發(fā)布時間：2019-05-11 00:21

【摘要】：中國李(Prunus salicina Lindl.)原產(chǎn)長江流域,經(jīng)長期栽培已在華南地區(qū)形成了一個獨(dú)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(shù)進(jìn)行果實(shí)、花、嫩莖葉和胚等進(jìn)行轉(zhuǎn)錄組測序,初步建立起三華李果實(shí)發(fā)育轉(zhuǎn)錄數(shù)據(jù)庫,挖掘果實(shí)發(fā)育成熟過程中差異表達(dá)基因,分析果實(shí)發(fā)育成熟過程中花青苷生物合成、果實(shí)成熟軟化相關(guān)的細(xì)胞壁代謝等差異表達(dá)基因,為今后果實(shí)著色改良、耐儲運(yùn)品種培育提供參考依據(jù)。主要研究結(jié)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(shí)(花后75 d、花后100d、花后130 d)為材料建立三華李開花結(jié)果時期轉(zhuǎn)錄組數(shù)據(jù)庫,獲得68,484條Unigene;并對其進(jìn)行GO、KEGG、NR、PlnTFDB等數(shù)據(jù)庫注釋,共有33,630條Unigene獲得注釋;并對1 kb以上的Unigene進(jìn)行SSR位點(diǎn)開發(fā),共發(fā)現(xiàn)7,984個SSR位點(diǎn)。2、基于4個不同發(fā)育時期的華蜜大蜜李果實(shí)樣品轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行差異表達(dá)基因的篩選,共獲得5,394個差異表達(dá)基因。對差異表達(dá)基因進(jìn)行轉(zhuǎn)錄因子預(yù)測、KEGG、表達(dá)模式GO富集分析發(fā)現(xiàn)一些時期高表達(dá)基因模塊;蛋白互作網(wǎng)絡(luò)分析發(fā)現(xiàn)果實(shí)發(fā)育成熟過程中的一些高蛋白互作集中模塊。對果實(shí)發(fā)育差異表達(dá)基因預(yù)測分析,發(fā)現(xiàn)大部分激素相關(guān)基因呈下調(diào)表達(dá)趨勢,并發(fā)現(xiàn)30個激素合成相關(guān)基因。對細(xì)胞壁代謝相關(guān)差異基因分析,發(fā)現(xiàn)41個細(xì)胞壁代謝相關(guān)基因;其中1個華蜜李幼果期(花后75 d)特異表達(dá)、2個華蜜李成熟果(花后130 d)時期特異表達(dá)基因。3、對三華李果實(shí)花青苷合成相關(guān)基因進(jìn)行分析,發(fā)現(xiàn)41個可能花青苷生物合成關(guān)鍵基因、26個可能參與花青苷轉(zhuǎn)運(yùn)、39個可能花青苷合成相關(guān)轉(zhuǎn)錄因子、3個果實(shí)色素積累而下調(diào)的基因。4、以特異引物擴(kuò)增條帶單一、溶解曲線峰值高、最高峰值溫度波動小為標(biāo)準(zhǔn)進(jìn)行篩選內(nèi)參基因,篩選結(jié)果表明HMBS2為華蜜大蜜李果實(shí)的基因表達(dá)量分析較合適的qRT-PCR內(nèi)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(zhuǎn)錄因子進(jìn)行qRT-PCR驗(yàn)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(shí)顏色深可能是受c23975.graph_c0(ANS)和c19863.graph_c0(UFGT)表達(dá)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(xiàn)c6572.graph_c0可能是影響果實(shí)花色苷形成的有效MYB轉(zhuǎn)錄因子之一。5、利用TA克隆技術(shù),得到了c6572.graph_c0的cDNA全長;并對cDNA進(jìn)行結(jié)構(gòu)預(yù)測,發(fā)現(xiàn)其含有蛋白R2R3結(jié)構(gòu)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(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S662.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 何業(yè)華;馮筠庭;胡桂兵;林順權(quán);劉成明;秦永華;楊向暉;葉自行;歐陽若;馬均;陳程杰;李少靈;莫有為;楊旭輝;許如記;;李新品種‘嶺溪李’[J];園藝學(xué)報;2015年09期

2 郭翠紅;何業(yè)華;馮筠庭;陳程杰;林文秋;欒愛萍;張雅芬;;廣東省李產(chǎn)業(yè)發(fā)展現(xiàn)狀調(diào)查[J];經(jīng)濟(jì)林研究;2015年01期

3 王延延;張新宇;周啟明;張曉靈;魏江春;;石果衣真菌(Endocarpon pusillum)比較轉(zhuǎn)錄組分析揭示其抗旱特性[J];中國科學(xué):生命科學(xué);2015年01期

4 李翠婷;張廣輝;馬春花;孟珍貴;陳軍文;楊生超;;野三七轉(zhuǎn)錄組中SSR位點(diǎn)信息分析及其多態(tài)性研究[J];中草藥;2014年10期

5 李炎林;楊星星;張家銀;黃三文;熊興耀;;南方紅豆杉轉(zhuǎn)錄組SSR挖掘及分子標(biāo)記的研究[J];園藝學(xué)報;2014年04期

6 王麗鴛;韋康;張成才;成浩;;茶樹花轉(zhuǎn)錄組微衛(wèi)星分布特征[J];作物學(xué)報;2014年01期

7 張楠;孫桂玲;戴均貴;楊艷芳;劉洪偉;邱德有;;銀杏細(xì)胞轉(zhuǎn)錄組高通量測序及分析[J];中國生物工程雜志;2013年05期

8 詹嘉紅;藍(lán)宗輝;魏小鳳;;黑布林李子皮色素的提取及穩(wěn)定性[J];食品研究與開發(fā);2011年05期

9 王化坤;陶建敏;渠慎春;房經(jīng)貴;馬瑞娟;章鎮(zhèn);婁曉鳴;;核果類果樹ITS序列分子進(jìn)化及系統(tǒng)發(fā)育關(guān)系研究[J];園藝學(xué)報;2010年03期

10 何業(yè)華;謝志亮;劉成明;林順權(quán);胡桂兵;歐陽若;葉自行;余小玲;韓景忠;徐永爐;何海波;何永勝;;南亞熱帶李新品種‘白脆雞麻李’[J];園藝學(xué)報;2009年12期

，

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