本文關(guān)鍵詞： TPS基因 Tre基因 海藻糖 組織特異性表達(dá) 可變多聚腺苷酸化　出處：《河北大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：海藻糖是由兩個D-吡喃葡萄糖以α1-α1糖苷鍵連接形成的非還原性二糖,在高溫、寒冷、缺氧、干燥、脫水和營養(yǎng)匱乏等惡劣環(huán)境脅迫下,昆蟲體內(nèi)海藻糖能有效地穩(wěn)定脫水酶,保護(hù)細(xì)胞膜和蛋白。本研究為海藻糖在提高昆蟲抗逆性方面的研究提供理論基礎(chǔ)�；诟咄哭D(zhuǎn)錄組數(shù)據(jù)篩選,從優(yōu)雅蟈螽中克隆TPS和Tre基因cDNA全長。旨在研究模擬野外溫度驟變和緩慢升降溫馴化對優(yōu)雅蟈螽Gampsocleis gratiosa海藻糖合成酶(trehalose-6-phosphate synthase,TPS)基因表達(dá)量、血淋巴海藻糖和總糖含量的影響和三者間的相關(guān)性,以及海藻糖水解酶(trehalase,Tre)基因組織特異性表達(dá)。采取模擬野外溫度驟變和緩慢升降溫兩種溫度馴化策略,以熱電偶溫度計(jì)測得優(yōu)雅蟈螽體溫與環(huán)境溫度最為接近的溫度值作為最適溫度,實(shí)時熒光定量PCR測定優(yōu)雅蟈螽TPS不同溫度表達(dá)和Tre表達(dá)的組織特異性,硫酸蒽酮法測定血淋巴海藻糖和總糖含量(以25℃最適溫度作為對照)�？寺〉玫揭粋�TPS基因,一個水溶性海藻糖水解酶(soluble trehalase,Tre1)基因和一個類膜結(jié)合型海藻糖水解酶(membrane-bound-like trehalase,Tre2-like)基因。TPS基因命名為GgTPS(GenBank登錄號:KU578006),全長3 225 bp,包含2 430 bp的開放閱讀框,5′-非編碼區(qū)(5′-untranslated region,5′-UTR)153 bp和3′-非編碼區(qū)(3′-untranslated region,3′-UTR)642 bp。共編碼809個氨基酸,預(yù)測的蛋白分子量(Mw)91.35 ku,理論等電點(diǎn)(p I)6.28。兩個Tre基因分別命名為GgTre1,GgTre2-like。分離到3個不同的GgTre1轉(zhuǎn)錄本(GenBank登錄號:KY400001~KY400003),全長分別為2 107,2 021,1 914 bp,5'-UTR 33 bp,3'-UTR長度分別為322,248,129 bp。轉(zhuǎn)錄本1和轉(zhuǎn)錄本3編碼區(qū)長度為1 752 bp,編碼583個氨基酸。蛋白分子量和等電點(diǎn)分別為67.88 ku和6.59。轉(zhuǎn)錄本2編碼區(qū)長度1 740 bp,編碼579個氨基酸,蛋白分子量和等電點(diǎn)分別為67.29 ku和6.34。得到3個不同的GgTre2-like轉(zhuǎn)錄本亞型(GenBank登錄號:KY400004~KY400006),全長分別為2 491,2 460,2 381 bp,5'-UTR 284 bp,3'-UTR分別為398,367,285 bp。開放閱讀框1 809 bp,編碼602氨基酸,蛋白分子量和等電點(diǎn)分別為67.88 ku和6.59。驟然降溫階段TPS相對表達(dá)量呈先升高后降低的變化趨勢,在15℃最高,與最適溫度25℃差異顯著。海藻糖相對含量在0℃顯著高于25℃�？偺窍鄬吭�0℃和10℃顯著高于25℃。驟然升溫階段,TPS相對表達(dá)量呈先降低后升高,再降低的變化趨勢,40℃達(dá)到最高且與25℃差異顯著。緩慢降溫階段TPS相對表達(dá)量呈先升高后降低的變化趨勢,在15℃和20℃顯著高于25℃;海藻糖含量表現(xiàn)出先降低后升高的變化趨勢,0℃顯著高于25℃,總糖含量在0℃、10℃和20℃與25℃差異顯著。緩慢升溫階段TPS相對表達(dá)量呈先降低后升高的變化趨勢,但與25℃無顯著差異。海藻糖相對含量在40℃顯著低于25℃�？偺窍鄬砍氏壬吆蠼档偷淖兓厔�,40℃和45℃顯著高于25℃。不同溫度馴化策略比較TPS相對表達(dá)量在15℃、40℃差異顯著。海藻糖相對含量在40℃和45℃差異顯著。總糖相對含量在10℃和20℃差異顯著。體溫在30℃差異顯著。GgTre1在雌性卵巢和雄性附腺表達(dá)量最高。GgTre2-like在卵巢中高表達(dá),在雄性肌肉和馬氏管高表達(dá)。實(shí)驗(yàn)結(jié)果表明,溫度馴化能夠提高昆蟲的抗逆能力,昆蟲體內(nèi)TPS表達(dá)量和海藻糖合成量與其所處環(huán)境溫度及作用時間密切相關(guān)。低、高溫條件下TPS的表達(dá)和海藻糖的積累模式不同�；蚪MDNA序列表明2個GgTre基因3'-UTR逐漸增長由可變多聚腺苷酸化(alternative polyadenylation,APA)引起。GgTre2-like較GgTre1有相對穩(wěn)定的表達(dá)趨勢。兩個GgTre基因表達(dá)的組織特異性不同。
[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.

【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q965;Q78

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