Screening,Cloning And Characterization Of 2C Protein Phosphatase Gene Related To Pre-Harvest Spouting In Wheat

2C Type protein phosphatase(PP2C)is a serine/threonine residue protein phosphatase,which plays an important role as a negative regulator in ABA-induced seed germination signaling pathway.In order to obtain PP2 C candidate genes which may be related to pre-harvest spouting(PHS),two wheat varieties with different PHS resistance were selected as materials in this study.Bioinformatics technology and fluorescence quantitative RT-PCR technology were used to search and screen candidate genes which were highly expressed in grains and expressed differently in two wheat varieties with different spike germination resistance.On this basis,three candidate genes were cloned and their expression patterns during seed development and germination were systematically analyzed.The main results are as follows:1.Using PP2 C domain "IPR001932" as the key word,50 PP2 C gene sequences of wheat with complete coding frame were obtained by searching Triticeae Full-Length CDS Data Base database.The tissue-specific expression analysis of these genes in roots,stems,leaves and grains showed that 12 genes were highly or specifically expressed in grains.Furthermore,the expression analysises of the above candidate genes during seed development using Huaimai 0360 and Zhongyou 9507 as test materials showed that there were six genes,named PP2C14,PP2C16,PP2C23,PP2C25,PP2C28 and PP2C34,which expressed differently in the two cultivars.It was speculated that these genes might play an important role in regulating PHS.These genes were used as candidate genes for research.2 The full-length sequences of three candidate genes,named TaPP2C16,TaPP2C25 and TaPP2C28,were isolated by RT-PCR and RACE,and located on the long arms of chromosomes 5,7 and 6,respectively.The full-length cDNAs of TaPP2C16 homologous genes TaPP2C16-5A,TaPP2C16-5B and TaPP2C16-5D were 1423 bp,1411 bp and 1416 bp,respectively.5’UTR was 6bp,3’UTR was 133 bp,124 bp and 127 bp,respectively.ORF was 1284 bp,1281 bp and 1284 bp,encoding 427,426 and 427 amino acids,respectively.The deduced amino acid sequences of the three homologous genes were different in 18 loci.The genome sequences of the three homologous genes are 2318 bp,2312 bp and 2289 bp in length,respectively.They all contain five exons and four introns,of which the first intron is the largest and the third intron is the smallest.The three sequences were relatively conserved except for 51 SNP differences and 9 base deletion or insertion mutations.The full length of TaPP2C25 homologous genes TaPP2C25-7A,TaPP2C-7B and TaPP2C-7D were 1038 bp,1037 bp and 1038 bp,respectively.The 5’UTR was 85 bp,84 bp and 84 bp,the3’UTR was 83 bp,83 bp and 84 bp,and contains a 870 bp open reading frame(ORF),encoding289 amino acids.There are many SNP differences in the coding region.The genome sequence was1955 bp,1945 bp and 2037 bp in length,respectively,with 8 exons and 7 introns.The full-length cDNA of TaPP2C28-6B gene is 1266 bp in length,contains a 1155 bp open reading frame,with 70 bp in the 5’ UTR and 41 bp in the 3’ UTR.TaPP2C28-6B was predicted to encode a 384 amino acid protein with a molecular mass of 39.63 k D and isoelectric point of 5.25.3.QRT-PCR analysis of cloned genes during seed development showed that the expression levels of TaPP2C16,TaPP2C25 and TaPP2C28 in two varieties with different PHS resistance increased gradually with grain filling process,and showed preferentially expression in both embryo and endosperm.However,there are some differences,such as the peak expression of TaPP2C25 A in Zhongyou 9507 was at 30 days after anthesis,and mainly in embryos at 25 days after anthesis,while the peak expression in Huaimai 0360 was at 15 days after anthesis,and mainly in embryos and endosperms at 15 days after anthesis;The expression of TaPP2C25 D in Zhongyou9507 was mainly in embryo and endosperm at 25 days after anthesis.Whilet the high expression of TaPP2C25 D in Huaimai 0360 was mainly in embryo at 25 days after anthesis.4.After treatment with ABA,GA3 and calcium chloride during seed germination,the expression levels of three homologous genes of TaPP2C16 and TaPP2C28 were firstly decreased and then increased.It was speculated that the expression of TaPP2C16 and TaPP2C28 genes were not affected by exogenous ABA and GA3,and might be related to the content of endogenous ABA and GA3.The expression levels of TaPP2C25 genes in GA3 treatment wwere same as that in TaPP2C16 treatment,but the expression levels of three genes in ABA treatment were different.TaPP2C25-B and TaPP2C25-D showed a trend of first increasing and then decreasing with the increase of treatment time,which indicated that the two genes had a certain response to exogenous ABA.Under Ca Cl2 treatment,except the expression of TaPP2C16-5D gene decreased with the increase of treatment time,the other genes showed a trend of first increasing and then decreasing,which indicated that most of the cloned genes were calcium-dependent.