Molecule Cloning And Expression Study Of Fosl2and Leptin Gene In Various Tissues From Yunan Black Pig

Fosl2(Fos-like antigen2)belongs to the activator protein-1transcription factor family,which arewidely expressed in various types of tissues and play an important role on aspects of mammaliangrowth、reproduction performance and the immune response.With the further research of recentyears,people found Fosl2gene were closely linked with fat metabolism、skeletal development andthe incidence of cancer;while Leptin gene was the earliest gene that found was correlated withenergy metabolism.On the basis of previous work,the cloning and bioinformatics analysis of Fosl2and Leptin gene with Yunan black pigs were studied; and in order to determine the role of these twogene play in different stages of pigs development from the perspective of molecular genetics,weanalysed the developmental expression of Fosl2gene and Leptin gene in different tissues. According tothe released mRNA sequence of human beings, mice,cattle and gorilla in GenBank of NCBI, theconservative area of Fosl2gene cDNA was found out with DNAstar and DNAMAN;meanwhile, RT-PCR methord was used for gene cloning with specific primers;then the acquired nucleotide sequenceas a probe homology was used in homology comparison in different species of NCBI;with the helpof bioinformatics software, the amino acid structure and sequence characteristics of two genes werepredicted and intelligible;at last, real-time quantitative polymerase chain reaction (PCR) was applyedto detect the development expression in mRNA level of two genes in different growth period ofYunan black pig’s heart, liver, spleen,lung,kidney, muscle, and backfat adipose tissue.The majorresults are as follows:1. Part of cDNA fragment of Fosl2gene by RT-PCR from placenta of Yunan black pig was acquiredwith the use of specific primers that was designed according to the compared conservative areaamong different species;and also, part of cDNA fragment of Leptin gene by RT-PCR from backfatadipose tissue in Yunan black pig was cloned. The results showed that the length of Fosl2gene is1427bp, in which the CDs area was984bp;the cloned length of Leptin gene was253bp.2.When the gene sequence was cloned in Yunan black pig’s organs according to the method1, thelength of770bp transcription of Fosl2was found unimagined.Compared with the cloned fragmentsfrom placenta, a44base-pairs(bp) missing in the end of the third exon occoured,at the same time,thefront area of the fourth exon also had a nucleotide missing with614bp.3.The nucleotide and amino acid homology of cloned Fosl2gene sequence and CDs of Leptin genewere analyzed by the bioinformatics information softwares:the nucleotide homology of the pig Fosl2gene with cattle,people,macaque,gorillas,rat,tree shrew,horse was91,89,89,89,89,88,90percentrespectively,while the amino acid identify was85,86,84,85,82,83,71percent respectively.Thisshowed a higher gene conserved among different species.The nucleotide homology of Leptin genewith human, cattle, mouse, seals, sea lions, dogs, gray seals, Mus musculus, macaque, domestic cats was88,92,85,83,87,92,83,85,88,91percent respectively.The amino acid identity with the above-described species was97,100,94,89,87,90,92,94,97,92percent respectively.The conservative of thisgene among mammals was very high.4. By real-time quantitative PCR,Yunan black pig Fosl2gene was detected widely expressed invarious tissues, although the relative expression of Fosl2gene of the7day age,30day age,60day ageand180day age in lung tissue showed a greater volatility,it has been maintained at the highestrelative expression level, followed by the relative expression in the spleen tissue. Leptin gene in the180day age was the highest in backfat adipose tissue.5. The expression of regularity in the same tissue of different growth periods between two genes wasshowed as follow: the mRNA level in lung tissue of Fosl2gene ranged first in7days of age, theexpression of lung,kidney and spleen were the highest in this time comparied with other time;muscletissue expressed the highest level in30days of age.The relative expression of Fosl2gene in the180days age was higher in heart and backfat adipose tissue than any other periods.The relativeexpression of Leptin gene in liver and muscle was higher in the7day age,spleen and lung of30daywas higher than other times,heart and liver of60day was advantaged.The relative expression inbackfat adipose tissue in180days age was significantly higher than other organizations.6.Fosl2gene and Leptin gene in backfat adipose tissue had a consistent regional variation trend indifferent development periods of pig:namely30days of age rised,60days of age fall.When it reached180day,the relative expression in two genes’ mRNA surged and reached the hightest level.