Polymorphisms Detection And Construction Of Recombinant Adenovirus Of Bovine NRIP1Gene

Nuclear receptor interacting protein1(NRIP1) is a nuclear protein that specificallyinteracts with the hormone-dependent activation domain AF2of nuclear receptors. Alsoknown as RIP140, this protein is a key regulator which modulates transcriptional activity of avariety of transcription factors. Previous work has demonstrated that NRIP1plays animportant role in regulating the expression of clusters of genes in the pathways of glucoseuptake, glycolysis, TCA cycle, fatty acid oxidation, mitochondrial biogenesis, and oxidativephosphorylation.Bovine NRIP1gene was chosen as candidate gene in this study. First, the singlenucleotide polymorphisms (SNPs) of bovine NRIP1gene were detected in1809cattle fromten cattle breeds by DNA pool, sequencing and Forced-PCR-RFLP. In addition, theassociation between SNPs and growth traits were analyzed was also carried out by SPSSsoftware. Further more, the coding sequence (CDS) of bovine NRIP1gene were cloned intorecombinant adenovirus. After that, shRNA for bovine NRIP1gene were designed. TheshRNAs with higher effect on bovine NRIP1gene silence were identified by fluorescencemicroscope. Collectively, all these work will support lots of work and reseach includingmarker-assisted selection (MAS), improvement and promotion of beef cattle, function onbovine NRIP1gene. The main results were shown below:1. Polymorphisms detection and population genetic analysis of bovine NRIP1geneIn the study, the single nucleotide polymorphisms (SNPs) of the coding sequences ofbovine NRIP1gene were detected in1809cattle from ten cattle breeds (Qinchuan, Nanyang,Luxi, Jiaxian, Caoyuan, Chinese Holstein, Angus, Hasake, Yak, and XD) by DNA pool,sequencing and Forced-PCR-RFLP. Two mutations (c.605A>G and c.1301T>C) wereidentified, both of which generated missense mutations (p.Asn202Ser and p.Pho434Ser) andrespectively located to repression domain1and repression domain2of NRIP1.By population genetics analysis, there were three genotypes (AA, AG and GG) inc.605A>G locus. The genotype AG was dominant in Luxi cattle breed whose genotypic frequency was0.4000. In the other nine cattle breeds, the genotype GG was predominant, andthe frequencies varied from0.6000~0.9259. In c.1301T>C locus, genotype AG and GG weredetected and GG genotype was predominant in all ten cattle breeds, the frequencies variedfrom0.9130~1.0000. It is proposed that the NRIP1gene is a conservative gene in cattlebreeds by all these findings.2. Association of polymorphisms in NRIP1gene and growth traits in Nanyang cattleThe association between two SNPs of NRIP1gene and growth traits (body weight, bodyheight, rump length, heart girth, huckebone width, average daily gain) in Nanyang cattle(birth,6,12,18and24months) was analyzed by SPSS software. The results showed thatc.605A>G locus was significantly associated with body weight and average daily gain inNanyang breed aged18months (p<0.05) in Nanyang cattle. The individuals with genotypeAG had greater than AA, and genotype AA had greater than GG (p<0.05). These will benefitfor the application of DNA maker related to the growth traits on marker-assisted selection(MAS), improvement and promotion of beef cattle.3.Construction of recombinant adenovirus containing bovine NRIP1geneIn this study，the coding sequences (CDS) of bovine NRIP1gene were cloned intorecombinant adenovirus. First of all, the recombinant plasmid pAdTrack-CMV-NRIP1whichhad a CMV promoter was constructed. Later, pAdTrack-CMV-NRIP1was linearized by PmeⅠ. Then pAdTrack-CMV-NRIP1was transformed into E.coli BJ5183completent cellscontaining pAdEasy-l which had conservative gene of adenovirus. There, recombinantplasmid pAd-NRIP1was achieved by the homogenous recombinance of two plamid(linearized pAdTrack-CMV-NRIP1and pAdEasy-l). After linearized by PacⅠ,pAd-NRIP1was transfected into293A package cells by lipofectamineTM2000. Finally, therecombinant adenovirus containing bovine NRIP1gene was constructed sucessfully.4. Selection of effective siRNA sequenceAccording to the bovine NRIP1gene sequence, four shRNA sequences(NRIP1-shRNA137, NRIP1-shRNA1445, NRIP1-shRNA2706, NRIP1-shRNA3285) whichtarget different area of bovine NRIP1gene were designed and synthesized. After annealing,four shRNA templates (NRIP1-shRNA137, NRIP1-shRNA1445, NRIP1-shRNA2706,NRIP1-shRNA3285) were respectively cloned into pENTR/CMV-GFP/U6plasmid whichcould express green fluorescent protein. In addition, four fragments of bovine NRIP1gene(RED-shRNA137, RED-shRNA1445, RED-shRNA2706, RED-shRNA3285) were clonedaccording to the distribution of shRNA sequences. And four recombinant plasmidspDsRed1-Express-N1respectively containing RED-shRNA137, RED-shRNA1445,RED-shRNA2706, RED-shRNA3285were constructed, which could express red fluorescent protein. By cotransfecting293T cell lines, the result showed that pDsRed1-Express-N1expressing NRIP1-shRNA1445, NRIP1-shRNA2706sequences caused an obviousinterference effect by fluorescence microscope observation. It was proposed thatNRIP1-shRNA1445and NRIP1-shRNA2706had higher effect on bovine NRIP1gene silenceaccording to red fluorescent protein level.