17α,20β双羟孕酮诱导鲤卵母细胞最终成熟相关基因表达内参基因的筛选

Reference gene validation for quantification of gene expression during final oocyte maturation induced by 17α, 20β-dihydroxy-4-pregnen-3-one in common carp(Cyprinus carpio)

本研究检测了18S rRNA(18S)、28S rRNA(28S)、组织蛋白酶Z(cathepsin Z,CTSZ)、延伸因子1-α(elongation factor 1-α,EF-1α)、3-磷酸甘油脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,gapdh)、β肌动蛋白(β-actin)6个候选内参基因在17α,20β双羟孕酮(DHP)诱导鲤(Cyprinus carpio)卵母细胞最终成熟过程的表达情况,并应用不同的内参分析软件对数据进行了分析。Bestkeeper软件分析表明EF-1α的变异系数和标准差是6个候选内参基因中最低的,且Bestkeeper指数在6个候选内参基因中最高,说明其表达稳定性最高,适合做为内参基因;geNorm软件分析认为需要同时使用EF-1α和CTSZ两个内参基因来校正目标基因的表达;NormFinder软件分析同样表明EF-1α是6个候选内参基因中表达最稳定的;最后通过RefFinder软件综合比较分析,确定EF-1α相对于其他5个候选内参基因,在17α,20β双羟孕酮诱导卵母细胞最终成熟阶段表达最为稳定,可作为内参校正17α,20β双羟孕酮诱导鲤卵母细胞最终成熟阶段各基因的表达情况。

Fina oocyte mauraion is akey step for successful spaning ad fertilizaion. Like most other vertebraes, teleosts hae full-grown postvitellogenic oocytes in the ovay tha ae physiologicaly arested a the G2/M border in the first meiotic prophae ad canot be fertilized. Shortly before ovulaion, the oocytes must become fully maure, which involves breadown of the germina vesicle（GVBD）, chromosome condensaion, asembly of the meiotic spindle, ad exclusion of the first pola body. Meiosis is aan arested a the second metahae. The time period from the resumption of meiosis to the second meiotic metahae ha been referred to a fina oocyte mauraion. Knowledge of the molecula events ad the role of vaious fators involved in fina oocyte mauraion is afocus of reseach in the field of reproductive biology. Quatitaive rea-time PCR （qPCR） is often used to quatify trascript aundace in such studies; however the technique is subject to considerale experimenta error ad vaiaion. A staly expressed housekeeping gene is typicaly used a areference gene to normaize this vaiaion. However a idea universa reference gene tha is stale under al experimenta circumstaces ha not been described. Reseachers should vaidae their reference genes before performing qPCR aaysis. We documented the expression of six cadidae reference genes： 18S rRNA（18S）, 28S rRNA（28S）, cahepsin Z （CTSZ）, elongaion fator, glyceradehyde-3-phosphae dehydrogenae （gadh）, ad β-atin during 17α 20β-dihydroxy-4-pregnen-3-one （DHP） induced fina oocyte mauraion in common cap （Cyprinus capio）. We used arage of softwae to evauae the expression staility of these reference genes. Anaysis with Bestkeeper reveaed tha EF-lαha the lowest CV% （2.65） ad STDEV （0.71） vaues, but ha the highest Bestkeeper index vaue （0.956） aong the six cadidae reference genes, suggesting tha EF-lαwa the best reference gene in the present study. Anaysis with geNorm reveaed tha EF-lαad CTSZ ha the lowest M （gene expression staility） vaues which indicaed tha they were the most stale genes aong the six. Additionaly, the vaue of parwise vaiaion of these two genes （V2/3） wa much lower tha the defalt （0.15）, suggesting tha EF-lαad CTSZ should be used concurrently a areference gene par to normaize the expression of the taget genes. Consistently, aaysis with NormFinder ad RefFinder demonstraed tha EF-lα was the most stale gene aong the six cadidaes. Hence, EF-lαca serve a areference gene to normaize the expression of taget genes during fina oocyte mauraion induced by DHP in common cap. The mRNA expression of luteinizing hormone receptor gene（LHR） during fina oocyte mauraion induced by DHP wa normaized by 6 cadidae reference genes respectively, ad the relaive expression wa vaied significatly depending on the reference gene tha wa used. Selection of astaly expressed reference gene is critica for al qPCR aaysis to ensure acurae taget gene mRNA exoression informaion.