优雅蝈螽海藻糖代谢相关基因cDNA全长克隆及表达分析

发布时间：2018-01-24 05:28

本文关键词： TPS基因 Tre基因海藻糖组织特异性表达可变多聚腺苷酸化　出处：《河北大学》2017年硕士论文　论文类型：学位论文

【摘要】：海藻糖是由两个D-吡喃葡萄糖以α1-α1糖苷键连接形成的非还原性二糖,在高温、寒冷、缺氧、干燥、脱水和营养匮乏等恶劣环境胁迫下,昆虫体内海藻糖能有效地稳定脱水酶,保护细胞膜和蛋白。本研究为海藻糖在提高昆虫抗逆性方面的研究提供理论基础。基于高通量转录组数据筛选,从优雅蝈螽中克隆TPS和Tre基因cDNA全长。旨在研究模拟野外温度骤变和缓慢升降温驯化对优雅蝈螽Gampsocleis gratiosa海藻糖合成酶(trehalose-6-phosphate synthase,TPS)基因表达量、血淋巴海藻糖和总糖含量的影响和三者间的相关性,以及海藻糖水解酶(trehalase,Tre)基因组织特异性表达。采取模拟野外温度骤变和缓慢升降温两种温度驯化策略,以热电偶温度计测得优雅蝈螽体温与环境温度最为接近的温度值作为最适温度,实时荧光定量PCR测定优雅蝈螽TPS不同温度表达和Tre表达的组织特异性,硫酸蒽酮法测定血淋巴海藻糖和总糖含量(以25℃最适温度作为对照)。克隆得到一个TPS基因,一个水溶性海藻糖水解酶(soluble trehalase,Tre1)基因和一个类膜结合型海藻糖水解酶(membrane-bound-like trehalase,Tre2-like)基因。TPS基因命名为GgTPS(GenBank登录号:KU578006),全长3 225 bp,包含2 430 bp的开放阅读框,5′-非编码区(5′-untranslated region,5′-UTR)153 bp和3′-非编码区(3′-untranslated region,3′-UTR)642 bp。共编码809个氨基酸,预测的蛋白分子量(Mw)91.35 ku,理论等电点(p I)6.28。两个Tre基因分别命名为GgTre1,GgTre2-like。分离到3个不同的GgTre1转录本(GenBank登录号:KY400001~KY400003),全长分别为2 107,2 021,1 914 bp,5'-UTR 33 bp,3'-UTR长度分别为322,248,129 bp。转录本1和转录本3编码区长度为1 752 bp,编码583个氨基酸。蛋白分子量和等电点分别为67.88 ku和6.59。转录本2编码区长度1 740 bp,编码579个氨基酸,蛋白分子量和等电点分别为67.29 ku和6.34。得到3个不同的GgTre2-like转录本亚型(GenBank登录号:KY400004~KY400006),全长分别为2 491,2 460,2 381 bp,5'-UTR 284 bp,3'-UTR分别为398,367,285 bp。开放阅读框1 809 bp,编码602氨基酸,蛋白分子量和等电点分别为67.88 ku和6.59。骤然降温阶段TPS相对表达量呈先升高后降低的变化趋势,在15℃最高,与最适温度25℃差异显著。海藻糖相对含量在0℃显著高于25℃。总糖相对含量在0℃和10℃显著高于25℃。骤然升温阶段,TPS相对表达量呈先降低后升高,再降低的变化趋势,40℃达到最高且与25℃差异显著。缓慢降温阶段TPS相对表达量呈先升高后降低的变化趋势,在15℃和20℃显著高于25℃;海藻糖含量表现出先降低后升高的变化趋势,0℃显著高于25℃,总糖含量在0℃、10℃和20℃与25℃差异显著。缓慢升温阶段TPS相对表达量呈先降低后升高的变化趋势,但与25℃无显著差异。海藻糖相对含量在40℃显著低于25℃。总糖相对含量呈先升高后降低的变化趋势,40℃和45℃显著高于25℃。不同温度驯化策略比较TPS相对表达量在15℃、40℃差异显著。海藻糖相对含量在40℃和45℃差异显著。总糖相对含量在10℃和20℃差异显著。体温在30℃差异显著。GgTre1在雌性卵巢和雄性附腺表达量最高。GgTre2-like在卵巢中高表达,在雄性肌肉和马氏管高表达。实验结果表明,温度驯化能够提高昆虫的抗逆能力,昆虫体内TPS表达量和海藻糖合成量与其所处环境温度及作用时间密切相关。低、高温条件下TPS的表达和海藻糖的积累模式不同。基因组DNA序列表明2个GgTre基因3'-UTR逐渐增长由可变多聚腺苷酸化(alternative polyadenylation,APA)引起。GgTre2-like较GgTre1有相对稳定的表达趋势。两个GgTre基因表达的组织特异性不同。
[Abstract]:Trehalose is a non reducing sugar two, by two D- pyran glucose to alpha 1- alpha 1 glycosidic bond formation at high temperature, cold, hypoxia, drying, dehydration and lack of nutrition and other adverse environmental stress, insect trehalose can effectively stabilize dehydratase, protect the cell membrane and protein. The research of trehalose in to provide a theoretical basis to improve the study of insect resistance. The high throughput screening of transcriptome data based on the cloning of full-length TPS and Tre gene cDNA from GAMPSOCLEIS gratiosa. To study and simulate field temperature change and slow temperature acclimation on g.gratiosa Gampsocleis gratiosa (trehalose-6-phosphate synthase TPS, trehalose synthase) gene expression, correlation of hemolymph trehalose content and total sugar content and between the three, and trehalose hydrolysing enzymes (trehalase, Tre) tissue specific expression gene. By simulating the field temperature change and slow rise Two kinds of cooling temperature acclimation strategy, with thermocouple thermometer g.gratiosa temperature and ambient temperature is close to the temperature value as the optimum temperature, real-time fluorescence quantitative PCR determination of GAMPSOCLEIS gratiosa TPS at different temperatures and expression of Tre tissue specificity, determination of hemolymph trehalose and total sugar content of sulfuric acid method (with the most the optimum temperature of 25 DEG C as control). Cloned a TPS gene, a water soluble trehalase (soluble trehalase Tre1) gene and a class of membrane-bound trehalase (membrane-bound-like trehalase Tre2-like).TPS gene, named GgTPS (GenBank accession number: KU578006), a total length of 3225 BP, contains 2430 BP open reading frame, 5 '- non encoding region (5' -untranslated region 5 '-UTR BP 3' - 153) and non encoding region (3 '-untranslated region 3', -UTR) of 642 bp. encoding 809 amino acids, forecast The molecular weight of the protein (Mw) 91.35 Ku, isoelectric point (P I) 6.28. two Tre genes were named GgTre1, GgTre2-like. were isolated from 3 different GgTre1 transcripts (GenBank accession number: KY400001~KY400003), full length is 2 107,2 021,1 914 BP, 5'-UTR 33 BP, 3'-UTR length were 322248129 bp. 1 transcripts and transcripts encoding 3 length is 1752 BP, encoding 583 amino acids. The protein molecular weight and isoelectric point were 67.88 Ku and 2 6.59. transcripts encoding length of 1740 BP, encoding 579 amino acids. The protein molecular weight and isoelectric point were 67.29 Ku and 6.34. are 3 different GgTre2-like isoforms (GenBank accession number: KY400004~KY400006), full length is 2 491,2 460,2 381 BP, 5'-UTR 284 BP, 3'-UTR respectively 398367285 bp. open reading frame of 1809 BP encoding 602 amino acids. The protein molecular weight and isoelectric point were 67.88 Ku and 6.59 Suddenly. The relative expression of TPS in the cooling stage then decreased, the highest in 15 degrees, and the optimum temperature of 25 significant differences. The relative content of trehalose in 0 C was significantly higher than that of 25 degrees. The relative content of total sugar was significantly higher than 25 DEG C in 0 degrees and 10 degrees. The sudden warming stage. The relative expression of TPS the amount was decreased at first and then increased, then decreased, and reached the highest 40 DEG and 25 DEG C respectively. The relative expression of TPS in slow cooling stage then decreased, significantly higher than 25 DEG C in 15 degrees and 20 degrees; the change trend of trehalose content showed first decreased and then increased 0. C was significantly higher than 25 DEG C, the total sugar content at 0 degrees, 10 degrees and 20 degrees and 25 degrees respectively. The slow heating stage the relative expression of TPS increased, but no significant difference with 25 degrees. The relative content of trehalose was significantly lower than 25 DEG C at 40 degrees. The relative content of total sugar The amount then decreased, 40 degrees and 45 degrees were significantly higher than 25 degrees Celsius. The relative expression of different temperature acclimation at 15 DEG C 40 TPS strategy, the difference was significant. The relative content of trehalose significantly at 40 degrees and 45 degrees difference. The relative content of total sugar was 10 degrees and 20 degrees difference in temperature. In 30 C.GgTre1 significant differences in female and male accessory gland ovary had the highest expression of high expression of.GgTre2-like in ovary, muscle and Malpighian tubules in male high expression. The experimental results show that temperature acclimation could improve the resistance of insects, insect TPS expression and production of trehalose synthase and its environment temperature and time close related. Low expression of TPS and trehalose accumulation mode under high temperature. The genomic DNA sequence of GgTre gene showed that 2 3'-UTR increased by a variable polyadenylation (alternative polyadenylation APA) by.GgTre2 -like has a relatively stable expression trend compared with GgTre1. The expression of two GgTre genes is different in tissue specificity.

【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q965;Q78

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