本文關(guān)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(nèi)蒙古大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甜瓜 ACO 基因家族 生物信息學(xué) 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(jì)價(jià)值。甜瓜豐富的種質(zhì)資源以及2012年甜瓜全基因組測(cè)序的完成,使得甜瓜成為除番茄之外的另一種研究果實(shí)發(fā)育成熟的模式植物。乙烯對(duì)呼吸躍變型果實(shí)的成熟起到重要的調(diào)控作用,ACC氧化酶(ACO)是乙烯生物合成途徑中的最后一個(gè)限速酶。本研究對(duì)甜瓜ACO基因家族進(jìn)行了全基因組分析,根據(jù)甜瓜轉(zhuǎn)錄組測(cè)序數(shù)據(jù)結(jié)果選擇候選基因,進(jìn)行克隆并構(gòu)建基因的超表達(dá)和CRISPR/Cas9編輯載體,通過子房注射法轉(zhuǎn)化甜瓜。主要研究結(jié)果如下:1)本研究從甜瓜全基因組鑒定得到了 9個(gè)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(gè)亞家族,9個(gè)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(jié)果顯示,CmACO1與CsACO3、CmACO3與CsACO2、CmACO6與CsACO1同源性最高。2)根據(jù)兩個(gè)不同時(shí)期甜瓜中果肉樣品轉(zhuǎn)錄組測(cè)序數(shù)據(jù),選取2個(gè)上調(diào)的差異表達(dá)基因進(jìn)行qPCR檢測(cè),qPCR檢測(cè)結(jié)果與轉(zhuǎn)錄組測(cè)序結(jié)果基本一致,均在成熟期表達(dá)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(zhǎng)度分別為1191bp 1131bp,分別編碼396、376個(gè)氨基酸,對(duì)兩個(gè)蛋白進(jìn)行生物信息學(xué)分析,結(jié)果表明兩個(gè)蛋白的結(jié)構(gòu)域包括DIOX_N和20G-Fe Ⅱ_Oxy,均為親水蛋白;無信號(hào)肽,均為非分泌蛋白;無跨膜結(jié)構(gòu),均不屬于跨膜蛋白。4)成功構(gòu)建了 CmACO7、CmACO8基因的超表達(dá)載體pPZP221-CmAC07、pPZP221-CmAC08 和編輯載體 pCRI-CmAC07、pCRI-CmAC08 并通過子房注射法轉(zhuǎn)化甜瓜,對(duì)T1代植株胚根進(jìn)行PCR檢測(cè),陽(yáng)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，

本文編號(hào)：1427908