绿盲蝽AOX、ATF和DES基因的克隆与功能分析

发布时间：2018-06-01 06:53

本文选题：绿盲蝽 + 性信息素　；参考：《华中农业大学》2016年硕士论文

【摘要】：绿盲蝽(Apolygus lucorum)是我国危害棉花的盲蝽蟓优势种之一,目前主要依赖化学防治,研究绿盲蝽体内重要基因的功能,对探寻绿色安全的防治措施具有重要意义。盲蝽性信息成分主要为短碳链的醛和酯组成,前人研究报道醇氧化酶(alcohol oxidase,AOX)、乙酰基转移酶(acetyltransferase,ATF)和去饱和酶(desaturase,DES)是醛和酯类物质的合成过程中的重要酶类,但它们在绿盲蝽中的功能并不清楚,因此本文开展了这三类基因在绿盲蝽体内的功能研究。RNA注射干扰是本文基因功能研究的重要手段,我们首先研究了RNA注射干扰绿盲蝽的最佳注射体积,结果表明羽化第一天的绿盲蝽成虫的最佳注射体积为100n L。本研究采用荧光定量PCR研究了8个ATF基因,2个AOX和1个DES基因在性信息素释放高峰期和未成熟雌雄虫臭腺中的表达差异,结果表明ATF基因NAA25在3龄绿盲蝽雌虫臭腺中特异性表达的特异性高表达,因此NAA25可能与绿盲蝽性信息素合成相关。本研究采用显微注射法对绿盲蝽的ATF基因NAA25、NAA20和NAA15进行了干扰,其中干扰NAA25后,性成熟绿盲蝽雌虫体内成熟卵子数量降低了87.1%(p0.005),干扰NAA20后,性成熟绿盲蝽雌虫体内成熟卵子数量降低了95.6%(p0.005),干扰NAA15后,性成熟绿盲蝽雌虫体内成熟卵子数量降低了98.8%(p0.005)。利用实时荧光定量检测了这3个基因从羽化第一天到性成熟时在全虫中的表达量,这3个基因均在羽化第一天和性成熟前一天高表达,由此我们推测这3个基因在卵巢发育中起着重要的功能。在本研究中,我们通过RACE技术获得2个N-alpha-acetyltransferase(NAA)基因的全长。其中N-alpha-acetyltransferase 25(NAA25)属于Nat B复合酶的非催化亚基,N-alpha-acetyltransferase 15(NAA15)属于Nat A复合酶的辅助亚基。其中NAA25序列全长为2898bp,ORF(open reading frame)区域为2064bp,编码687个氨基酸。NAA15序列全长为1103bp,ORF区域为759bp,编码252个氨基酸。
[Abstract]:Apolygus lucorum (Apolygus lucorum) is one of the dominant species that harm cotton in China. At present, it is very important to study the function of important genes in the plant by chemical control. It is of great significance to explore the control measures of green safety. The sex information components of stink bug mainly consist of aldehydes and esters of short carbon chain. Previous studies have reported that alcohol oxidase, acetyltransferase (ATF) and desaturase (DES) are important enzymes in the synthesis of aldehydes and esters, and some previous studies have reported that alcohol oxidase, acetyltransferase (ATF) and desaturase (DES) are important enzymes in the synthesis of aldehydes and esters. However, the function of these three genes in green bug is not clear. RNA injection interference is an important method to study the gene function in this paper. We first studied the optimal injection volume of RNA to interfere with the green bug. The results showed that the best injection volume of the adult was 100nL on the first day of Eclosion. In this study, the expression of 8 ATF genes, 2 AOX genes and 1 DES gene were studied by fluorescence quantitative PCR in the osmidine glands of male and female females during the peak period of sex pheromone release. The results showed that the specific expression of ATF gene NAA25 was highly specific in the stink glands of the third instar female bugs, so NAA25 might be related to the synthesis of sex pheromones. In this study, the microinjection method was used to interfere with the ATF gene NAA25, NAA20 and NAA15. After interfering with NAA25, the number of mature eggs decreased by 87.1% (P 0.005) and NAA20 (P < 0.05), respectively. The number of mature eggs decreased by 95.6% (p 0.005), and the number of mature eggs decreased by 98.8% (P 0.005) after interfering with NAA15. The expression of these three genes in the whole insect from the first day of emergence to the first day of sexual maturity was detected by real-time fluorescence quantitative analysis. The three genes were highly expressed on the first day of emergence and the day before sexual maturation. Therefore, we speculate that these three genes play an important role in ovarian development. In this study, we obtained two full length N-alpha-acetyltransferase NAA genes by RACE technique. Among them, N-alpha-acetyltransferase 25 (NAA25) belongs to the non-catalytic subunit N-alpha-acetyltransferase 15 (NAA15) of Nat B complex enzyme. It belongs to the auxiliary subunit of Nat A complex enzyme. The total length of NAA25 sequence is 2898bpSORF open reading frame region is 2064bp. the total length of NAA25 sequence encoding 67-amino acid. NAA15 sequence is 1103bpHORF region is 759bp, encoding 252amino acid.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433

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