甜瓜ACO基因家族成员的鉴定及CmACO7和CmACO8基因的克隆

发布时间：2018-01-15 10:19

  本文关键词：甜瓜ACO基因家族成员的鉴定及CmACO7和CmACO8基因的克隆　出处：《内蒙古大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 甜瓜 ACO 基因家族 生物信息学 CRISPR/Cas9

【摘要】：甜瓜(Cucumis melo L.)属于葫芦科,是全世界广泛栽培的水果作物,具有重要的经济价值。甜瓜丰富的种质资源以及2012年甜瓜全基因组测序的完成,使得甜瓜成为除番茄之外的另一种研究果实发育成熟的模式植物。乙烯对呼吸跃变型果实的成熟起到重要的调控作用,ACC氧化酶(ACO)是乙烯生物合成途径中的最后一个限速酶。本研究对甜瓜ACO基因家族进行了全基因组分析,根据甜瓜转录组测序数据结果选择候选基因,进行克隆并构建基因的超表达和CRISPR/Cas9编辑载体,通过子房注射法转化甜瓜。主要研究结果如下:1)本研究从甜瓜全基因组鉴定得到了 9个ACO基因,分别命名为CmACO1-CmACO9,其中CmACO2基因存在可变剪接现象,可编码3种mRNA。甜瓜ACO基因家族分为2个亚家族,9个ACO蛋白序列同源性为19.95%-75.54%,同组蛋白氨基酸序列同源性均在40%以上,组间蛋白的同源性仅为20%左右。甜瓜ACO基因家族与其他物种ACO基因的系统发育分析结果显示,CmACO1与CsACO3、CmACO3与CsACO2、CmACO6与CsACO1同源性最高。2)根据两个不同时期甜瓜中果肉样品转录组测序数据,选取2个上调的差异表达基因进行qPCR检测,qPCR检测结果与转录组测序结果基本一致,均在成熟期表达量上升,选定CmAC07、CmACO8作为候选基因。3)从甜瓜中克隆得到了 CmAC07、CmACO8基因cDNA,长度分别为1191bp 1131bp,分别编码396、376个氨基酸,对两个蛋白进行生物信息学分析,结果表明两个蛋白的结构域包括DIOX_N和20G-Fe Ⅱ_Oxy,均为亲水蛋白;无信号肽,均为非分泌蛋白;无跨膜结构,均不属于跨膜蛋白。4)成功构建了 CmACO7、CmACO8基因的超表达载体pPZP221-CmAC07、pPZP221-CmAC08 和编辑载体 pCRI-CmAC07、pCRI-CmAC08 并通过子房注射法转化甜瓜,对T1代植株胚根进行PCR检测,阳性率分别为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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