Cloning Of Different IGF-I Transcriptons And Expression Pattern For Different Tissues In Swine

IGF-â… is a single copy gene,due to alternative splicing,and the use of multiple polyadenylation signals and different promoters,the IGF-â… gene and its mRNAs are complicated. Spliced variants of insulin-like growth factor 1(IGF-1),a small peptide with a critical role in metabolism and growth,have been identified in various vertebrate species.IGF-â… play an important role in muscle growth and development,myoblast fusion and differentiation,and improve muscle grow and meat quality.Therefore,it has important significance for studying on pig's candidate genes of economic trait that molecular genetic construction and space-time expression of IGF-â… gene is studied by modern molecular biological technology.This research applied to Songliao Black Pig as the object of studying,we cloned the pig two new IGF-â… mRNAs and analyzed structure and characteristics of them by bioinformatics.Levels of different spliced variants of IGF-â… in different tissues,at different ages,in adipose muscle tissues of different breeds were measured by Semi Quantitative Reverse Transcription-Polymerase Chain Reaction(SQ RT-PCR),and analyzed the genetic correlation between the levels of different spliced variants of IGF-â… and meat quality traits by SPSS.We detected the polymorphisms of the 5'-regulatory region of IGF-â… genes by PCR-SSCP(single-strand conformation polymorphism),sequencing and microsatellite methods in the population of Songliao black pig,and analyzed the genetic correlation between the polymorphisms and carcass and meat quality traits by SPSS.The main results are as follows:1.The cDNA sequences of Songliao Black pig two spliced variants of IGF-â… gene,including exon five,were cloned by RACE.They were named type Eb of classâ… and type Eb of classâ…¡-IGF-â… genes,type Eb of classâ… - IGF-â… gene contains a 564bp open reading frame(ORF) encoding 187 amino acids while type Eb of classâ…¡- IGF-â… gene contains a 516bp ORF encoding 171 amino acids.2.The alignment analysis of CDS sequences of type Eb of classâ… and type Eb of classâ…¡-IGF-â… genes among human,sheep,cows,goats,chimpanzees,rhesus monkeys and horses shows that the consistency of type Eb of classâ… - IGF-â… gene in Songliao Black pig and other mammals is more than 91%,while the comparability of type Eb of classâ…¡- IGF-â… gene is more than 90%.The comparability of the protein sequences of type Eb of classâ… and type Eb of classâ…¡-IGF-â… genes of Songliao Black pig with other mammals are more than 96%and 93%respectively.It verified that type Eb of classâ… and type Eb of classâ…¡- IGF-â… genes are highly conserved in mammals.3.We predicted the basic features,protein structure and molecular function of porcine type Eb of classâ… and type Eb of classâ…¡proteins with bioinformatics methods.The results indicate that porcine type Eb of classâ… and type Eb of classâ…¡- IGF-â… genes belong to IGFs family,type Eb of classâ… and type Eb of classâ…¡proteins structure also contains the same IGFBPs,typeâ… andâ…¡receptor domain.Bioinformatics results show that porcine type Eb of classâ… and type Eb of classâ…¡proteins are very conservative in the key structural and functional region.This suggests that porcine type Eb of classâ… and type Eb of classâ…¡proteins should have the similar function with some known proteins.4.The expression characteristic of four spliced variants of insulin-like growth factor-â… gene was investigated by RT-PCR.The results showed that the three spliced variants of insulin-like growth factor-â… gene expressed in heart,liver,spleen,lung,kidney,lung,stomach,duodenum,ileum, colon,appendix,brachi -triceps,longissimus muscle of back,biceps femoris,testicle of Songliao Black Pig at the age of birth,one-month,three- month and six-month:There is much difference in the same tissue at different growing period.5.Three spliced variants of insulin-like growth factor-â… gene have the similar expression curves,which is the "S"curve or clock curve,in the same tissue at different growing period.The expression level of type Ea of classâ… -IGF-â… gene in longissimus of Songliao Black pig at one-month and six-month are significantly more than that birth and three-month.;The expression level of type Eb of classâ… -IGF-â… gene in longissimus of Songliao Black pig at one-month are significantly more than that birth,three-month and six-month;The expression level of type Eb of classâ…¡-IGF-â… gene in longissimus of Songliao Black pig at birth and one-month are significantly more than three-month and six-month.6.The relationship of four spliced variants of insulin-like growth factor-â… gene expression with pork quality was different at different growing stage:At one-month the type Ea of classâ… -IGF-â… gene mRNA level and meat color was negative correlation;the type Eb of classâ… -IGF-â… gene mRNA level and PH value were positive correlation.The type Ea of classâ… -IGF-â… gen mRNA level was extremely positive correlation with meat color and marbling at three-month.At six-month the type Ea of classâ… -IGF-â… gene mRNA level and the ratio of cook was positive correlation;the type Eb of classâ… -IGF-â… gen mRNA level was extremely positive correlation with meat color and marbling;the type Ea of classâ… -IGF-â… gene mRNA level was negative correlation with PH value;the type Eb of classâ…¡-IGF-â… gene mRNA level and drop ratio were negative correlation.7.The expression characteristic of four spliced variants of insulin-like growth factor-â… gene was investigated by RT-PCR.The results showed that the three spliced variants of insulin-like growth factor-â… gene expressed in heart,liver,spleen,lung,kidney,lung, stomach,duodenum,ileum,colon,appendix,brachi -triceps,longissimus muscle of back,biceps femoris,testicle of Songliao Black Pig,Crossed and Landrace:There is much difference in the same tissue at different breed.8.Class 1 IGF-â… mRNA(type Ea or type Eb) appeared to be much more abundant than class 2 IGF-â… mRNA in Songliao Black pig and Crossed.But type Eb of Classâ…¡-IGF-â… mRNA appeared to be much more abundant than class 1 IGF-â… mRNA in Landrace.9.The relationship of four spliced variants of insulin-like growth factor-â… gene expression with pork quality was different at different breeds:In Songliao Black pig:the type Ea of classâ… -IGF-â… gene mRNA level and the ratio of cook was positive correlation;the type Eb of classâ… -IGF-â… gen mRNA level was extremely positive correlation with meat color and marbling; the type Ea of classâ… -IGF-â… gene mRNA level was negative correlation with PH value:the type Eb of classâ…¡-IGF-â… gene mRNA level and drop ratio were negative correlation.In Crossed the type Ea of classâ… -IGF-â… gene mRNA level and cooked ratio were positive correlation;the type Eb of classâ…¡-IGF-â… gene mRNA level and drop ratio were negative correlation.The type Eb of classâ…¡-IGF-â… gen mRNA level was extremely positive correlation with PH value in Landrace.10.The polymorphism of the 5'-regulatory region of IGF-â… gene in Songliao black pig was analyzed by PCR-SSCP,sequencing and microsatellite methods.The results showed that the polymorphisms were not detected by PCR-SSCP in the 5' -regulatory region of the IGF-â… gene. Four alleles and six genotypes(AA,BB,AB,CC,DD,CD) were detected in Songliao Black pig populations.The association analysis-found the individual of AA genotypes have better meat quality and allele A had positive additive effects on meat quality traits.It could be preliminarily deduced that IGF-â… gene was probably a major gene or a QTL linked gene which associated with meat quality and carcass traits in pig.