| Wool is one of the main by-products of the sheep breeding industry and one of the important economic sources of herdsmen’s income.China is a big country that has many of sheep breeds and a large sheep-breeding power.Wool industry is an important industry in China’s pastoral areas and has a decisive position in the pastoral economy.As a natural material in textile fibers,wool has irreplaceable position in the textile industry due to its many excellent characteristics.Fine wool(13～24 μm in diameter)is an indispensable material for many high-end textiles.However,two thirds of the wool needed in China’s textile industry depends on imports,of which superfine wool is more dependent on imports.China’s sheep farming is distributed in many provinces such as Northeast,Northwest and Inner Mongolia,but most of them are mutton sheep,the number of fine wool sheep is small,and the production of high-end fine wool is low.China Merino fine wool sheep is an early breed of fine wool sheep in our country,but the breed resource is single and the quantity is insufficient.Therefore,it is of great scientific and economic significance to excavate the fine genes of fine wool sheep and improve the quality and yield of wool in China,which is also a major problem to improve the income of herdsmen and the scientific and technological content of sheep industry.Studies have shown that one of the differences in the composition of coarse and fine hair lies in the difference in the content and distribution of proteins called high glycine-lysine(HGTPs).Studies have also shown that the genetic polymorphism affects the bending and fineness of wool.Sheep HGTPs are a multi-gene coding keratin family,whose members mainly include KAP6,KAP7 and KAP8 three different keratin associated proteins(KAP).In view of this,based on the previous research on the polymorphism of the three gene coding regions,this paper mainly used three kinds of sheep(Chinese Merino fine wool sheep,Suffolk sheep,Hu sheep),using PCR cloning,RACE cloning,Sequencing and bioinformatics analysis were used to compare and analyze the sequence differences of three sheep HGTP family gene promoter regulatory elements and upstream regulatory regions,so as to understand the differences of KAP6,KAP7 and KAP8 gene promoter regions in different sheep.It provides a certain theoretical reference for fine wool sheep breeding.The results are summarized as follows:(1)Verification and promoter prediction of KAP6,KAP7 and KAP8 gene upstream sequences obtained by walking method TAIL-PCR(thermal asymmetric interlaced PCR)The upstream clones of KAP6,KAP7 and KAP8 obtained by TAIL-PCR were verified by fragment amplification of the splicing region using DNA samples from Chinese Merino sheep,Hu sheep and goats.Three sequences of sheep were submitted and the Gen Bank accession numbers MT084114,MT084115 and MT090064 were obtained.The sequence of the three clones of the three sheep near the CDS(Coding sequence)region was consistent,indicating that the sequence of the KAP6,KAP7 and KAP8 keratin genes in the 400 ～ 500 bp region near the upstream of the CDS region may be highly conserved in sheep,and had nothing to do with the breed of sheep.At the same time,a preliminary promoter prediction analysis was performed on the verified sequences,and the promoters were predicted in each sequence.The promoter of KAP6,kap7 and KAP8 genes was predicted to be located approximately in the range of-40 ～-1000 bp,-800 ～-1500 bp and-100 ～-900 bp upstream of their respective transcription starting points respectively based on the comprehensive score.At the same time,it also predicted the binding sites of TATA box and related transcription factors such as AP1,TFIID,SRF,NF-KB,CREB,SP1 and so on.(2)KAP6 and KAP8 gene full-length c DNA clone and transcription start point confirmationThe 5′-end RACE(Rapid Amplification of c DNA Ends)and 3′-end RACE clones of KAP6 and KAP8 genes were sequenced and spliced to obtain two gene full-length c DNA sequences with lengths of 606 bp and 559 bp respectively,which were highly consistent with the corresponding sheep sequences published by NCBI.The lengths of their CDS sequences were 252 bp and 189 bp respectively.The5′-UTR(Untranslated Region)regions were obtained in the upstream of CDS,with lengths of 43 bp and 50 bp respectively,and the transcription start points of the two genes and their specific positions and bases in the upstream clone sequences were determined.(3)Cloning and polymorphism analysis of KAP6 and KAP8 upstream regionsIn the preliminary amplification of a small sample size of the upstream promoter sequence of KAP6 and KAP8 genes(selecting Chinese Merino sheep,Hu sheep,and goats as experimental materials),the sequences of about 1600 bp upstream of the KAP6 gene of Chinese merino sheep and Hu sheep were almost identical after interspecies comparison,and no individual polymorphism differences were found among sheep breeds.In the interspecific comparison of KAP8 gene amplified sequences,Chinese Merino and Hu goats had an insertion polymorphism of about 7base length fragments relative to goats,while Chinese Merino and Hu goats and goats had a repetitive(CA)3 base sequence of different lengths.In population testing of KAP6 and KAP8 gene promoter sequences(selecting Chinese Merino sheep and Suffolk sheep as experimental materials),a total of 37 complete sequences were obtained for the KAP6 gene(including 22 Chinese Merino sheep and 15 Suffolk sheep,all of which are around 1000 bp in length).A total of 7polymorphic loci were found,and there were 7 haplotypes in the results.Two haplotypes only appeared in a single sheep breed and belonged to a variety-specific haplotype.A total of 32 complete sequences were obtained for the KAP8 gene(including 21 Chinese Merino sheep and 11 Suffolk sheep,all of which are around2,500 bp in length).A total of 74 SNP(single nucleotide polymorphism)polymorphism sites and fragment insertion-deletion polymorphic sites were found,and there were 32 haplotypes.No species-specific sequences were found in the polymorphisms obtained in the upstream amplified region of the coding region of the KAP8 gene,and 12 representative sequences were selected as representative sequences of the KAP6 and KAP8 genes of Chinese Merino sheep,Suffolk sheep based on the variety-specific haplotypes and mutation points(4)Bioinformatics analysis and interspecies comparison of KAP6 and KAP8 upstream regulatory sequencesThe KAP6 and KAP8 gene representative sequences of Chinese merino sheep and Suffolk sheep were used to predict the transcription factor binding sites and compared within species and between species.The results showed that in the cross-species comparison of the predicted sites of the KAP6 gene,the two sheep breeds had a combination of 12 common transcription factors.Secondly,in the upstream region of the KAP6 gene of Suffolk sheep,six predicted binding sites of transcription factors specifically possessed by this breed were found,namely POU5F1,SMAD1,MBD3,TRIM28,POU2F1 and CREBBP.Similarly,the upstream region of the KAP8 gene also has common transcription factors with 16 binding sites common to both cultivars.Three species-specific binding sites of KAP8 gene were found in Merino sheep,namely CDK9,SMAD4 and TRIM28,in which CDK9 had stable transcriptional elongation and TRIM28 had inhibitory effect.Suffolk sheep have 10 species-specific binding sites,which are STAT3,USF1,SMAD3,STAT1,EP300,CREB1,ETV4,SIN3 A,POLR3A,among which CREB1 has the effect of enhancing transcription,SIN3 A may have the role of transcriptional inhibition.The transcriptional regulation of genes is a more complicated process.The comparative analysis of predicted sites provides a theoretical reference for further study of the gene regulation mechanism of KAP6 and KAP8 and the selection of specific promoter... |
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