太行鸡CCNB2基因剪接异构体的克隆鉴定及其在卵泡发育中的表达模式分析

doi:10.16431/j.cnki.1671-7236.2022.10.004

摘要/Abstract

摘要： 【目的】试验旨在克隆鸡细胞周期素B2(cyclin B2,CCNB2)基因不同剪接异构体,并对其进行生物信息学和组织表达分析。【方法】采用反转录PCR技术对太行鸡CCNB2基因不同剪接异构体进行扩增和克隆,采用生物信息学软件对CCNB2基因不同剪接异构体的部分核苷酸序列进行比对,比较不同物种CCNB2不同剪接异构体的氨基酸序列相似性并构建系统进化树,对其进行亚细胞定位及二级结构预测。利用实时荧光定量PCR技术分析CCNB2基因不同剪接异构体在太行鸡卵巢和不同发育时期卵泡中的相对表达量。【结果】太行鸡CCNB2基因存在3种剪接异构体,即CCNB2-AS1、CCNB2-AS2和CCNB2-AS3,分别编码390、383和401个氨基酸。太行鸡CCNB2-AS1、CCNB2-AS2和CCNB2-AS3 3个剪接体之间的氨基酸序列相似性均为97.00％,这3个剪接体均与原鸡的相似性最高,分别为99.71％、99.71%和100％。3个剪接异构体在进化上与原鸡亲缘关系最近,其次为雉鸡。亚细胞定位结果表明,3个剪接异构体均主要定位于细胞质;蛋白质二级结构分析显示,3种剪接异构体均以α-螺旋为主;CCNB2-AS1、CCNB2-AS2和CCNB2-AS3在太行鸡不同发育时期卵泡中的相对表达量存在差异,并且不同剪接异构体在同一时期卵泡中的表达趋势也存在差异,其中CCNB2-AS1在等级前卵泡中高表达;而CCNB2-AS2在大白卵泡中表达量显著高于其他时期(P＜0.05),且在其他时期均以较低水平表达;CCNB2-AS3的表达则与CCNB2-AS1呈现相反的表达趋势,在等级卵泡中高表达。【结论】本研究成功鉴定到太行鸡CCNB2基因的3种剪接异构体,其在各时期卵泡中均有表达,但在等级前卵泡中的表达趋势存在差异。

关键词: 太行鸡; CCNB2基因; 剪接异构体; 生物信息学分析; 组织表达

Abstract: 【Objective】 The aim of the experiment was to clone different splice isomers of cyclin B2 (CCNB2) gene in chicken,and analyze their bioinformatics and tissue expression.【Method】 Reverse transcription PCR was used to amplify and clone the different splice isomers of CCNB2 gene in Taihang chickens.Partial nucleotide sequences of different splice isomers of CCNB2 gene were compared by bioinformatics software, the amino acid sequence similarity of different splice isomers of CCNB2 in different species was compared, the phylogenetic tree was constructed,and its subcellular localization and secondary structure were predicted.The relative expression of different splice isoforms of CCNB2 gene in ovaries and follicles of Taihang chickens at different developmental stages were analyzed by Real-time quantitative PCR.【Result】 In Taihang chickens,the CCNB2 gene had three splicing isomers:CCNB2-AS1,CCNB2-AS2 and CCNB2-AS3,which encoded 390,383 and 401 amino acids,respectively.The amino acid sequence similarity among the three splices CCNB2-AS1,CCNB2-AS2 and CCNB2-AS3 of Taihang chickens was 97.00%,the three spliceosomes had the highest similarity with Gallus,which were 99.71%,99.71% and 100%, respectively.The three splice isomers were evolutionarily closest to Gallus gallus,followed by Phasianus colchicus.Subcellular localization showed that the three splice isoforms were mainly localized in the cytoplasm.Protein secondary structure analysis showed that the three splice isomers were alpha helix.The relative expression levels of CCNB2-AS1,CCNB2-AS2 and CCNB2-AS3 in the follicles of Taihang chickens at different developmental stages were different,and the expression trends of different splice isomers in the follicles at the same stage were also different. CCNB2-AS1 was highly expressed in pregrade follicles.The expression of CCNB2-AS2 in great white follicles was significantly higher than that in other stages (P＜0.05).The expression of CCNB2-AS3 showed an opposite trend to that of CCNB2-AS1,and was highly expressed in graded follicles.【Conclusion】 In this study,three splice isoforms of CCNB2 gene in Taihang chickens were successfully identified,which were expressed in follicles at all stages,but there were differences in the expression trend in pregrade follicles.

Key words: Taihang chickens; CCNB2 gene; splicing isoforms; bioinformatics analysis; tissue expression

中图分类号:

S831.2

魏佳荣, 张贝贝, 马腾壑, 李梦霄, 李雪男, 王斌, 付冠华, 王红娜, 刘超. 太行鸡CCNB2基因剪接异构体的克隆鉴定及其在卵泡发育中的表达模式分析[J]. 中国畜牧兽医, 2022, 49(10): 3726-3735.

WEI Jiarong, ZHANG Beibei, MA Tenghe, LI Mengxiao, LI Xuenan, WANG Bin, FU Guanhua, WANG Hongna, LIU Chao. Cloning and Identification of Splice Isoforms of CCNB2 Gene and Their Expression Patterns in Follicular Development in Taihang Chickens[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3726-3735.

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