發(fā)布時間：2018-06-25 08:23

  本文選題：森林草莓 + 黃果�。� 參考：《沈陽農業(yè)大學》2017年博士論文

【摘要】：果色是影響草莓果實外觀品質和商品價值的核心指標,草莓果色是花青苷積累的結果,目前對于草莓花青苷積累調控機制的研究還不充分。森林草莓(Fragaria vesca)是多年生草本植物,基因組小,是研究草莓屬植物以及薔薇科植物的重要模式材料。草莓的黃果性狀由c位點基因控制。為了研究與森林草莓黃果表型相關的關鍵基因,我們首先利用黃果森林草莓'Yellow Wonder'驗證FvF3H(報道稱與c位點基因共分離)的基因功能,隨后,結合轉錄組高通量測序、遺傳圖譜構建和比較基因組分析進一步篩選c位點可能的候選基因。主要結果如下:1.利用HPLC方法測定森林草莓'Yellow Wonder'(YW)和'Ruegen'(RG)果實中花青苷含量和組分,結果顯示紅果類型RG中花青苷含量隨果實成熟而持續(xù)增加,而黃果類型YW果實成熟過程中花青苷含量變化很小。RG果實中花青苷組分以Cy 3-glucoside和Pg3-glucoside為主,還有Pg3-rutinoside和Pg3-malonylglucoside;YW中能夠檢測到少量的Cy 3-glucoside、Pg 3-glucoside 和 Pg 3-malonylglucoside 三類花青苷。2.開展FvF3H基因功能驗證,發(fā)現(xiàn)在YW中過量表達紅果的FvF3H基因沒有引起果實花青苷含量的增加,也沒有引起果色變紅。3.采用Illumina測序技術對森林草莓YW和RG轉熟期果實進行轉錄組高通量測序。經de novo裝配得到75,426條Unigene,鑒定獲得595個差異表達基因,其中224個在YW中上調表達,371個下調表達。4.分析轉錄組測序結果和qRT-PCR結果,發(fā)現(xiàn)6個花青苷合成催化酶的編碼基因(C4H、CHS、CHI、F3H、DFR和ANS)在YW中同時下調表達;多個調控花青苷合成的轉錄因子也存在顯著表達差異,其中MYB1、MADS等轉錄因子在YW中下調表達,MYB1R在YW中上調表達。對花青苷合成相關結構基因和轉錄因子的序列分析發(fā)現(xiàn)RG和YW的MYB10基因CDS序列中存在一個SNP差異。5.利用構建的F2群體材料繪制了森林草莓c位點的基因連鎖圖譜,確定包含c位點基因候選區(qū)域的物理距離是718 kb。6.整合圖譜數(shù)據與擬南芥、森林草莓全基因組水平的比較基因組分析數(shù)據,發(fā)現(xiàn)轉錄因子FvMYB10位于c位點基因的候選區(qū)域內。7.發(fā)現(xiàn)FvMYB10基因編碼區(qū)存在TGG→TCG的SNP差異,氨基酸序列也存在相應差異Trp→Ser,且該SNP差異與森林草莓果色表型共分離。因此,推斷FvMYB10是c位點基因候選基因。8.分析發(fā)現(xiàn)FvMYB10的突變位點TGG→TCG(Trp→Ser)位于MYB R2 DNA結合區(qū)域,該區(qū)域對調控花青苷合成基因的表達有重要作用。因此,FvMYB10 R2區(qū)域的錯義突變(Trp→Ser)可能是黃果表型產生的關鍵所在。9.試驗發(fā)現(xiàn)YW和RG間FvMYB10的表達量并沒有顯著的差異。說明FvMYB10的SNP變異影響果色表型的作用機理可能更多的是通過影響其對花青苷合成基因的調控作用,而不是通過影響自身基因表達來實現(xiàn)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.
【學位授予單位】：沈陽農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S668.4

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