本文選題：香葉醇乙酰基轉(zhuǎn)移酶 + 基因克隆��； 參考：《云南農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：香葉醇存在于很多植物性精油中,是玫瑰香氣的主要揮發(fā)性成分,也是重要的萜烯類香氣成分。香葉醇為單萜類物質(zhì),通過甲戊二羥酸途徑進(jìn)行生物合成,香葉醇/香茅醇乙酰轉(zhuǎn)移酶(Geraniol acetyl transferase,AAT)以乙酰輔酶A為乙�；w,主要以香葉醇/香茅醇為底物,催化乙酸香葉酯和乙酸香茅酯的合成�！略路邸�(Rose chinensis‘Pallida’)是中國古老月季,對(duì)現(xiàn)代月季育成有重要作用,具有大多數(shù)現(xiàn)代月季沒有的香味。本研究以中國古老月季‘月月粉’為研究對(duì)象,采用同源克隆結(jié)合‘月月粉’全基因組序列,獲得了RcAAT基因全長,采用實(shí)時(shí)熒光定量PCR對(duì)該基因在‘月月粉’不同部位和不同發(fā)育時(shí)期的表達(dá)進(jìn)行分析,利用病毒誘導(dǎo)沉默系統(tǒng)(VIGS),完成RcAAT在植株內(nèi)的瞬時(shí)功能表達(dá)。具體研究結(jié)果如下:(1)根據(jù)GenBank已發(fā)表其它物種的香葉醇乙�；D(zhuǎn)移酶基因,獲取序列并設(shè)計(jì)引物,擴(kuò)增出長度為188bp的片段,結(jié)合‘月月粉’全基因組序列和PCR擴(kuò)增,獲得了RcAAT基因的cDNA全長,對(duì)其進(jìn)行生物信息分析,結(jié)果顯示RcAAT是一個(gè)1641bp的核苷酸序列,其核苷酸序列包含1個(gè)68bp的5′-UTR,1個(gè)1371bp的OFR,1個(gè)202bp的3′-UTR。該基因在69~71位為起始密碼子ATG,下游有同框終止密碼子TAA和polyA尾。氨基酸同源序列對(duì)比發(fā)現(xiàn),月季RcAAT與玫瑰和草莓具有較高的同源性。其中與玫瑰的同源性為87%,與草莓同源性為67%。(2)采用實(shí)時(shí)熒光定量PCR對(duì)RcAAT在‘月月粉’不同發(fā)育時(shí)期和不同組織部位的表達(dá)進(jìn)行分析。研究表明,在不同組織部位中RcAAT表達(dá)量差異顯著,莖的表達(dá)量最低,雄蕊表達(dá)量最高;在花朵不同發(fā)育時(shí)期中,RcAAT表達(dá)量隨著花瓣開放程度表達(dá)量逐漸增加,在花蕾期幾乎無表達(dá),在盛開期花瓣表達(dá)量到達(dá)頂峰,而后下降。(3)構(gòu)建病毒誘導(dǎo)的基因沉默載體RcAAT-TRV2�！略路邸斞恐糜诰褐谐檎婵胀瓿汕秩�,將侵染枝條嫁接到砧木上,4-5周后嫁接芽開花,利用氣象色譜質(zhì)譜聯(lián)用儀(GC/MS),對(duì)成功沉默RcAAT的‘月月粉’花瓣進(jìn)行花香成分分析。結(jié)果表明,香葉醇及乙酸香葉酯等含量被相應(yīng)的降低,表明RcAAT基因主要參與了香葉醇和乙酸香葉酯的合成。
[Abstract]:Geraniol is the main volatile component of rose aroma and an important terpene aroma component in many essential oils. Geraniol / citronellol acetyltransferase (Geraniol acetyl transferase AATA) was used as acetyl donor, and vanillin / citronellol was used as substrate, while geraniol / citronellol was used as substrate for biosynthesis of geraniol and citronellol acetyltransferase A as monoterpenoids. Catalytic synthesis of citronella acetate and citronella acetate. Rose chinensis (Pallida) is an ancient Chinese rose, which plays an important role in the development of modern rose, and has the fragrance that most modern rose does not have. In this study, the full-length RcAAT gene was obtained by homologous cloning combined with the whole genome sequence of Chinese ancient Chinese rose 'lunar powder'. Real-time fluorescent quantitative PCR was used to analyze the expression of RcAAT in different parts and different developmental stages of 'Moon Powder'. The transient functional expression of RcAAT in plants was accomplished by virus induced silencing system. The specific results are as follows: (1) according to the gene of geraniol acetyltransferase of other species published in GenBank, the sequence was obtained and primers were designed to amplify the fragment of 188bp, which was combined with the whole genome sequence of 'Moon Powder' and PCR amplification. The full-length cDNA of RcAAT gene was obtained, and its biological information was analyzed. The results showed that RcAAT was a nucleotide sequence of 1641bp. The nucleotide sequence of RcAAT contained a 68bp's 5- UTR-UTRs, a 1371bp's OFR, and a 202bp's 3r-UTR. At the 69-71 position, the gene is the start codon ATG, and the codon terminating codon TAA and polyA tail are found downstream. The comparison of amino acid homology showed that RcAAT had high homology with rose and strawberry. The expression of RcAAT in different development stages and tissues of 'Moon Powder' was analyzed by real-time fluorescence quantitative PCR. The results showed that there were significant differences in RcAAT expression in different tissues, the lowest in stem and the highest in stamens, and the expression of RcAAT increased with the opening of petals in different flower development stages. The expression of RcAAT-TRV2 was almost non-expressed at bud stage, and peaked at blooming stage, then decreased to .t3) to construct the virus-induced gene silencing vector RcAAT-TRV2. The terminal bud of 'Yueyue' was placed in the liquid of bacteria to complete the infection. The infecting branches were grafted onto the rootstock for 4-5 weeks and then the grafting buds bloomed. The flower aroma components of the petals of 'Moon Powder' which were successfully silenced by RcAAT were analyzed by means of GC / MS. The results showed that the contents of geraniol and vanillin were decreased, which indicated that RcAAT gene was mainly involved in the synthesis of geraniol and germane acetate.
【學(xué)位授予單位】：云南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S685.12

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1 李晉華;月月粉乙�；D(zhuǎn)移酶基因RcAAT的克隆及功能分析[D];云南農(nóng)業(yè)大學(xué);2017年

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本文編號(hào)：2022998