Genome-wide Analysis Of BFT And APs Genes In Cotton

BROTHER OF FT AND TFL1(BFT)is known as the brother of FLOWERING LOCUS T(FT)and TERMINAL FLOWER1(TFL1),and they play an important role in regulating flowering transition.Under salt stress,Arabidopsis BFT protein interacts with b ZIP transcription factor FD in shoot apical meristem to form a complex to regulate flowering transition.A yeast two-hybrid c DNA library of Xinluzao 33 leaf tissue was previously constructed,and an aspartic proteinase(APs)interacted with the Gh FT1 protein of upland cotton was screened by yeast two-hybrid(Y2H).The protease gene plays an important role in plant growth and development,disease resistance and stress.Object: Genome-wide identification of cotton BFT and Gh APs genes in this study.Through bioinformatics analysis,the possible biological functions of Gh APs were preliminarily predicted.The function of Gh BFT in flowering regulation were explored by protein interaction and transgenic Arabidopsis assays.Methods: Cotton BFT and Gh APs genes were identified by genome-wide analysis according to the recently updated cotton genome database.Gh BFT1 and Gh BFT2 were cloned by RT-PCR.the expression profiles of Gh BFT1 and Gh BFT2 were detected by quantitative real-time RT-PCR(q RT-PCR).The interaction between Gh BFT protein and upland cotton FD was studied by Y2 H and bimolecular fluorescence complementary(Bi FC)assays.Plant expression vector 35S:Gh BFT1 was constructed and transformed into wild-type Arabidopsis thaliana by Agrobacterium mediated floral-dip method.Results: 1.Genome-wide analysis of cotton BFT gene:(1)A total of 12 BFT genes were identified by genome-wide analysis according to the cotton genome database,including four BFT in tetraploid G.hirsutum and G.barbadense,and two BFT in diploid G.arboretum and G.raimondii,respectively.(2)Cotton BFT gene consists of four exons and three introns,and exons 1 and 4 are relatively large;the physical and chemical properties of BFT proteins are similar,and they have the same conserved motif and domain(D-P-D-x-P;G-x-H-R).(3)The cloned Gh BFT1 and Gh BFT2 genes all come from D subgenome with the ORF length of 525 bp and 522 bp,respectively.(4)The expression levels of Gh BFT1 and Gh BFT2 were the highest in upland cotton leaves,followed by flowers,and the expression level of Gh BFT1 in each tissue was significantly higher than that of Gh BFT2.(5)Gh BFT1 and Gh BFT2 were induced in response to salt,drought,ABA,high temperature and low temperature stress with varied degrees,and both had similar expression patterns.(6)Y2H and Bi FC assays showed that both Gh BFT1 and Gh BFT2 could interact with upland cotton Gh FD1,Gh FD2,Gh FD3 and Gh FD4 proteins;(7)Overexpression of 35S:Gh BFT1 in Arabidopsis thaliana delayed flowering and increased the number of rosette leaves.2.Genome-wide analysis of cotton APs gene:(1)87 Gh APs were identified in upland cotton,of which 83 had two complete ASP conserved sites.(2)Phylogenetic tree analysis divided them into three categories: typical(A),nucellin-like(B)and atypical(C),and the members of each subgroup have some differences in gene structure,conservative motif and amino acid sequence.(3)Chromosome mapping showed that Gh APs genes were distributed on all chromosomes except A04,and five pairs of tandem repeat genes were found.(4)Collinearity analysis showed that there were a lot of collinearity among Gh APs genes,and there was a collinear relationship between each Gh APs gene and 1–4 genes.(5)Promoter prediction showed that Gh APs gene promoters contained a number of cis-acting elements of light,hormone(ABA,GA,IBA and Me JA),stress(drought,low temperature,antioxidation,defense and stress),circadian rhythm,meristem expression,endosperm expression and cell cycle regulation.(6)Tissue expression analysis showed that Gh APs were expressed in root,stem,leaf,pistil,sepal,torus,petal,anther and filament of upland cotton and in ovule and fiber at different developmental stages.Conclusion: 1.There are 12 BFT genes in cotton,four in G.hirsutum and G.barbadense,two in G.arboretum and G.raimondii,respectively.Gh BFT1 and Gh BFT2 were highly expressed in cotton leaves and flowers,and responded to salt,drought,ABA,high and low temperature stress.Gh BFT1 and Gh BFT2 proteins interacted with four FD homologous proteins in upland cotton.Ectopic overexpression of Gh BFT1 in Arabidopsis thaliana resulted in delayed flowering of transgenic Arabidopsis,indicating that Gh BFT might compete with FT to bind FD protein,thus inhibiting flowering transition.2.In upland cotton,87 possible Gh APs gene family members were identified and divided into three subgroups: A,B and C.There were differences in gene structure,conserved motifs and amino acid sequences among members of each subgroup,indicating that they may have functional diversity.Gh APs may have gene replication events during the process of evolution.They were expressed in all tissues and development stages of cotton,suggesting that Gh APs may be involved in the regulation of seed germination,ovule development and anther formation of upland cotton.The sequences of Gh APs promoters are rich in elements related to drought,low temperature,wound stress,ABA,GA,Me JA and other hormone related elements,indicating that Gh APs play an important role in response to a variety of stress processes.