奶牛乳腺CSN1S1基因线状与环状可变剪接体鉴定分析及功能探讨

Identification and Functional Analysis of CSN1S1 Gene Linear and Circular Alternative Transcripts in Bovine Mammary Gland

CSN1S1基因是调控奶牛乳汁中酪蛋白合成的一个重要基因。为鉴定分析奶牛CSN1S1基因可变剪接体种类及分布,验证其环状结构及表达能力,本研究根据牛CSN1S1基因m RNA序列(NM_181029.2)和不同外显子序列分别设计线状及环状引物,用PCR和RT-PCR技术克隆CSN1S1基因可变剪接体,筛选分析不同线状和环状可变剪接体及特征。结果发现:40个阳性克隆中,除全长CSN1S1 mRNA外,共获得12种CSN1S1线状可变剪接体,通过NCBI数据库比对分析确定了其序列组成及所占比重;检测到3种具有完整反向拼接的序列,同时发现多种滚环式结构,进一步确认环形RNA的存在;为验证环状可变剪接体能否表达,实验构建一种过表达载体pEGFP-CSN1S1-12～16 (包含CSN1S1基因12～16号外显子序列),转染293T细胞,48 h后在荧光显微镜下发现有绿色荧光效应,证明其具有翻译潜能。本研究成功筛选出不同线状与环状可变剪接体,并对其表达能力进行验证,为探讨如何提高牛奶酪蛋白合成提供新的依据。

The CSN1 S1 gene is an important gene in the regulation of casein synthesis in milk.We aimed to identify and analyze the distribution and content of alternative spliceosomes of CSN1 S1 gene,and to verify its ring structure and expression ability.In this study designed primers based on the CSN1 S1 mRNA sequence(NM181029.2).PCR was used to clone and screen different linear alternative spliceosomes.At the same time,the circular primers were designed according to different exons’sequences of this gene.RT-PCR was used to clone and verify the different circular sequences and their characteristics.As a result,among the 40 positive clones,12 CSN1 S1 linear alternative spliceosomes were obtained in addition to full length of CSN1 S1 cDNA.The sequence composition and its proportion were determined by NCBI database alignment analysis.Three sequences with complete exon splicing were identified in the sequencing of circular alternative spliceosomes.The discovery of the rolling ring structure further confirmed the presence of circular RNA.In order to verify whether circular alternative spliceosomes whic h we have found out have the ability to express,a kind of expression plasmid of circular alternative spliceosome pEGFP-CSN1 S1-12~16 was constructed.Expression plasmid pEGFP-CSN1 S1-12~16 consisted of exon 12~16 of the CSN1 S1 gene.After expression plasmid transformed into 293 T cells,we found a green fluorescent effect during 48 h observation by fluorescence microscopy.This phenomenon proved that it has translation potential.In this experiment,different linear and circular alternative spliceosomes were successfully screened,and their expression ability was validated,which provided the basis for improving the content of milk casein.