Cloning, Expression And Protein Identification Of A CAP Gene In Gossypium Arboreum L.

Two cotton germplasm(Gossypium arboreum L.),DPL971 and its lint-fuzzlessmutant(DPL972), were used as materials in this study.The cDNA and DNA sequences of adenylyl cyclase-associated protein(CAP)were obtained from DPL971 and DPL972.To gain insight on the CAP role in cotton fiber development,the cloned CAP cDNA was expressed and purified.The expression level in earlier stages of fiber development and protein characters of this CAP gene were analyzed in this research. The main results are as follows:1.The primary structure of 253 fiber(trichome)development associated-genes were compared between fuzzless mutant(DPL972)and wild-type(DPL971),and the target gene(cap)closely related to the fuzzless mutant was found.2.The whole cDNA sequence of the cap genes(GaCAP/GaCAPm)were cloned between DPL971 and DPL972 by RT-PCR technology.This gene possess an open reading frame(ORF)of totally 1416 nt encoded a protein of 471 amino acids,and the molecular weight of its protein is 50.6kDa.The deducible nucleotide sequence and amino acid sequence of GaCAP/GaCAPm are highly homologous to the CAP genes of other species.3.The expression patterns of GaCAP and GaCAPm were identified in different tissues from DPL971 and DPL972 using semi quantitative reverse transcriptase PCR(RT-PCR)and confirmed by Northern blot analysis.The results showed that CAP transcripts maintained a persistent high level during the early ovule development(from -1 to 4 DPA)in both DPL971 and DPL972.While the level of CAP transcripts in the mutant decreased gradually as the ovule developed(4 to 9 DPA)and hardly any transcripts were detected in the 9 DPA ovule.In addition,a moderate level of CAP was detected in leaves,hypocotyls,and roots during the different developmental stages.These results indicated the CAP gene specifically expressed in ovules during the fiber developmental stages compared with that in other tissues over various vegetative periods.The comparison of gene expression profiles between wild type and fuzzless mutant also provided the evidence that CAP was involved in fuzz formation.4.The specific primers were designed by the GaCAP cDNA sequence isolated from cotton.Using the leaf DNA of Gossypium arboreum L.as template,GaCAP DNA sequence was obtained by PCR. The gene's sequence was analyzed by means of DNAStar.The coding region of the GaCAP gene contains ten exons and nine introns.The intron size is approximately ranged from 100 to 1000 bp.The CAP proteins in G.arboreum(471 amino acids)and A.thaliana(476 amino acids)are similar in size. However,the genome sequence of GaCAP(～4kb)was much larger than AtCAP gene(～2.9kb)which indicated that the sequence of intron is more complex in G.arboreum.5.The alignment of the amino acid sequences between G.arboreum CAP and the CAPs from other species showed that the identity between the C-terminal domain of G.arboreum L.CAPs and those of A. thaliana,Medicago truncatula and Oryza sativa CAP is 83%,while the N-terminal domain of G arboreum L.CAP is 74%,73%and 63%identical with the corresponding regions of A.thaliana,M. truncatula and O.sativa CAP,respectively.So the C-terminal domain of G.arboreum L.CAP shows higher homology with other plant CAPs than the N-terminal domain.The CAP key motifs(e.g.Arginine, Leucine and Glutamic acid repeats motif,actin binding domain,SH3 motif and multimerization domain) were consistently found in the putative GaCAP protein sequence.Intriguingly,the nonpolar amino acid Alanine was substituted by a polar amino acid Threonine at conserved position 44 of GaCAPm.This may result in the different structure and function of GaCAPm from other CAP homologs.6.To determine the copy number of GaCAP gene homologue in cotton genomic DNA,Southern blotting hybridization was conduced by applying GaCAP cDNA probe.The result indicated that there was only one copy in cotton genome.7.The Prokaryota expression vector was constructed,the protein was expressed in vitro and purified to study the characterization of objective protein.The optimal temperature of the recombinant CAP was 30-60℃with a best temperature at 36℃,and its optimal pH was 5.0～6.0.The GaCAP maintained 80%activity after treated 30 min in the buffer with pH 4.0,and this reveal the protein is acid tolerance.