【摘要】 为了探究马鹿(Cervus elaphus)鹿茸软骨生成过程中TMEM183A(transmembrane protein 183A)基因可变剪接转录本的结构特征及不同转录本在前软骨和软骨组织间的表达差异，试验采集鹿茸前软骨和软骨组织，提取RNA并将其反转录为cDNA,采用PCR方法对TMEM183A基因可变剪接转录本的外显子3～8区域及整个编码序列(coding sequence, CDS)区进行扩增，测序后进行组装，获得各可变剪接转录本序列，将其与野生型(WT)转录本序列(登录号为XM＿043924710.1)的外显子进行比对，根据结构特征对可变剪接转录本进行分类，采用实时荧光定量PCR方法检测两种组织中TMEM183A基因不同类型可变剪接转录本表达丰度。结果表明：共获得9个新的TMEM183A基因可变剪接转录本(除已知的可变剪接转录本),分别命名为TMEM183A X10～18(已上传至GenBank中，登录号分别为PP816184、PP824820、PP831015、PP839277、PP839278、PP839279、PP839280、PP831016、PP831017)。针对某个可变剪接转录本来说，马鹿TMEM183A基因外显子3,8,9较少或不发生可变剪接，外显子跳跃(ES)多发生于外显子4～7区域范围内。其中外显子1存在3种不同长度，外显子3,4,7,8存在2种不同长度。这些可变剪接转录本与野生型之间的差异主要集中于中间区域及C端；其中7种新的TMEM183A基因可变剪接转录本F-Box结构域丢失。WT表达量在前软骨和软骨中均最高，且均显著高于其他类型(P<0.05);转录本一共可分为9类[野生型(wild type, WT)、外显子4～7跳跃(4-7ES)、外显子2,4～7跳跃(2,4-7ES)、外显子4～6跳跃，外显子7 5′可变剪接A5SS(4-6ES-7A5SS)、外显子4～6跳跃(4-6ES)、外显子5,6跳跃(5,6ES)、外显子6跳跃(6ES)、外显子6,7跳跃(6,7ES)、外显子4,6,7跳跃(4,6,7ES)]。在两种组织中，野生型转录本的相对表达丰度均显著高于其他8种类型可变剪接转录本(P<0.05)。在软骨组织中，6,7ES和6ES的相对表达丰度显著高于4-7ES、2,4-7ES、4-6ES-7A5SS、4-6ES和5,6ES(P<0.05)。除4-6ES-7A5SS的相对表达丰度在前软骨和软骨中差异不显著外(P>0.05),其他8个转录本的相对表达丰度在前软骨和软骨间差异显著或极显著(P<0.05或P<0.01、P<0.001),其中4-7ES、2,4-7ES、5,6ES在前软骨中的相对表达丰度极显著高于软骨(P<0.01、P<0.001),WT、6-4ES、6ES、6,7ES、4,6,7ES在软骨中的相对表达丰度显著或极显著高于前软骨(P<0.05或P<0.001)。说明马鹿鹿茸TMEM183A基因不同类型可变剪接转录本的表达在前软骨和软骨中存在组织特异性，推测TMEM183A可能参与调控鹿茸软骨的生成。更多还原

【Abstract】 In order to explore the structural characteristics of the alternative splicing isoforms of the TMEM183A(Transmembrane Protein 183A) gene transcript during the cartilage formation process of Cervus elaphus deer antler and the expression differences of different isomers between the anterior cartilage and cartilage tissue. Precartilage and cartilage tissue from deer antler were collected. RNA was extracted and reverse transcribed into cDNA. The range of exon 3 to 8 and CDS region of alternative splicing isoforms of the TMEM183A gene transcript were amplified by PCR. After sequencing, the sequences of each isoform were assembled. The exons were compared with those of the wild-type(WT) transcript sequence(XM＿04392477.1), and the alternative splicing isoforms of the transcripts were classified based on structural characteristics. The expression levels of different alternative splicing isoforms of the TMEM183A gene in the two tissues was detected by real-time fluorescence quantitative PCR. The results showed that a total of 9 new alternative splicing isoforms of the TMEM183A gene transcript were obtained(excluding the known alternative splicing isoforms of the transcript), named TMEM183A X10～18, respectively. These isoforms have been uploaded to GenBank and assigned the following accession numbers: PP816184, PP824820, PP831015, PP839277, PP839278, PP839279, PP839280, PP831016 and PP831017. For a certain alternative splicing transcript, alternative splicing occurred infrequently or not at all in exons 3, 8 and 9 of the TMEM183A gene in Cervus elaphus, while exon skipping(ES) mostly happened within the range of exon 4 to 7. Among them, exon 1 had three different lengths, and exon 3, 4, 7 and 8 had two different lengths. The differences between these isoforms and the wild type were mainly concentrated in the middle and C-terminal. The F-Box domains were lost in the alternative splicing isoforms of the 9 new identified TMEM183A gene transcripts. The expression level of the wild-type(WT) was the highest in both anterior cartilage and cartilage, significantly higher than that in other types(P<0.05). Alternative splicing isoforms could be classified into 9 types: wild type(WT), exon 4-7 skipping(4-7ES), exon 2,4-7 skipping(2,4-7ES), exon 4-6 skipping with exon 7 5′ alternative splicing A5SS(4-6ES-7A5SS), exon 4-6 skipping(4-6ES), exon 5,6 skipping(5,6ES), exon 6 skipping(6ES), exon 6,7 skipping(6,7ES), and exon 4,6,7 skipping(4,6,7ES). In both tissues, the relative expression abundance of wild-type transcripts was significantly higher than that of the other eight types of alternative splicing transcripts(P<0.05). In cartilage tissue, the relative expression abundances of 6,7ES and 6ES were significantly higher than those of 4-7ES, 2,4-7ES, 4-6ES-7A5SS, 4-6ES and 5,6ES(P<0.05).Except for the 4-6ES-7A5SS isoform, which showed no significant expression difference between the anterior cartilage and the cartilage(P>0.05), the other 8 transcript alternative splicing isomers showed significant or extremely significant expression differences between the anterior cartilage and the cartilage(P<0.05 or P<0.01, P<0.001). Among them, the relative expression abundance of 4-7ES, 2,4-7ES, and 5,6ES in the anterior cartilage were extremely significantly higher than those in the cartilage(P<0.001), whereas the relative expression abundance of WT, 6-4ES, 6ES, 6,7ES, and 4,6,7ES in the cartilage were significantly or extremely significantly higher than those in the anterior cartilage(P<0.05 or P<0.001). These results suggested that the expression of alternative splicing isoforms of different types of TMEM183A gene transcripts in deer antler was tissue-specific in the anterior cartilage and cartilage. It was speculated that TMEM183A might be involved in regulating the formation of deer antler cartilage.更多还原