Study Of Genetic Variation, Alternative Splicing, Cloning And Expression Within ANGPTL6Gene In Chinese Native Cattle

Angiopoietin-like protein6, which is encoded by ANGPTL6gene （also known asangiopoietin growth factor, AGF）, has been extensively characterized with regard to itsproposed functions as angiogenesis and energy metabolism. In model animals, it has beenproved that the genetic variations within ANGPTL6gene have great effects on growth anddevelopment. However, up to now, it is very regretful that the genetic variation andexpression of bovine ANGPTL6gene is still vacant. Therefore, the purpose was to revealgenetic variation characteristics, alternative splicing and expression of ANGPTL6gene andprovide some basal information for molecular breeding of Chinese native cattle in this study.The main methods, results and conclusions were listed as follows:1. Genetic variations of ANGPTL6gene and their associations with growth traits andslaughter traits in Chinese native cattleIn this experiment, genetic variation within ANGPTL6gene was detected by DNA poolsequencing and PCR-RFLP methods in a total of1652cows belonging to five differentChinese native cattle breeds （including Qinchuan cattle, Nanyang cattle, Jiaxian Red cattle,Luxi cattle and Caoyuan Red cattle）. The results showed three novel SNPs （namelyI2-2359:g.T>C, I2-2403:g.C>A and I4-3258:g.G>T） within the intronic region of ANGPTL6gene were identified in cattle for the first time. Chi-square test showed I2-2359:g.T>C andI2-2403:g.C>A loci were in Hardy-Weinberg equilibrium （P>0.05） except I4-3258:g.G>Tlocus in the analyzed population. Analysis of genetic diversity indicated I2-2359:g.T>C andI4-3258:g.G>T were at intermediate polymorphic status, and I2-2403:g.C>A locus was at lowpolymorphic status. Further, the linkage disequilibrium and haplotypes were analyzed amongthree SNPs, suggested that they were all little linked. Interestingly, Haplotype with TCG（wild-type） was dominant with frequency of>40.0%among eight different haplotypes. Moreover, independent χ2-test of genotypic distribution showed the three variants were allsignificantly different in five cattle breeds （P<0.05）.In Qinchuan cattle, statistical analysis showed body height （BH）, hucklebone width（Hu-W）, chest girth （CG）, chest depth （CD） and chest breadth （CB） were significantlyassociated with I2-2359:g.T>C and I2-2403:g.C>A loci. The analysis of combined effectsshowed the different combined loci were associated with BH, CG, CD, CB and Hu-W.Slaughter test also revealed that the effect of the combined genotypes was extremelysignificant on the carcass weight （P<0.01）.In Nanyang cattle, association analysis showed the I2-2403:g.C>A and I4-3258:g.G>Tloci were significantly associated with chest girth index of24months old （CGI-24） and bodyhindquarters index of6months old （BHI-6） of individuals, and the I2-2359:g.T>C locus wassignificantly associated with body length index of6months old （BLI-6）（P<0.05）. Further,combined analysis revealed the average daily gain of18months old （ADG-18） of individualswith genotypic TC-CC-GG was higher than other combined genotypes in ScaI-VspI-RsaIlocus. Moreover, chest girth of18months old （CG-18） with TC-GG and ADG-18with TT-GTwere preponderant in ScaI-RsaI locus.In Jiaxian cattle, the I2-2403:g.C>A locus had a significant association with its growthtraits. Combined analysis showed the different combined loci were significantly associatedwith height of hip cross （HHC）, BL, CC, CCB and body weight （BW）（P<0.05or P<0.01）.In Luxi cattle, the variance analysis showed that the different genotypes of single andcombined sites were all significantly or extremely significantly associated with its growthtraits and the growth traits of heterozygous individuals were all higher than the homozygousindividuals. For I2-2359:g.T>C locus, the BH and CC of individuals with heterozygousgenotype TC were significantly higher than homozygous genotypes TT and CC （P<0.05）. ForI2-2403:g.C>A locus, the BW, BH and CC of genotype TC were extremely significantdifferent from genotypes TT and CC （P<0.01）.In conclusion, the present results provided evidence that polymorphisms of ANGPTL6gene were associated with growth and slaughter traits, and may apply to Chinese native cattlebreeding program as a possible candidate for marker-assisted selection （MAS）.2. The identification of alternative splicing within ANGPTL6gene and associations withcarcass weight in Qinchuan cattleFor this study, the alternative splicing within ANGPTL6gene was detected in78Qinchuan cows by cloning sequencing method. Further, the associations between differentvariants and carcass weight were analyzed to explore their effects on body weight ofQinchuan cattle. Additionally, the relative expression levels of different variants were performed by both semi-RT-PCR and RT-qPCR methods.The present results showed the occurrence of alternative splicing by intron retention （IR）event in the bovine ANGPTL6gene. By BLAST alignment, we have verified that thebANGPTL6gene has a splice variant generated due to the retention of its partial intron3. Thecomputational analysis of the bANGPTL6genomic sequence showed that its intron3has ahigh percentage of GC （62.31%） and a length of199nt, characteristics that have beenassociated with an IR event. The IR event does not interfere with the coding region as thebANGPTL6prepropeptide, which is entirely coded in the third exon. Additionally, both theintronless （namely, bANGPTL6α） and intron-retaining （namely, bANGPTL6β） ANGPTL6transcripts are constitutively co-expressed in the bovine liver. To verify the effects of thebANGPTL6gene on body weight, the association analysis was performed between differentsplice variants and carcass weight on Qinchuan cattle. Statistical results showed thebANGPTL6β effects on carcass weight as a dominant negative isoform. Further, the relativeexpression level of bANGPTL6β is significantly higher than bANGPTL6α in bovine liver.Overall, our findings will be helpful for studies on the molecular mechanism of IRevents and the functions of ANGPTL6gene. They could be applied to molecular breedingindustry for improving the meat performance of Qinchuan cattle. Specially, bANGPTL6β geneprobably also contributes to a new target for treatment of obesity and obesity-related diseases.3. Bioinformatics analysis, cloning and prokaryotic expression of bovine ANGPTL6geneBased on some bioinformatics knowledge, online software （ProtParam, TMHMM andPredictProtein, etc.） were carried out to analyze bANGPTL6α and bANGPTL6β gene and theirproteins. The analyzed results showed the structure and properties of ANGPTL6α andbANGPTL6β proteins were generally consistent. The forty discrepant amino acids arecomposed of extend strand （e）, random coil （c） and β-corner, except α-helix. These resultssupport the conclusion that bANGPTL6is a secreted-related protein. Further, they could behelpful for exploring and understanding the structures and functions of bovine ANGPTL6αand bANGPTL6β proteins.The coding sequences of bANGPTL6α and bANGPTL6β gene were obtained from livertissue of Qinchuan cattle by PCR and sequencing methods. Then they were cloned intopGM-T vector, and the sequencing results of the positive clones were same as the sequence inGenBank （NM₀01128070.1）. The positive combined plasmids were digested with EcoR Iand Hind III, and the digested target DNA fragments were constructed to pET28a （+） vector.The positive recombined plasmids pET28a （+）-ANGPTLα and pET28a （+）-ANGPTLβ weretransformed into E.coli BL21（DE3） and induced with IPTG. SDS-PAGE results showed no fusion protein6His-ANGPTLα or ANGPTLβ was expressed in E.coli.For this reason, we also analyzed the frequency of the rare or low-usage codons withinbANGPTL6α and bANGPTL6β gene. The statistic analysis suggested the frequency reach upto17%, and their internal sequences contain the bunchy AGG and GGA codons. Thus, therare codons could be a main factor that caused unsuccessful expression of bovine ANGPTL6in this study. Based on the previous reports, the inducible expression of bANGPTL6α andbANGPTL6β could be explored through the two ways: the rare or low-usage codons arereplaced by the common codons （synonymous mutations）; choosing bacterial strainRossetta-gami （DE3）, which has abundant rare codons itself.