本文關鍵詞：甜瓜ACO基因家族成員的鑒定及CmACO7和CmACO8基因的克隆　出處：《內蒙古大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 甜瓜 ACO 基因家族 生物信息學 CRISPR/Cas9

【摘要】：甜瓜(Cucumis melo L.)屬于葫蘆科,是全世界廣泛栽培的水果作物,具有重要的經(jīng)濟價值。甜瓜豐富的種質資源以及2012年甜瓜全基因組測序的完成,使得甜瓜成為除番茄之外的另一種研究果實發(fā)育成熟的模式植物。乙烯對呼吸躍變型果實的成熟起到重要的調控作用,ACC氧化酶(ACO)是乙烯生物合成途徑中的最后一個限速酶。本研究對甜瓜ACO基因家族進行了全基因組分析,根據(jù)甜瓜轉錄組測序數(shù)據(jù)結果選擇候選基因,進行克隆并構建基因的超表達和CRISPR/Cas9編輯載體,通過子房注射法轉化甜瓜。主要研究結果如下:1)本研究從甜瓜全基因組鑒定得到了 9個ACO基因,分別命名為CmACO1-CmACO9,其中CmACO2基因存在可變剪接現(xiàn)象,可編碼3種mRNA。甜瓜ACO基因家族分為2個亞家族,9個ACO蛋白序列同源性為19.95%-75.54%,同組蛋白氨基酸序列同源性均在40%以上,組間蛋白的同源性僅為20%左右。甜瓜ACO基因家族與其他物種ACO基因的系統(tǒng)發(fā)育分析結果顯示,CmACO1與CsACO3、CmACO3與CsACO2、CmACO6與CsACO1同源性最高。2)根據(jù)兩個不同時期甜瓜中果肉樣品轉錄組測序數(shù)據(jù),選取2個上調的差異表達基因進行qPCR檢測,qPCR檢測結果與轉錄組測序結果基本一致,均在成熟期表達量上升,選定CmAC07、CmACO8作為候選基因。3)從甜瓜中克隆得到了 CmAC07、CmACO8基因cDNA,長度分別為1191bp 1131bp,分別編碼396、376個氨基酸,對兩個蛋白進行生物信息學分析,結果表明兩個蛋白的結構域包括DIOX_N和20G-Fe Ⅱ_Oxy,均為親水蛋白;無信號肽,均為非分泌蛋白;無跨膜結構,均不屬于跨膜蛋白。4)成功構建了 CmACO7、CmACO8基因的超表達載體pPZP221-CmAC07、pPZP221-CmAC08 和編輯載體 pCRI-CmAC07、pCRI-CmAC08 并通過子房注射法轉化甜瓜,對T1代植株胚根進行PCR檢測,陽性率分別為21.5%、20.8%、10.5%、12.3%。
[Abstract]:Cucumis melo L.) belongs to Cucurbitaceae and is a widely cultivated fruit crop all over the world. It has important economic value. The rich germplasm resources of muskmelon and the complete genome sequencing of muskmelon in 2012. It makes melon become another model plant to study fruit development and maturation besides tomato. Ethylene plays an important role in regulating the ripening of respiration type fruit. ACC oxidase (ACO) is the last rate-limiting enzyme in ethylene biosynthesis pathway. In this study, the whole genome of ACO gene family in muskmelon was analyzed. According to the data of melon transcriptome sequencing, candidate genes were selected for cloning and construction of gene overexpression and CRISPR/Cas9 editing vector. The main results are as follows: 1) in this study, 9 ACO genes were identified from the whole genome of muskmelon, named CmACO1-CmACO9. Among them, CmACO2 gene has variable splicing phenomenon, which can encode three kinds of mRNA.The ACO gene family of melon is divided into two subfamilies. The homology of 9 ACO proteins was 19.95- 75.54.The homology of amino acid sequence of homohistone was above 40%. The homology of intergroup protein was only about 20%. Phylogenetic analysis of ACO gene family and ACO gene of other species showed that CmACO1 and CsACO3. CmACO3 and CsACO2CmACO6 have the highest homology with CsACO1. Two up-regulated differentially expressed genes were selected for qPCR detection. The results of qPCR were consistent with the results of transcriptome sequencing. The expression of CmAC07 was increased in the mature stage, and CmAC07 was selected. CmAC07CmACO8 gene was cloned from muskmelon as candidate gene. The length of CmAC07CmACO8 gene was 1191bp 1131bp. Two proteins were analyzed by bioinformatics, encoding 396,376 amino acids respectively. The results showed that the domains of the two proteins included DIOX_N and 20G-Fe 鈪，

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