Bioinformatic Analysis Of Cotton CIN TCP Transcriptional Factors And Their Functions In Gossypol Gland Formation

TCP(TEOSINTE-BRANCHED1/CYCLOIDEA/PCF)genes encode plant-specific transcriptional factors containing conserved TCP domain.The TCP domain is a bHLH motif consisting of 60 amino acids which are necessary for DNA binding and protein–protein interactions.It is suggested that TCP transcriptional factors play crucial roles in multiple developmental processes,including leaf development,flower development and seed germination in Arabidopsis,and the elongation of cotton fibers as well.Cotton(Gossypium)is an important commercial crop,and cotton fibers are the most widely used natural fibers in textile industry,which occupies a pivotal position in the national economy.The completion of genome sequencing of diploid cottons G.raimondii,G.arboretum and tetraploid cottons G.hirsutum and G.barbadense,offered a good opportunity for the comprehensive identification of the members of cotton TCP gene family.It is of great significance to fully identify the members of the TCP gene family in cotton and carry out their corresponding functional studies.In this study,comprehensive identification of the TCP transcriptional factor family in tetraploid cotton G.hirsutum and G.barbadense were performed.On this basis,we conducted following analysis in G.hirsutum and G.barbadense including identification of TCP genes,chromosomal distribution and gene expression level detection in different tissues.Meanwhile,we employed virus-induced gene silencing(VIGS)to down-regulate the expression of CIN TCP genes GhTCP2A/2D,GhTCP24A/24 D and GhTCP3A/3D,which were predominantly expressed in most cotton tissues.The main results are as followed.1 Identification of TCP protein family members of G.hirsutum and G.barbadense and phylogenetic analysis of cotton TCP referencesTo identify all members of TCPs in upland cotton(G.hirsutum)genome and sea-island cotton(G.barbadense)genome,we performed a BLASTp search against upland and sea-island cotton protein database using the TCP protein sequences of G.raimondii and G.arboretum as probes.A total of 73 and 69 encoding genes of TCP transcriptional factors were identified in the assembled genome of upland cotton and sea-island cotton,respectively.Class I subfamily contains the largest number of members of TCP genes,accounting for more than 60% of total TCPs.In class II subfamilies,the number of CYC/TB1 cluster members is the least.The number of CIN cluster members is twice as much as that of CYC/TB1 cluster members.Based on the comparative analysis of the members of the TCP protein family of G.raimondii,G.arboretum,G.hirsutum and G.barbadense,40 TCP references of the Gossypium species were finally obtained.We further conducted phylogenetic analysis using TCP protein sequences of Gossypium species by employing MEGA 6 software.The results showed that the TCP transcriptional factor family of the Gossypium species could be divided into 7 groups(A to G).2 Chromosomal distributions of the TCP genes in Gossypium speciesUsing the TCP genes in G.raimondii as a reference,the chromosomal distributions of TCP genes of upland cotton and sea-island cotton were analyzed by MapInspect software.It was demonstrated that the chromosomal distribution of TCP genes in upland cotton and sea-island cotton is roughly of the same.Except the chromosome 6 of tetraploid cotton,all the rest chromosomes contained TCP genes,while the chromosome 12 had largest number of TCP genes.3 Expression profiles of CIN TCP genes in Gossypium speciesThe expression of CIN TCP genes in three developmemtal stages of G.hirsutum and G.barbadense leaves were analyzed.The expression level of the CIN TCP genes in these two tetraploid cotton species showed no remarkable change.The changing trend with leaf development of expression level of TCP2A/2D and TCP24A/24 D was opposite and the expression level of TCP3A/3D showed no significant change in leaves of different developmental stages.Based on RNA-Seq data of various tissues of upland cotton,the expression level of GhTCP2A/2D,GhTCP24A/24 D and GhTCP3A/3D are the predominantly expressed CIN TCP genes.Quantitative RT-PCR analysis indicated that the expression level of CIN TCP genes in leaves and petals was higher than in root,stem and fibers.4 The number of gossypol glands increased in cotton leaves with GhTCP2A/2D or GhTCP24A/24 D silencedVirus-induced gene silencing(VIGS)was employed to down-regulate the expression level of CIN TCP genes GhTCP2A/2D,GhTCP24A/24 D and GhTCP3A/3D.In the mature leaves with GhTCP2A/2D or GhTCP24A/24 D down-regulated,the gossypol gland numbers increased significantly.Whereas,down-regulation of GhTCP3A/3D resulted in the curly leaves showing no significant change in gland numbers.Yeast two hybrid experiment showed that GhTCP2 A,but not GhTCP24 A,interacted with GoPGF,the key transcriptional factor of cotton gland differentiation.5 Transcriptomic analysis of immature TRV: GhTCP2 A leavesRNA-sequencing was conducted to compare gene expression profiles of GhTCP2-silenced immature leaves.The results showed that the hormone transduction pathway was significantly changed.Especially,the jasmonic acid synthase genes were significantly up-regulated,and the jasmonic acid content increased in the immature leaves.We envisioned that GhTCP2 and/or GhTCP24 might regulate gossypol gland formation via jasmonic acid pathway.Based on the results,40 TCP reference genes of the Gossypium species were obtained.Meanwhile,it was also found that GhTCP24A/24 D was a negative regulator of cotton gland differentiation.