CAJ | 학술논문

旨在根据GenBank 上公布的原鸡GnRHR基因序列设计2对引物，采用RT-PCR方法，从京海黄鸡肌肉组织中克隆GnRHR基因的编码区序列，并使用多种生物软件和在线工具对目的序列进行生物信息学分析，分析GnRHR基因与其他物种的同源性、蛋白质的理化性质、蛋白质序列的跨膜区、亚细胞定位、亲水性、潜在的磷酸化位点、保守结构域及该基因编码蛋白的二级结构、三级结构等。同时为GnRHR基因和β-actin基因各设计1对引物，采用RT-qPCR的方法研究GnRHR基因在京海黄鸡12个组织和器官中的表达情况。最终成功克隆了京海黄鸡GnRHR基因完整的编码区序列和部分侧翼序列，Blast结果显示，京海黄鸡GnRHR基因与原鸡、番鸭、藏羚羊、野猪、马、小鼠、大鼠、家兔、绵羊、人、牛、黑猩猩和斑马鱼的同源性分别为99．7％，86．7％，55．7％，54．6％，52％，51．6％，50．8％，50％，49．9％，49.6％，49．4％，47．4％和39．3％，并成功构建系统发育树。蛋白质结构分析显示，GnRHR蛋白分子量为45．432 kDa，理论等电点为9．55，包含20种氨基酸，其中，亮氨酸含量最高，占13．8％，赖氨酸含量最低，占0．7％；不稳定系数为65．35，显示该蛋白不稳定；脂溶指数为95．54，总平均疏水指数为0．312，为非水溶性蛋白；该蛋白不属于分泌型蛋白，主要存在于胞膜上，没有信号肽结构，存在16个潜在磷酸化位点和11个糖基化位点；保守结构域分析显示存在2个低复杂度序列和7段跨膜结构，跨膜分析显示该蛋白为7次跨膜蛋白，二级结构预测结果显示，在GnRHR二级结构中，α-螺旋占29．83％，β-折叠占17．18％，无规则卷曲占52．98％，GnRHR蛋白三级结构为复杂的7次跨膜螺旋结构，且为单链蛋白。组织表达分析显示，GnRHR基因主要在垂体中高表达，表达量显著高于其他组织，在其他11个组织和器官中表达量较低。旨在根據GenBank 上公佈的原鷄GnRHR基因序列設計2對引物，採用RT-PCR方法，從京海黃鷄肌肉組織中剋隆GnRHR基因的編碼區序列，併使用多種生物軟件和在線工具對目的序列進行生物信息學分析，分析GnRHR基因與其他物種的同源性、蛋白質的理化性質、蛋白質序列的跨膜區、亞細胞定位、親水性、潛在的燐痠化位點、保守結構域及該基因編碼蛋白的二級結構、三級結構等。同時為GnRHR基因和β-actin基因各設計1對引物，採用RT-qPCR的方法研究GnRHR基因在京海黃鷄12箇組織和器官中的錶達情況。最終成功剋隆瞭京海黃鷄GnRHR基因完整的編碼區序列和部分側翼序列，Blast結果顯示，京海黃鷄GnRHR基因與原鷄、番鴨、藏羚羊、野豬、馬、小鼠、大鼠、傢兔、綿羊、人、牛、黑猩猩和斑馬魚的同源性分彆為99．7％，86．7％，55．7％，54．6％，52％，51．6％，50．8％，50％，49．9％，49.6％，49．4％，47．4％和39．3％，併成功構建繫統髮育樹。蛋白質結構分析顯示，GnRHR蛋白分子量為45．432 kDa，理論等電點為9．55，包含20種氨基痠，其中，亮氨痠含量最高，佔13．8％，賴氨痠含量最低，佔0．7％；不穩定繫數為65．35，顯示該蛋白不穩定；脂溶指數為95．54，總平均疏水指數為0．312，為非水溶性蛋白；該蛋白不屬于分泌型蛋白，主要存在于胞膜上，沒有信號肽結構，存在16箇潛在燐痠化位點和11箇糖基化位點；保守結構域分析顯示存在2箇低複雜度序列和7段跨膜結構，跨膜分析顯示該蛋白為7次跨膜蛋白，二級結構預測結果顯示，在GnRHR二級結構中，α-螺鏇佔29．83％，β-摺疊佔17．18％，無規則捲麯佔52．98％，GnRHR蛋白三級結構為複雜的7次跨膜螺鏇結構，且為單鏈蛋白。組織錶達分析顯示，GnRHR基因主要在垂體中高錶達，錶達量顯著高于其他組織，在其他11箇組織和器官中錶達量較低。
지재근거GenBank 상공포적원계GnRHR기인서렬설계2대인물，채용RT-PCR방법，종경해황계기육조직중극륭GnRHR기인적편마구서렬，병사용다충생물연건화재선공구대목적서렬진행생물신식학분석，분석GnRHR기인여기타물충적동원성、단백질적이화성질、단백질서렬적과막구、아세포정위、친수성、잠재적린산화위점、보수결구역급해기인편마단백적이급결구、삼급결구등。동시위GnRHR기인화β-actin기인각설계1대인물，채용RT-qPCR적방법연구GnRHR기인재경해황계12개조직화기관중적표체정황。최종성공극륭료경해황계GnRHR기인완정적편마구서렬화부분측익서렬，Blast결과현시，경해황계GnRHR기인여원계、번압、장령양、야저、마、소서、대서、가토、면양、인、우、흑성성화반마어적동원성분별위99．7％，86．7％，55．7％，54．6％，52％，51．6％，50．8％，50％，49．9％，49.6％，49．4％，47．4％화39．3％，병성공구건계통발육수。단백질결구분석현시，GnRHR단백분자량위45．432 kDa，이론등전점위9．55，포함20충안기산，기중，량안산함량최고，점13．8％，뢰안산함량최저，점0．7％；불은정계수위65．35，현시해단백불은정；지용지수위95．54，총평균소수지수위0．312，위비수용성단백；해단백불속우분비형단백，주요존재우포막상，몰유신호태결구，존재16개잠재린산화위점화11개당기화위점；보수결구역분석현시존재2개저복잡도서렬화7단과막결구，과막분석현시해단백위7차과막단백，이급결구예측결과현시，재GnRHR이급결구중，α-라선점29．83％，β-절첩점17．18％，무규칙권곡점52．98％，GnRHR단백삼급결구위복잡적7차과막라선결구，차위단련단백。조직표체분석현시，GnRHR기인주요재수체중고표체，표체량현저고우기타조직，재기타11개조직화기관중표체량교저。
Based on the published mRNA nucleotide sequence of Gallus gallus GnRHR gene,two pair of prim-ers were designed to clone the GnRHR gene coding sequence of Jinghai yellow chicken by RT-PCR.A variety of software and online tools were used to analyze the homology among different species ,physical and chemical proper-ties,transmembrane region,subcellular localization,hydrophilic,potential phosphorylation locus,conserved domain database ,secondary structure and tertiary structure of GnRHR protein .Two pair of primers were designed to detect the tissue expression of GnRHR gene in twelve tissues of Jinghai yellow chicken by RT-qPCR.Finally,GnRHR gene was cloned which contained CDS region ,part of promoter region and 3′region.Result of Blast showed that GnRHR gene of Jinghai yellow chicken shared 99.7%,86.7%,55.7%,54.6%,52%,51.6%,50.8%,50%,49.9%, 49.6%,49.4%,47.4%and 39.3%identity with Gallus gallus,Cairina moschata,Pantholops hodgsoni,Sus scro-fa,horse,mice,rats,rabbits,sheep,cows,chimpanzees and zebrafish .Phylogenetic tree was constructed .Analysis of GnRHR protein structure showed that the molecular weight was 45 .432 kDa and pI was 9 .55 .GnRHR protein was consisted of twenty kinds of amino acids ,in which leucine accounted for the highest content of 13.8%and Lysine accounted for the lowest content of 0 .7%.The instability index was computed to be 65 .35 which classified the pro-tein as unstable .The grand average of hydropathicity was 0 .312 which showed that the protein was Non-water-solu-ble protein.GnRHR protein did not belong to secreted protein ,mainly presented on membrane ,with no signal peptide and contained 16 phosphorylation sites and 11 glycosylation sites .Analysis of conserved domains showed that GnRHR protein included two low complexity sequences and seven transmembrane segments which agreed with the transmem -brane analysis .The secondary structure of GnRHR was mainly composed of random coil .The tertiary structure of do-main area of GnRHR protein showed a helix and single strand structure .The result of tissue expression showed that GnRHR was mainly expressed in pituitary .The expression quantity of GnRHR in other eleven tissues was low .