猪CHPT1基因的发现及其表达谱分析

Detection and expression profile of procine CHPT1 gene

【目的】对猪脂肪组织RNA的Solexa测序结果进行分析整理,发现猪新转录本CHPT1基因。研究CHPT1在猪不同组织及脂肪细胞不同分化阶段的表达谱,以便为后续的基因功能研究奠定基础。【方法】取猪的背部脂肪组织提取RNA,建立cDNA文库,进行Solexa测序,对测序结果进行生物信息学分析。利用反转录聚合酶链式反应(RT-PCR)及实时荧光定量PCR,克隆猪CHPT1基因的CDs区部分序列,并对其在不同组织及脂肪细胞不同分化阶段的表达谱进行分析。【结果】由Solexa测序分析结果发现,猪存在新转录本基因CHPT1,其对应的Tag表达量在180日龄大白猪与240日龄荣昌猪的脂肪组织中无明显差异,在3日龄与240日龄荣昌猪脂肪组织中表达差异达到20倍以上。克隆获得了其CDs区193bp的cDNA序列。实时荧光定量PCR检测结果表明,CHPT1基因在3日龄和180日龄大白猪的各个组织中均有表达,其中在肝脏和肾脏中表达量较高,且表达量有差异。3日龄与180日龄大白猪相比,肾脏和脂肪组织中CHPT1的表达量均有显著差异。在猪前体脂肪细胞的时序表达结果中,CHPT1表达量呈先增加后降低的趋势,其中第2天表达量明显升高,于第6天达到最大值,之后降低。【结论】猪存在CHPT1基因,其在猪的各个组织中都有表达,对猪的脂肪沉积可能发挥着重要作用。

[Objective] By arranging the result of Solexa sequencing,we discovered the new transcrip- tion of porcine CHPT1. Swine＇s different tissue and adipocyte at different differentiation stage expression profile of the CHPT1 was examined to provide an experiment material of gene founction subsequent re- search. [Method] Bioinformatics analysis of the Solexa sequence expression was studied by Reverse-tran- scription polymerase chain reaction（RT-PCR） and realtime fluorescence quantitative PCR. We discovered and cloned a part of CDs sequence of CHPT1 ,analyzed its tissue expression profile in swine＇s different tis- sue and adipocyte different differentiation stages. [Result] Through analyzing the Solexa sequence,we dis- covered a new transcription of procine CHPT1 ,adipose tissue expression of the gene＇s counterpart Tag on 180-day-old Large White and 240-day-old Rongchang pig hadn＇t difference, the adipose tissue expression of the gene＇s counterpart Tag on 3-day-old and 240-day-old Rongchang pig had as high as 20 multiple＇ s differences. The sequence of CDs area was cloned, CDs fragment 193 bp long was obtained. The realtime fluorescence quantitative PCR detection result showed that the gene is widely expressed in 3-day-old and 180-day-old Large White. The different tissues have different expressions, and high expression exists inkidney and liver. By comparision, there are remarkable differences in the kidney tissue and adipose tissue expression was significantly different. The control expression of CHPT1 in porcine preadipocytes results showed that the expression of CHPT1 increased in the beginning, and then became lower. The expression increased obviously on the second day,the highest expression level was on the sixth day. [Conclusion] The experimental results demonstrate that the gene of CHPT1 has expressed in swine＇s every tissue. CHPT1 may play an important role in swine fatty deposition.