猪hnRNPUL1基因克隆及变异剪接体鉴定

Cloning and Identification of Splice Variants of the Porcine hnRNPUL1 Gene

旨在克隆民猪hnRNPUL1基因全长编码区(complete coding sequence, CDS)序列并进行可变剪接分析,研究其组织表达和亚细胞定位情况。本研究以3头3月龄民猪为试验材料,利用RT-PCR技术克隆hnRNPUL1基因CDS及可变剪接体,应用qRT-PCR检测其在心、肝、脾、肺、肾、胃、大肠、小肠、背肌、腿肌等组织中的表达情况,同时,在生物信息学预测的基础上,通过融合表达绿色荧光蛋白的方法鉴定核定位序列。研究结果表明,猪hnRNPUL1基因(GenBank accession No.:MN399154) CDS全长2 580 bp,预期编码的多肽链存在着hnRNPs家族的特征性结构域--SAP、SPRY和AAA;猪hnRNPUL1普遍表达于所检测的各组织中,其中在脾脏中表达量最高,在腿肌中表达量最低;核定位序列(NLS)位于多肽链的133~430 aa处,与生物学信息学预测的结果存在着偏差;同时获得了2个变异剪接体和11种可变剪接片段。本研究结果对进一步揭示猪hnRNPUL1基因功能及转录调控机制提供了基础。

The study was conducted to clone the complete coding sequence(CDS) and transcript variants of porcine hnRNPUL1, and to analyze its tissue expression and subcellular localization. In this study, three 3-month-old Min pigs were used. The CDS and transcript variants of porcine hnRNPUL1 were cloned using RT-PCR method, and the expression was detected in tissues including heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, back muscle and leg muscle by qRT-PCR. At the same time, the subcellular localization was analyzed by inserting the fragments of CDS into pEGFP-N1 vector on the basis of bioinformatics prediction. It was found that the CDS of porcine hnRNPUL1 gene(GenBank accession No.: MN399154) was 2 580 bp of length, and the predicted polypeptide contained SAP, SPRY, and AAA domains, the characteristic domains of hnRNPs family. Porcine hnRNPUL1 was ubiquitously expressed in all detected tissues, with the highest level in the spleen but the lowest in leg muscle. The nuclear localization sequence(NLS) was located at 133-430 aa of the polypeptide chain, which was different from the result predicted by the bioinformatics methods. In addition, 2 splice variants and 11 alternative splicing fragments were identified. The results have provided the basis for further revealing the function and transcriptional regulation mechanism of porcine hnRNPUL1.