月月粉乙酰基转移酶基因RcAAT的克隆及功能分析

发布时间：2018-06-15 18:06

  本文选题：香叶醇乙酰基转移酶 + 基因克隆　； 参考：《云南农业大学》2017年硕士论文

【摘要】：香叶醇存在于很多植物性精油中,是玫瑰香气的主要挥发性成分,也是重要的萜烯类香气成分。香叶醇为单萜类物质,通过甲戊二羟酸途径进行生物合成,香叶醇/香茅醇乙酰转移酶(Geraniol acetyl transferase,AAT)以乙酰辅酶A为乙酰基供体,主要以香叶醇/香茅醇为底物,催化乙酸香叶酯和乙酸香茅酯的合成。‘月月粉’(Rose chinensis‘Pallida’)是中国古老月季,对现代月季育成有重要作用,具有大多数现代月季没有的香味。本研究以中国古老月季‘月月粉’为研究对象,采用同源克隆结合‘月月粉’全基因组序列,获得了RcAAT基因全长,采用实时荧光定量PCR对该基因在‘月月粉’不同部位和不同发育时期的表达进行分析,利用病毒诱导沉默系统(VIGS),完成RcAAT在植株内的瞬时功能表达。具体研究结果如下:(1)根据GenBank已发表其它物种的香叶醇乙酰基转移酶基因,获取序列并设计引物,扩增出长度为188bp的片段,结合‘月月粉’全基因组序列和PCR扩增,获得了RcAAT基因的cDNA全长,对其进行生物信息分析,结果显示RcAAT是一个1641bp的核苷酸序列,其核苷酸序列包含1个68bp的5′-UTR,1个1371bp的OFR,1个202bp的3′-UTR。该基因在69~71位为起始密码子ATG,下游有同框终止密码子TAA和polyA尾。氨基酸同源序列对比发现,月季RcAAT与玫瑰和草莓具有较高的同源性。其中与玫瑰的同源性为87%,与草莓同源性为67%。(2)采用实时荧光定量PCR对RcAAT在‘月月粉’不同发育时期和不同组织部位的表达进行分析。研究表明,在不同组织部位中RcAAT表达量差异显著,茎的表达量最低,雄蕊表达量最高;在花朵不同发育时期中,RcAAT表达量随着花瓣开放程度表达量逐渐增加,在花蕾期几乎无表达,在盛开期花瓣表达量到达顶峰,而后下降。(3)构建病毒诱导的基因沉默载体RcAAT-TRV2。‘月月粉’顶芽置于菌液中抽真空完成侵染,将侵染枝条嫁接到砧木上,4-5周后嫁接芽开花,利用气象色谱质谱联用仪(GC/MS),对成功沉默RcAAT的‘月月粉’花瓣进行花香成分分析。结果表明,香叶醇及乙酸香叶酯等含量被相应的降低,表明RcAAT基因主要参与了香叶醇和乙酸香叶酯的合成。
[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.
【学位授予单位】：云南农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S685.12

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1 李晋华;月月粉乙酰基转移酶基因RcAAT的克隆及功能分析[D];云南农业大学;2017年

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