子的序列分析发现RG和YW的MYB10基因CDS序列中存在一个SNP差异。5.利用构建的F2群体材料绘制了森林草莓c位点的基因连锁图谱,确定包含c位点基因候选区域的物理距离是718 kb。6.整合图谱数据与拟南芥、森林草莓全基因组水平的比较基因组分析数据,发现转录因子FvMYB10位于c位点基因的候选区域内。7.发现FvMYB10基因编码区存在TGG→TCG的SNP差异,氨基酸序列也存在相应差异Trp→Ser,且该SNP差异与森林草莓果色表型共分离。因此,推断FvMYB10是c位点基因候选基因。8.分析发现FvMYB10的突变位点TGG→TCG(Trp→Ser)位于MYB R2 DNA结合区域,该区域对调控花青苷合成基因的表达有重要作用。因此,FvMYB10 R2区域的错义突变(Trp→Ser)可能是黄果表型产生的关键所在。9.试验发现YW和RG间FvMYB10的表达量并没有显著的差异。说明FvMYB10的SNP变异影响果色表型的作用机理可能更多的是通过影响其对花青苷合成基因的调控作用,而不是通过影响自身基因表达来实现的。
[Abstract]:Fruit color is the core index that affects the appearance quality and commodity value of strawberry fruit. Strawberry fruit color is the result of anthocyanin accumulation. At present, the regulation mechanism of strawberry anthocyanin accumulation is not enough. Fragaria vesca is a perennial herbaceous plant with small genome. It is an important model material for the study of Strawberry and Rosaceae plants. The yellow fruit traits of strawberry were controlled by c locus gene. In order to study the key genes associated with the phenotype of forest strawberry yellow Wonder', we first used yellow Wonder'to verify the gene function of FvF3H (reported co-isolation with c locus), and then combined with high throughput sequencing of transcriptome. Genetic map construction and comparative genomic analysis were used to screen possible c locus candidate genes. The main results are as follows: 1. The contents and components of anthocyanin in the fruit of Yellow Wonder'(YW) and Ruegen'(RG) were determined by HPLC. The results showed that the content of anthocyanin in red fruit type RG increased with the fruit ripening. However, the content of anthocyanin in YW fruit changed little during maturation. The components of anthocyanin in RG fruit were mainly Cy 3-glucoside and Pg3-glucoside, and a small amount of Cy 3-glucoside PG 3-glucoside and PG 3-malonylglucoside were detected in YW, and a small amount of Cy 3-glucoside PG 3-glucoside and PG 3-malonylglucoside were detected in YW. It was found that FvF3H gene overexpressed in YW did not increase anthocyanin content or red fruit color. The transcriptional high-throughput sequencing of forest strawberry YW and RG was carried out by Illumina sequencing technique. 75426 Unigene genes were obtained by de novo assembly, and 595 differentially expressed genes were identified, of which 224 genes were up-regulated and 371down-regulated genes were expressed in YW. By analyzing the results of transcriptome sequencing and qRT-PCR, it was found that six genes encoding anthocyanin synthase (C _ 4H _ (4) H _ (CHS) CHIF _ (3H) DFR and ans were down-regulated simultaneously in YW, and that there were significant differences in the expression of several transcription factors regulating anthocyanin synthesis. The expression of MYB1R was down-regulated in YW, and MYB1R was up-regulated in YW. Sequence analysis of structural genes and transcription factors associated with anthocyanin synthesis revealed that there was a SNP difference between RG and YW MYB10 gene CDS sequences. The genetic linkage map of c locus of forest strawberry was plotted by using F2 population material, and the physical distance of candidate region containing c locus was 718 kb. 6. The integration map data and the comparative genomic analysis data of Arabidopsis thaliana and forest strawberry showed that the transcription factor FvMYB10 was located in the candidate region of c locus gene. It was found that there were SNP differences of TGG and TCG in the coding region of FvMYB10 gene, and there was also a corresponding difference in amino acid sequence, and the SNP difference was coisolated from the color phenotype of forest strawberry fruit. Therefore, it is inferred that FvMYB10 is a candidate gene of c locus. It was found that the mutation site of FvMYB10, TGG TCG (TRP Ser), was located in the DNA binding region of MYB R2, which played an important role in regulating the expression of anthocyanin synthesis gene. Therefore, the missense mutation of FvMYB10 R2 region (TRP Ser) may be the key to the phenotypic production of yellow fruit .9. The results showed that there was no significant difference in FvMYB10 expression between YW and RG. The results suggested that the mechanism of SNP variation of FvMYB10 affecting fruit color phenotype may be more by affecting its regulation on anthocyanin synthesis gene than by affecting its own gene expression.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S668.4

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