【摘要】：為比較甘油醛-3-磷酸脫氫酶(glyceraldehyde 3-phosphate dehydrogenase,GAPDH)、18S rRNA和β-actin基因在脊尾白蝦(Exopalaemon carinicauda)作內(nèi)參基因的優(yōu)劣,本研究采用同源克隆和RACE技術(shù),克隆了脊尾白蝦GAPDH基因全長cDNA序列(GenBank登錄號:KX893516),通過實(shí)時(shí)熒光定量PCR(quantative real-time PCR,qPT-PCR)技術(shù),檢測3種基因在脊尾白蝦不同組織及不同蛻殼后時(shí)間點(diǎn)的表達(dá)量變化,在此基礎(chǔ)上進(jìn)行內(nèi)參穩(wěn)定性分析。結(jié)果顯示,脊尾白蝦GAPDH基因全長1514 bp,開放讀碼框1002 bp,編碼333個(gè)氨基酸,二級結(jié)構(gòu)預(yù)測顯示GAPDH蛋白具有一個(gè)高度保守的NAD~+結(jié)合功能域(NAD binding domain)和行使糖運(yùn)輸和代謝的催化功能域。分析qRT-PCR結(jié)果并結(jié)合ge Norm、Norm Finder和Best Keeper 3種軟件的分析發(fā)現(xiàn),在不同組織和不同蛻殼后時(shí)間點(diǎn),3種內(nèi)參基因的穩(wěn)定性由高到低依次為18S r RNA、GAPDH、β-actin。因此,在脊尾白蝦不同組織和不同蛻殼后時(shí)間點(diǎn)的定量分析中,選取單內(nèi)參基因時(shí),推薦使用18S rRNA為內(nèi)參基因,雙內(nèi)參時(shí)推薦18S rRNA和GAPDH,而18S rRNA、β-actin和GAPDH在其他生理?xiàng)l件下作內(nèi)參基因的穩(wěn)定性還有待進(jìn)一步研究。
[Abstract]:In order to compare the advantages and disadvantages of 18s rRNA and 尾 -actin gene of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal reference gene in Exopalaemon carinicauda, homologous cloning and race techniques were used in this study. The full-length cDNA sequence of GAPDH gene (GenBank accession number: KX893516) was cloned. The expression changes of three genes in different tissues and time points after molting were detected by real-time fluorescence quantitative PCR (quantative real-time PCR-qPT-PCR). On this basis, the stability of internal parameters is analyzed. The results showed that the GAPDH gene was 1514 BP in length and 1002 BP in the open reading frame, encoding 333 amino acids. The secondary structure prediction showed that the GAPDH protein had a highly conserved NAD- binding domain (NAD binding domain) and a catalytic domain for glucose transport and metabolism. By analyzing the results of qRT-PCR and combining with three kinds of software, GE Norm Finder and Best Keeper, it was found that the stability of the three internal reference genes was 18s r RNA-GAPDH, 尾 -actinin from high to low in different tissues and at different time points after molting. Therefore, in the quantitative analysis of different tissues and post-molting time points of white shrimp, 18s rRNA is recommended to be used as internal reference gene when single internal reference gene is selected. The stability of 18s rRNA, 尾 -actin and GAPDH genes under other physiological conditions should be further studied.
【作者單位】： 淮海工學(xué)院海洋生命與水產(chǎn)學(xué)院江蘇省海洋生物技術(shù)重點(diǎn)實(shí)驗(yàn)室;江蘇省海洋生物產(chǎn)業(yè)技術(shù)協(xié)同創(chuàng)新中心;江蘇省農(nóng)業(yè)種質(zhì)資源保護(hù)與利用平臺;中國水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所;
【基金】：江蘇省農(nóng)業(yè)科技支撐計(jì)劃項(xiàng)目(BE2013363) 江蘇省高�！扒嗨{(lán)工程”培養(yǎng)基金項(xiàng)目 江蘇省2015年度普通高校研究生科研創(chuàng)新計(jì)劃項(xiàng)目(KYLX15_1486) 連云港市產(chǎn)學(xué)研合作項(xiàng)目(CXY1517) 淮海工學(xué)院江蘇省海洋生物技術(shù)重點(diǎn)實(shí)驗(yàn)室研究基金項(xiàng)目(2015HS001)
【分類號】：S917.4

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