CAJ | 학술논문

以健康白绒乌骨鸡肌肉、肾、肝、肌胃和皮肤为试验素材,应用实时荧光定量聚合酶链反应技术探讨β‐肌动蛋白(beta‐actin ,ACTB)、3‐磷酸甘油醛脱氢酶(glyceraldehyde‐3‐phosphate dehydrogenase ,GA PDH)和18S核糖体RNA(18S ribosomal RNA ,18S rRNA )3个内参基因的组织表达稳定性及其对酪氨酸酶( tyrosinase ,TYR)、小眼畸形相关转录因子(microphthalmia‐associated transcription factor ,MITF)和刺鼠信号蛋白(agouti signaling pro tein ,A S I P)等目的基因在白绒乌骨鸡各组织表达水平的影响,并采用紫外分光光度法分析各组织黑色素沉积规律,从中筛选出理想的内参基因.geNorm和NormFinder软件分析结果显示,内参基因GA PD H在白绒乌骨鸡各组织表达稳定性最高,ACTB表达稳定性最低.相对定量和黑色素沉积结果表明,GA PDH作为内参基因时,各组织 MITF、TYR基因的相对表达量与黑色素沉积规律基本一致,表现为皮肤＞肾＞肌胃＞肝＞肌肉；ASIP基因的相对表达量与黑色素沉积规律正好相反,表现为肌肉＞肝＞肌胃＞肾＞皮肤,这与各目的基因对黑色素沉积的调控功能相符,即 MITF和 TYR 基因高表达有利于黑色素沉积,ASIP基因高表达则抑制黑色素沉积.而18S rRNA和ACTB作为内参基因时,各目的基因表达量未表现出上述规律,与其各自对黑色素沉积的调控功能不相吻合,没有达到理想的校正效果.表明 GA PD H基因稳定性最好,且能真正体现目的基因对黑色素沉积的表达调控,是校正目的基因在白绒乌骨鸡不同组织中mRNA表达量的最优内参基因.
이건강백융오골계기육、신、간、기위화피부위시험소재,응용실시형광정량취합매련반응기술탐토β‐기동단백(beta‐actin ,ACTB)、3‐린산감유철탈경매(glyceraldehyde‐3‐phosphate dehydrogenase ,GA PDH)화18S핵당체RNA(18S ribosomal RNA ,18S rRNA )3개내삼기인적조직표체은정성급기대락안산매( tyrosinase ,TYR)、소안기형상관전록인자(microphthalmia‐associated transcription factor ,MITF)화자서신호단백(agouti signaling pro tein ,A S I P)등목적기인재백융오골계각조직표체수평적영향,병채용자외분광광도법분석각조직흑색소침적규률,종중사선출이상적내삼기인.geNorm화NormFinder연건분석결과현시,내삼기인GA PD H재백융오골계각조직표체은정성최고,ACTB표체은정성최저.상대정량화흑색소침적결과표명,GA PDH작위내삼기인시,각조직 MITF、TYR기인적상대표체량여흑색소침적규률기본일치,표현위피부＞신＞기위＞간＞기육；ASIP기인적상대표체량여흑색소침적규률정호상반,표현위기육＞간＞기위＞신＞피부,저여각목적기인대흑색소침적적조공공능상부,즉 MITF화 TYR 기인고표체유리우흑색소침적,ASIP기인고표체칙억제흑색소침적.이18S rRNA화ACTB작위내삼기인시,각목적기인표체량미표현출상술규률,여기각자대흑색소침적적조공공능불상문합,몰유체도이상적교정효과.표명 GA PD H기인은정성최호,차능진정체현목적기인대흑색소침적적표체조공,시교정목적기인재백융오골계불동조직중mRNA표체량적최우내삼기인.
Summary Silky Fowl is a precious local breed containing a large amount of melanin . Therefore ,it is considered to have high edible and medicinal value . Study on the related gene expression is helpful to reveal the internal regulation mechanism of melanin deposition . TYR (tyrosinase) , MITF (microphthalmia‐associated transcription factor) and ASIP(agoutisignalingprotein) areimportantcandidategenesfortheformationofpigment.Real‐time fluorescent quantitative polymerase chain reaction ( RT‐qPCR) has been widely applied to analyze gene expression as a convenient approach , and the selection of appropriate reference genes according to specific samples or conditions is critical to ensure the accuracy and reliability of gene expression . The objectives of this study are to screen the ideal reference genes and to provide a necessary basis for further study on gene expression regulation of melanin deposition and other important traits in Silky Fowl through investigating the expression stability of reference genes ( ACTB , GA PDH and 18S rRNA ) and their effects on the expression of melanin genes ( TYR , MITF and ASIP) in tissues of Silky Fowl . RT‐qPCR was adopted to investigate mRNA expression levels of the related genes in the muscle , kidney , liver , gizzard and skin of Silky Fowl , and the ultraviolet spectrophotometry was used to measure the melanin content in tissues of Silky Fowl . GeNorm and NormFinder softwares were applied to evaluate the expression stability of reference genes , and 2- △ △ CT method was applied to calculate the relative expression of target gene . The results from RT‐qPCR showed that the melting curve displayed an obvious single peak and standard curves had good linear relationship . Comprehensive evaluation of geNorm and NormFinder indicated that the expression stability of GA PDH was the highest , while ACTB was the lowest among the three reference genes in tissues . Relative quantity and melanin content analysis demonstrated that taking GA PD H as a reference gene , the expressionlevelof TYR,ASIPandMITFgeneswiththemelanincontentintissueshadthesametrend:skin >kidney > gizzard > liver > muscle , but the expression level of A SIP gene was opposite to melanin deposition content , which implied that each target gene expression was matched with their respective function on regulating melanin deposition . For instance , high expression of TYR and MITF genes promoted melanin deposition , while the ASIP gene inhibited melanin deposition . Taking 18S rRNA and ACTB as reference genes , the expression of TYR,MITFandASIPgeneswerenotrelatedtothemelanindeposition,andanidealcalibrationresultcouldnot be obtained . In conclusion , reference gene plays an important role in the expression of target gene . In this paper , GA PD H gene not only had highest expression stability in tissues , but also could truly reflect the regulation effect of the targetgenes(TYR,MITFandASIP) onmelanindeposition.Thus,GAPDHgeneisthemostsuitablereference gene for normalizing the mRNA expression of genes in tissues of Silky Fowl .