Cloning And Functional Analysis On Splice Variants Of Porcine ZNF280D Gene

Zinc finger protein is one of the most widely distributed transcription factors in eukaryotes.It is closely related to cell proliferation,differentiation,growth and the occurrence and development of certain diseases.The organism makes it possible to produce multiple transcripts by alternative splicing,which greatly enriches the diversity and complexity of the genome.Different transcripts express different proteins,and are coordinated and refined to meet the needs of the organism different growth and development stage of the higher demand,can be regarded as an important way of biological evolution.ZNF280D gene of Zinc finger protein is prevalent in a variety of organisms in nature which with different splicing structure and quantity in different species.ZNF280D gene,a member of the zinc finger family,would have a great potential regulation with the growth and development of organisms.The aim of this study was to investigate the types of splice isoforms of ZNF280D gene in Mashen pig and Large white pig,and their expression characteristics in different organs and tissues of different pig breeds and the developmental expression pattern in longissimus dorsi,psoas muscle and biceps femoris.The Based on the transcriptase sequencing predicted results about ZNF280D gene splicing site in pig,these splicing sites were validated by PCR amplification and cloning techniques.The different splice structure of the gene was obtained and computational analysis.The mRNA expression spectrum of ZNF280D in 11 kinds of tissues of two pigs at 3 months of age,just as the liver,spleen,lung,kidney,pancreas,stomach,small intestine,cerebellum,abdominal fat and muscle were detected by real-time fluorescence quantitative PCR(qRT-PCR).The developmental regularity of ZNF280D gene mutation in the longissimus dorsi,psoas muscle and biceps femoris(totally about 18 organizations)from this two type pigs who were at the three developmental stage.The main results are as follows:(1)ZNF280D gene have two types of transcripts in Mashen pig and Large white pig because of selective shear was defined as ZNF280D1 and ZNF280D2.ZNF280D1 is a conventional transcript of this gene.ZNF280D2 is the mutant which lost the most part of 5'sequecne of the eighth exon,and 74 amino acids less than normal peptide chain,and a C2H2 zinc finger domain is added to the encoded protein.The tC2H2 turned into the maC2H2 type of zinc.But the new zinc finger domain is not easy to bind to DNA,which can enhance the avidity of adjacent zinc finger structure with DNA,and improve the stability of the binding product.(2)The relative expression levels of ZNF280D2 were significantly or most significantly higher than those of ZNF280D1 in most organizations of Mashen pigs and Large White pigs(P<0.05;P<0.01),suggest that the new spliceosome ZNF280D2 play a major role.The expression quantity of ZNF280D mRNA in pancreas(mainly ZNF280D2),stomach,and muscles(include ZNF280D1 and ZNF280D2)are significantly higher than those in other tissues(P<0.05).ZNF280D2 may have an important effect on the growth and development process of digestive system and muscle in pigs.In addition,the relative expression levels of large white ZNF280D1 in the cerebellum significantly higher than ZNF280D2(P<0.01),and significantly higher than that of in other organizations(P<0.01),explain ZNF280D1 may also have some function in the cerebellum.(3)With the increase of age,the relative expression of ZNF280D2 in longissimus dorsi,psoas and biceps femoris muscle in Large White pigs and Mashen pigs are obviously increased.The expression of ZNF280D1 in muscle of Large White pigs are gradually reduced,and in the muscle of Mashen pigs are firstly decreases,then small amplitude increases,but the expression of ZNF280D1 at 6 months of age is still lower than the expression of birth.The relative expression of ZNF280D2 in almost muscle tissues(except longissimus dorsi of Large white at birth)are significantly higher than that of ZNF280D1(P<0.01),which mean the ZNF280D2 play a major role and ZNF280D1 auxiliary regulation.The expression quantity trends of two ZNF280D transcripts are not affected by the type of muscle,and just ZNF280D1 affected by the pig species.The developmental rule of ZNF280D genes expression in pigs related to the genetic background of different pig breeds.