本文選題：果實(shí)香氣 + 超表達(dá)載體　； 參考：《揚(yáng)州大學(xué)》2016年碩士論文

【摘要】：與果實(shí)品質(zhì)有重要關(guān)系的脫輔基類胡蘿卜素香氣成分形成有關(guān)的CCOs(類胡蘿卜素裂解氧化酶)主要是CCD1和CCD4分支,課題組前期研究表明柿果實(shí)中確實(shí)存在脫輔基類胡蘿卜素香氣成分,但其形成是否與CCDs相關(guān)還不確定。根據(jù)已經(jīng)克隆的DkCCD1基因,本研究構(gòu)建了含有DkCCD1基因的超表達(dá)載體和RNAi表達(dá)載體,確定了 Micro-Tom番茄的遺傳轉(zhuǎn)化體系,并以農(nóng)桿菌葉盤轉(zhuǎn)化法將上述兩個(gè)載體成功導(dǎo)入番茄中。主要研究結(jié)果如下:1、CTAB法提取'小方柿'果肉總RNA,根據(jù)帶有不同酶切位點(diǎn)的引物獲得目的基因,將擴(kuò)增的目的基因序列與NCBI登錄的基因序列進(jìn)行基因序列比對(duì)和氨基酸序列比對(duì),發(fā)現(xiàn)基因序列相似度均超過99%,氨基酸序列相似度均為100%,由此得到用于后續(xù)實(shí)驗(yàn)的目的基因;將得到的目的基因與雙元表達(dá)載體pCAMBIA1301連接,成功構(gòu)建了pCAMBIA1301-CCD1超表達(dá)載體和具有發(fā)卡結(jié)構(gòu)的pCAMBIA1301-CCD1-RNAi表達(dá)載體,通過凍融法將載體導(dǎo)入農(nóng)桿菌EHA105,-80°C保存?zhèn)溆谩?、在Micro-Tom番茄再生體系的基礎(chǔ)上,對(duì)載體上所攜帶的標(biāo)記基因HygB和篩選過程中的抑菌劑特美汀(Tim)進(jìn)行壓力篩選。結(jié)果發(fā)現(xiàn),Hyg的濃度越高,對(duì)外植體的分化影響越大,愈傷誘導(dǎo)、芽分化和生根篩選培養(yǎng)過程中使用的濃度分別為7 mg/L、9 mg/L和5 mg/L;Tim對(duì)外植體的生長沒有較大的抑制作用,濃度為450 mg/L時(shí),對(duì)愈傷和芽分化有小幅度影響,濃度為500 mg/L時(shí),對(duì)生根有小幅度影響,結(jié)合成本考慮,篩選培養(yǎng)過程中抑菌劑濃度均選擇400 mg/L。根據(jù)以上結(jié)果確定愈傷誘導(dǎo)選擇培養(yǎng)基為MS + 2 mg/L6-BA+0.2mg/LNAA+7mg/LHyg + 400 mg/LTim + 0.7%瓊脂 +3%蔗糖,芽分化選擇培養(yǎng)基為 MS + 1 mg/L ZT + 9 mg/L Hyg + 400 mg/L Tim + 0.7%瓊脂 + 3%蔗糖,生根選擇培養(yǎng)基為 MS + 0.1 mg/LNAA + 5mg/L Hyg + 400 mg/L Tim + 0.7%瓊脂 + 3%蔗糖。3、根據(jù)表達(dá)載體上的潮霉素基因設(shè)計(jì)引物,目的條帶大小為729 bp,提取抗性株的DNA和RNA分別進(jìn)行PCR和RT-PCR檢測,確定目的基因DkCCD1已成功導(dǎo)入番茄植株并進(jìn)行表達(dá)。以最終RT-PCR檢測陽性為轉(zhuǎn)化成功的標(biāo)準(zhǔn),本實(shí)驗(yàn)中超表達(dá)植株和RNAi表達(dá)植株的子葉轉(zhuǎn)化率均為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 擄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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S665.2;Q943.2

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