柿果实CCD1基因超表达和RNAi载体构建及其农杆菌转化Micro-Tom番前的研究

发布时间：2018-04-24 21:22

本文选题：果实香气 + 超表达载体　；参考：《扬州大学》2016年硕士论文

【摘要】：与果实品质有重要关系的脱辅基类胡萝卜素香气成分形成有关的CCOs(类胡萝卜素裂解氧化酶)主要是CCD1和CCD4分支,课题组前期研究表明柿果实中确实存在脱辅基类胡萝卜素香气成分,但其形成是否与CCDs相关还不确定。根据已经克隆的DkCCD1基因,本研究构建了含有DkCCD1基因的超表达载体和RNAi表达载体,确定了 Micro-Tom番茄的遗传转化体系,并以农杆菌叶盘转化法将上述两个载体成功导入番茄中。主要研究结果如下:1、CTAB法提取'小方柿'果肉总RNA,根据带有不同酶切位点的引物获得目的基因,将扩增的目的基因序列与NCBI登录的基因序列进行基因序列比对和氨基酸序列比对,发现基因序列相似度均超过99%,氨基酸序列相似度均为100%,由此得到用于后续实验的目的基因;将得到的目的基因与双元表达载体pCAMBIA1301连接,成功构建了pCAMBIA1301-CCD1超表达载体和具有发卡结构的pCAMBIA1301-CCD1-RNAi表达载体,通过冻融法将载体导入农杆菌EHA105,-80°C保存备用。2、在Micro-Tom番茄再生体系的基础上,对载体上所携带的标记基因HygB和筛选过程中的抑菌剂特美汀(Tim)进行压力筛选。结果发现,Hyg的浓度越高,对外植体的分化影响越大,愈伤诱导、芽分化和生根筛选培养过程中使用的浓度分别为7 mg/L、9 mg/L和5 mg/L;Tim对外植体的生长没有较大的抑制作用,浓度为450 mg/L时,对愈伤和芽分化有小幅度影响,浓度为500 mg/L时,对生根有小幅度影响,结合成本考虑,筛选培养过程中抑菌剂浓度均选择400 mg/L。根据以上结果确定愈伤诱导选择培养基为MS + 2 mg/L6-BA+0.2mg/LNAA+7mg/LHyg + 400 mg/LTim + 0.7%琼脂 +3%蔗糖,芽分化选择培养基为 MS + 1 mg/L ZT + 9 mg/L Hyg + 400 mg/L Tim + 0.7%琼脂 + 3%蔗糖,生根选择培养基为 MS + 0.1 mg/LNAA + 5mg/L Hyg + 400 mg/L Tim + 0.7%琼脂 + 3%蔗糖。3、根据表达载体上的潮霉素基因设计引物,目的条带大小为729 bp,提取抗性株的DNA和RNA分别进行PCR和RT-PCR检测,确定目的基因DkCCD1已成功导入番茄植株并进行表达。以最终RT-PCR检测阳性为转化成功的标准,本实验中超表达植株和RNAi表达植株的子叶转化率均为0.23%。
[Abstract]:CCOs (carotenoid lytic oxidase) related to the formation of aroma components of carotenoids, which have important relationship with fruit quality, are mainly CCD1 and CCD4 branches. The previous study showed that the aroma components of carotenoids were present in persimmon fruit, but it was uncertain whether the aroma components were related to CCDs in persimmon fruit. Based on the cloned DkCCD1 gene, the superexpression vector and RNAi expression vector containing DkCCD1 gene were constructed, the genetic transformation system of Micro-Tom tomato was established, and the two vectors were successfully introduced into tomato by Agrobacterium tumefaciens leaf disk transformation. The main results were as follows: the total RNA of 'Xiao Fang' persimmon pulp was extracted by the method of 1: 1 CTAB, and the target gene was obtained according to the primers with different enzyme cutting sites. The amplified target gene sequence was compared with the gene sequence registered in the NCBI for gene sequence alignment and amino acid sequence alignment. It was found that the similarity of gene sequence and amino acid sequence were all more than 99 and 100, respectively, and the target gene was obtained for further experiment. The target gene was connected with the binary expression vector pCAMBIA1301. PCAMBIA1301-CCD1 superexpression vector and pCAMBIA1301-CCD1-RNAi expression vector with hairpin structure were successfully constructed. The vector was transferred into Agrobacterium tumefaciens EHA105 ~ 80 掳C by freezing and thawing method to preserve spare. 2. On the basis of regeneration system of Micro-Tom tomato, the vector was transferred to Agrobacterium tumefaciens by freezing and thawing. The marker gene HygB carried on the vector and the inhibitor of termetin in the screening process were screened under pressure. The results showed that the higher the concentration of Hyg was, the greater the effect on the differentiation of explants. The callus induction, bud differentiation and rooting culture were 7 mg / L ~ 9 mg/L and 5 mg / L ~ (-1) Tim had no significant inhibitory effect on the growth of explants, respectively. When the concentration was 450 mg/L, the callus and bud differentiation were slightly affected, and when the concentration was 500 mg/L, the rooting was slightly affected. Considering the cost, the concentration of antimicrobial agents was 400 mg / L in the process of screening and culture. According to the above results, the selected medium for callus induction was MS 2 mg/L6-BA 0.2mg/LNAA 7mg/LHyg 400 mg/LTim 0.7% Agar 3% sucrose. The optimal medium for bud differentiation was MS 1 mg/L ZT9 mg/L Hyg 400 mg/L Tim 0.7% Agar 3% sucrose. The rooting selection medium was MS 0.1 mg/LNAA 5mg/L Hyg 400 mg/L Tim 0.7% Agar 3% sucrose. 3 primers were designed according to the hygromycin gene on the expression vector. Objective to extract the DNA and RNA of resistant plants for PCR and RT-PCR detection, and confirm that the target gene DkCCD1 has been successfully introduced into tomato plants and expressed. In this experiment, the cotyledon conversion rate of the overexpressed plants and the RNAi expressed plants was 0.2323, taking the final RT-PCR positive as the standard of successful transformation.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S665.2;Q943.2

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