初探lncRNA-Tubb4b的蛋白编码能力及其对微管基因的调控

doi:10.11843/j.issn.0366-6964.2022.01.018

摘要/Abstract

摘要： 旨在探索长链非编码Tubb4b基因（lncRNA-Tubb4b）是否具有蛋白编码能力及其对Tubb4b基因的调控作用。本研究在lncRNA-Tubb4b中添加起始密码子ATG、0/1/2个的T尾和Flag标记，通过构建载体、脂质体转染和Western blot等技术对lncRNA-Tubb4b蛋白编码能力进行分析；随后，构建过表达载体和合成siRNA，通过脂质体转染和实时荧光定量PCR等技术检测lncRNA-Tubb4b对微管基因Tubb4b的影响。结果表明，我们扩增了添加0、1和2个T尾的Flag片段，其与lncRNA-Tubb4b融合后，成功构建到pcDNA3.1（-）载体中；转染小鼠精原细胞后发现，添加0、1和2个T尾的lncRNA-Tubb4b均没有FLAG蛋白条带；以pcDNA 3.1（-）-EGFP载体为对照，在小鼠精原细胞中过表达lncRNA-Tubb4b时，对照组细胞有荧光产生，试验组Tubb4b基因的mRNA表达水平极显著地降低（P<0.01）；在小鼠精原细胞中干扰lncRNA-Tubb4b表达后，极显著地提高Tubb4b基因的mRNA表达水平（P<0.01）。这表明，lncRNA-Tubb4b确实不编码蛋白质，过表达或干扰lncRNA-Tubb4b的表达会极显著地降低或升高Tubb4b基因的表达。

关键词: lncRNA-Tubb4b, 微管, Tubb4b, 蛋白编码, 小鼠

Abstract: The study aimed to explore whether the long non-coding RNA of Tubb4b (lncRNA-Tubb4b) has protein coding capability and the regulatory effect of lncRNA-Tubb4b on the Tubb4b gene. The ATG, 0/1/2 T tails and Flag sequence were added to lncRNA-Tubb4b to analyze the coding ability of lncRNA-Tubb4b by constructing vector, liposome transfection and Western blot; Then, overexpression vector of lncRNA-Tubb4b was constructed and the siRNA of lncRNA-Tubb4b was synthesized, while the liposome transfection and real-time fluorescent quantitative PCR technology were used to detect the influence of lncRNA-Tubb4b on the Tubb4b gene.The results showed that the Flag fragments with 0, 1, and 2 T-tail, was amplified in this study, were fused with lncRNA-Tubb4b, then were successfully constructed into the pcDNA3.1(-) vector; After transfection of mouse spermatogonia, it was found that lncRNA-Tubb4b with 0, 1, and 2 T-tail had no FLAG protein bands. With pcDNA 3.1(-)-EGFP vector as a control, overexpression of lncRNA-Tubb4b significantly reduced the mRNA expression level of Tubb4b in mouse spermatogonia (P<0.01), while there was fluorescence in control cells. And interfering the expression of lncRNA-Tubb4b significantly increased the mRNA expression level of Tubb4b in mouse spermatogonia (P<0.01). It is indicated that lncRNA-Tubb4b does not encode protein, and overexpression or interference with the expression of lncRNA-Tubb4b will extremely significantly reduce or increase the expression of Tubb4b.

Key words: lncRNA-Tubb4b, tubule, Tubb4b, protein coding, mouse

中图分类号:

S814.1

冯美莹, 卫恒习, 李莉, 张守全. 初探lncRNA-Tubb4b的蛋白编码能力及其对微管基因的调控[J]. 畜牧兽医学报, 2022, 53(1): 179-187.

FENG Meiying, WEI Hengxi, LI Li, ZHANG Shouquan. The Preliminary Research of the Protein Coding Ability of lncRNA-Tubb4b and Its Regulation on Microtubule Genes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 179-187.

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