三華李果實發(fā)育轉錄組分析及其花青苷生物合成相關基因表達分析
發(fā)布時間：2019-05-11 00:21
【摘要】：中國李(Prunus salicina Lindl.)原產長江流域,經長期栽培已在華南地區(qū)形成了一個獨特的南亞熱帶栽培群體,并成為廣東省第一大落葉果樹,主要有三華李類(紅皮紅肉)、綠皮白肉類(竹絲李、紅線李等)和紅皮白肉類(三月李等)等三種類型,其中三華李類品種栽培面積約占廣東全省李面積80%以上。本研究以三華李類品種‘華蜜大蜜李’和‘白脆雞麻李’為材料,通過RNA-Seq技術進行果實、花、嫩莖葉和胚等進行轉錄組測序,初步建立起三華李果實發(fā)育轉錄數據庫,挖掘果實發(fā)育成熟過程中差異表達基因,分析果實發(fā)育成熟過程中花青苷生物合成、果實成熟軟化相關的細胞壁代謝等差異表達基因,為今后果實著色改良、耐儲運品種培育提供參考依據。主要研究結果如下:1、以華蜜李大蜜李的葉芽、花朵、胚和不同發(fā)育時期的果實(花后75 d、花后100d、花后130 d)為材料建立三華李開花結果時期轉錄組數據庫,獲得68,484條Unigene;并對其進行GO、KEGG、NR、PlnTFDB等數據庫注釋,共有33,630條Unigene獲得注釋;并對1 kb以上的Unigene進行SSR位點開發(fā),共發(fā)現7,984個SSR位點。2、基于4個不同發(fā)育時期的華蜜大蜜李果實樣品轉錄組數據進行差異表達基因的篩選,共獲得5,394個差異表達基因。對差異表達基因進行轉錄因子預測、KEGG、表達模式GO富集分析發(fā)現一些時期高表達基因模塊;蛋白互作網絡分析發(fā)現果實發(fā)育成熟過程中的一些高蛋白互作集中模塊。對果實發(fā)育差異表達基因預測分析,發(fā)現大部分激素相關基因呈下調表達趨勢,并發(fā)現30個激素合成相關基因。對細胞壁代謝相關差異基因分析,發(fā)現41個細胞壁代謝相關基因;其中1個華蜜李幼果期(花后75 d)特異表達、2個華蜜李成熟果(花后130 d)時期特異表達基因。3、對三華李果實花青苷合成相關基因進行分析,發(fā)現41個可能花青苷生物合成關鍵基因、26個可能參與花青苷轉運、39個可能花青苷合成相關轉錄因子、3個果實色素積累而下調的基因。4、以特異引物擴增條帶單一、溶解曲線峰值高、最高峰值溫度波動小為標準進行篩選內參基因,篩選結果表明HMBS2為華蜜大蜜李果實的基因表達量分析較合適的qRT-PCR內參基因。通過對10個候選花青苷生物合成基因、9個候選MYB轉錄因子進行qRT-PCR驗證表明,相比華蜜大蜜李葉和花,華蜜大蜜李成熟果(花后130 d)果實顏色深可能是受c23975.graph_c0(ANS)和c19863.graph_c0(UFGT)表達的影響;與白脆雞麻李成熟果(花后130 d)相比較,華蜜大蜜李成熟果(花后130 d)果肉顏色更深可能是受c21951.graph_c0(CHS2)和c19863.graph_c0(UFGT)的影響。并發(fā)現c6572.graph_c0可能是影響果實花色苷形成的有效MYB轉錄因子之一。5、利用TA克隆技術,得到了c6572.graph_c0的cDNA全長;并對cDNA進行結構預測,發(fā)現其含有蛋白R2R3結構域。
[Abstract]:Chinese Li (Prunus salicina Lindl.) Native to the Yangtze River Basin, after long-term cultivation, it has formed a unique subtropical cultivation population in South China, and has become the largest deciduous fruit tree in Guangdong Province, mainly Sanhua plum (red skin and red meat), green skin and white meat (bamboo silk plum), There are three types of red plum and red skin and white meat (March plum, etc.). Among them, the cultivation area of Sanhua plum varieties accounts for more than 80% of the total plum area in Guangdong Province. In this study, Sanhua plum varieties' Huami big honey plum 'and' white crispy chicken plum 'were used as materials to sequence the fruit, flowers, tender stems, leaves and embryos by RNA-Seq technique, and the transcription database of fruit development of Sanhua plum was established. The differentially expressed genes during fruit development and ripening were excavated, and the differentially expressed genes such as anthocyanin biosynthesis and cell wall metabolism related to fruit ripening were analyzed, so as to improve the fruit coloring in the future. The cultivation of varieties resistant to storage and transportation provides a reference basis. The main results are as follows: 1. The database of leaf bud, flower, embryo and fruit at different developmental stages (75 d after anthesis, 100 d after anthesis, 130 d after anthesis) was established. Get 68484 Unigene;. GO,KEGG,NR,PlnTFDB and other database comments were carried out, and a total of 33630 Unigene comments were obtained. A total of 7984 SSR loci were found in Unigene with more than 1 kb. 2. 5394 differentially expressed genes were screened based on the data of four different developmental stages of plum fruit samples, and a total of 5394 differentially expressed genes were obtained. the results showed that 5394 differentially expressed genes were obtained by screening the differentially expressed genes based on the data of four different developmental stages of Prunus mandshurica fruit samples. The transcription factor prediction of differentially expressed genes was carried out. GO enrichment analysis of KEGG, expression pattern found some high expression gene modules, and protein interaction network analysis found some high protein interaction concentration modules during fruit development and maturation. Based on the prediction and analysis of differentially expressed genes in fruit development, it was found that most of the hormone-related genes were down-regulated and 30 genes related to hormone synthesis were found. 41 genes related to cell wall metabolism were found by analyzing the differentially related genes of cell wall metabolism. Among them, one was specifically expressed at the young fruit stage (75 days after anthesis) and two genes were specifically expressed at the mature fruit stage (130 days after anthesis). 3. The anthocyanin synthesis related genes in the fruit of Sanhua plum were analyzed. It was found that 41 key genes of anthocyanin biosynthesis, 26 of which may be involved in anthocyanin transport, 39 of which may be involved in anthocyanin synthesis related transcription factors, and 3 genes down-regulated by pigment accumulation in fruit. 4. The bands were amplified with specific primers. The peak value of dissolution curve was high and the maximum peak temperature fluctuation was small as the standard for screening internal reference genes. The results showed that HMBS2 was a suitable qRT-PCR internal reference gene for gene expression analysis of honey plum fruit. The results of qRT-PCR verification of 10 candidate anthocyanin biosynthesis genes and 9 candidate MYB transcription factors showed that compared with Huamei plum leaves and flowers, The color depth of the ripe fruit of plum (130d after anthesis) may be affected by the expression of c23975.graph_c0 (ANS) and c19863.graph_c0 (UFGT). Compared with the ripe fruit of white crispy chicken plum (130 d after anthesis), the darker flesh color of the ripe fruit (130 d after anthesis) was probably affected by c21951.graph_c0 (CHS2) and c19863.graph_c0 (UFGT). It was found that c6572.graph_c0 may be one of the effective MYB transcription factors affecting anthocyanin formation in fruit. The full length of c6572.graph_c0 cDNA was obtained by TA cloning technique, and the structure of cDNA was predicted, and it was found that cDNA contained protein R2R3 domain.
【學位授予單位】：華南農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S662.3
本文編號：2474111
[Abstract]:Chinese Li (Prunus salicina Lindl.) Native to the Yangtze River Basin, after long-term cultivation, it has formed a unique subtropical cultivation population in South China, and has become the largest deciduous fruit tree in Guangdong Province, mainly Sanhua plum (red skin and red meat), green skin and white meat (bamboo silk plum), There are three types of red plum and red skin and white meat (March plum, etc.). Among them, the cultivation area of Sanhua plum varieties accounts for more than 80% of the total plum area in Guangdong Province. In this study, Sanhua plum varieties' Huami big honey plum 'and' white crispy chicken plum 'were used as materials to sequence the fruit, flowers, tender stems, leaves and embryos by RNA-Seq technique, and the transcription database of fruit development of Sanhua plum was established. The differentially expressed genes during fruit development and ripening were excavated, and the differentially expressed genes such as anthocyanin biosynthesis and cell wall metabolism related to fruit ripening were analyzed, so as to improve the fruit coloring in the future. The cultivation of varieties resistant to storage and transportation provides a reference basis. The main results are as follows: 1. The database of leaf bud, flower, embryo and fruit at different developmental stages (75 d after anthesis, 100 d after anthesis, 130 d after anthesis) was established. Get 68484 Unigene;. GO,KEGG,NR,PlnTFDB and other database comments were carried out, and a total of 33630 Unigene comments were obtained. A total of 7984 SSR loci were found in Unigene with more than 1 kb. 2. 5394 differentially expressed genes were screened based on the data of four different developmental stages of plum fruit samples, and a total of 5394 differentially expressed genes were obtained. the results showed that 5394 differentially expressed genes were obtained by screening the differentially expressed genes based on the data of four different developmental stages of Prunus mandshurica fruit samples. The transcription factor prediction of differentially expressed genes was carried out. GO enrichment analysis of KEGG, expression pattern found some high expression gene modules, and protein interaction network analysis found some high protein interaction concentration modules during fruit development and maturation. Based on the prediction and analysis of differentially expressed genes in fruit development, it was found that most of the hormone-related genes were down-regulated and 30 genes related to hormone synthesis were found. 41 genes related to cell wall metabolism were found by analyzing the differentially related genes of cell wall metabolism. Among them, one was specifically expressed at the young fruit stage (75 days after anthesis) and two genes were specifically expressed at the mature fruit stage (130 days after anthesis). 3. The anthocyanin synthesis related genes in the fruit of Sanhua plum were analyzed. It was found that 41 key genes of anthocyanin biosynthesis, 26 of which may be involved in anthocyanin transport, 39 of which may be involved in anthocyanin synthesis related transcription factors, and 3 genes down-regulated by pigment accumulation in fruit. 4. The bands were amplified with specific primers. The peak value of dissolution curve was high and the maximum peak temperature fluctuation was small as the standard for screening internal reference genes. The results showed that HMBS2 was a suitable qRT-PCR internal reference gene for gene expression analysis of honey plum fruit. The results of qRT-PCR verification of 10 candidate anthocyanin biosynthesis genes and 9 candidate MYB transcription factors showed that compared with Huamei plum leaves and flowers, The color depth of the ripe fruit of plum (130d after anthesis) may be affected by the expression of c23975.graph_c0 (ANS) and c19863.graph_c0 (UFGT). Compared with the ripe fruit of white crispy chicken plum (130 d after anthesis), the darker flesh color of the ripe fruit (130 d after anthesis) was probably affected by c21951.graph_c0 (CHS2) and c19863.graph_c0 (UFGT). It was found that c6572.graph_c0 may be one of the effective MYB transcription factors affecting anthocyanin formation in fruit. The full length of c6572.graph_c0 cDNA was obtained by TA cloning technique, and the structure of cDNA was predicted, and it was found that cDNA contained protein R2R3 domain.
【學位授予單位】：華南農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S662.3
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