Alternative Splicing Type And Expression Pattern For Different Transcripts Of IGF-Ⅰ Gene In Bos Taurus And Sika Deerr

Insulin-like growth factor-I (IGF-Ⅰ) is a polypeptide protein, which is similarwith insulin in the structure, and is a major mediator in the growth-promoting activityof growth hormone. Due to alternative splicing of different start sites and terminationsites, IGF-Ⅰ gene formed many kinds of mRNA transcripts.In the present study, we examined the alternative splicing type of IGF-Ⅰ gene,analyzed the homology among different species and predicted the IGF-Ⅰ function,which are performed by clone different start sites and termination sites of IGF-Ⅰ genein Bos taurus and Sika deer using RACE. Two transcription start sites of IGF-Ⅰ gene5'ends were cloned by5'RACE in Sika deer. As the leader exons, exon1and exon2coding type I and type II IGF-Ⅰ mRNA sequence, respectively. Different terminationsites of IGF-Ⅰ gene3'ends were cloned in Bos taurus and Sika deer by3'RACE. Ourresult revealed that there are two different termination sites in3'ends of Bos taurus,which are exon6and exon5coding Ea and Eb IGF-Ⅰ mRNA, respectively. There arethree different termination sites in3'ends of Sika deer, which are exon6, exon5andexon5-6coding Ea, Eb and Ec IGF-Ⅰ mRNA, respectively. The expression pattern ofIGF-Ⅰ mRNA transcripts in different tissues were measured by semi-quantitative PCR.At the same time, we detected polymorphism of IGF-Ⅰ5′UTR by sequencing andsingle nucleotide polymorphisms (SNP), then analyzed the impact of sequencemutation in IGF-Ⅰ gene promoter and transcription factor binding sites structure. Thisstudy will lay a molecular biology foundation for further understanding of the IGF-Ⅰaction mechanism and a foundation for the research of germplasm characteristicsamong different species.According to the previous reports about class I and class II IGF-Ⅰ mRNA in Bos Taurus, our study has obtained four kinds of transcripts in Bos taurus and six kinds oftranscripts in Sika deer. The multiple alignment analysis of nucleic acids and aminoacid sequences of IGF-Ⅰ mRNA transcripts were performed in Bos taurus, Sika Deerand other species. The results shown that different transcripts in IGF-Ⅰ gene amongspecies were highly conserved. The homology of class I-Ea and class II-Ea sequencebetween Bos taurus and Sika deer are higher than other species. The homology ofClass I-Eb and Class II-Eb sequence in Bos taurus and Sika deer are more than92%,but the homology between Swine and Sika deer are as high as96%. Compare maturepeptid of amino acid sequence among different species shown that the amino acidsequences are exactly the same in people, Bos taurus, Horse, Swine, et al. Twodifferent amino acid residues were found at positions D67and D69in Sika Deer.We analyzed the protein function of all kinds of IGF-Ⅰ transcripts in Bos taurusand Sika deer using bioinformatics. The results indicates that all IGF-Ⅰ mRNAtranscripts protein belong to secretory protein. So the IGF-Ⅰ need to combined withIGF-ⅠR which is a transmembrane transport process.The expression characteristic were investigated by Semi-quantitative PCR aboutfour IGF-Ⅰ mRNA transcripts in Red steppes different tissues, including heart, liver,spleen, lung, kidney, duodenum, rumen, testicle, triceps, gluteus medius, longissimusmuscle of the back, semitendinosus, latissimus dorsi muscle, musculus tensor fasciaelatae, musculus deltoideus and musculus biceps femoris. The results revealed that theexpressions have a difference among different transcripts in different tissues, but alltranscripts were expressed at the highest level in liver, meanwhile, shown a positivecorrelation. The expression levels in the spleen and testis are relatively high in fourtranscripts. We detected the expression of IGF-Ⅰ mRNA transcripts in nine muscletissues of Red steppes. Class I-Ea has obvious expression and the other threetranscripts expressed very weak or no expression. The results implied that class-I EaIGF-Ⅰ mRNA may play an important role on muscle traits.We analyzed the expressions of IGF-Ⅰ transcripts in six tissues including heart,liver, spleen, lung, kidney and duodenum of adult and fetal Sika deer by Semi-quantitative PCR. The results revealed that all transcripts are expressed at thehighest level in liver, and shown positive and negative correlation among sixtranscripts. While class I-Ea, class I-Eb and class II-Ea are expressed at a relativelyhigh level in the spleen, other transcripts are low or no express. All transcripts wereexpressed at a low level or no expression in heart, lung, kidney and duodenum. Indifferent tissues of fetal Sika deer, we only detect the expression of class I-Ea IGF-ⅠmRNA. Other transcripts have not expression in fetal Sika deer. Analysis theexpression of Class I-Ea between adult and fetal Sika deer shown that the expressionin liver and spleen of adult Sika deer were strong than in fetal Sika deer, while theexpression in lung, kidney and stomach were weak than in fetal Sika deer. They alldid not express in heart.Sequence sizes of1954bp and1833bp were obtained from IGF-Ⅰ5′UTR in Bostaurus and Sika deer using clone, respectively. Bioinformatics analysis shown that the5′UTR exist multiple promoters and transcription factor binding sites, which have animportant role in gene start, transcription process and expression levels.The polymorphism of IGF-Ⅰ gene in Bos taurus indicated that there was onesingle nucleotide mutation: C→T in the5′UTR at a distance of exon1510bp. Thegenetic analysis revealed that AB genotype was the predominant genotype in fourgroups of134Bos taurus. Charolais, Simmental and Limousin groups keep aHardy-Weinberg balanced condition but the red steppes were not. Bioinformaticsanalysis revealed that the structure of promoter and transcription factor binding siteswas not changed by mutation site. The polymorphisms were not detected in the IGF-Ⅰgene5′UTR in Sika deer. It could be deduced that IGF-Ⅰ gene5′UTR is highlyconserved during evolutionary history.