Cloning, Expression And Bioinformatic Analysis Of BHLH Protein Genes In Cotton

BHLH (basic helix-loop-helix transcription factor) proteins are an important class of transcription factors found throughout plants and involved in a wide range of physiological processes, such as photomorphogenesis, light signal transduction, secondary metabolites biosynthesis, and so on. Flavonoids contain a lot of secondary metabolites, including anthocyanins, proanthocyanidins, flavone, flavonols, etc. which are the primary pigment with important physiological functions and also affect the quality of nutrition and appearance of agricultural products. Anthocyanins and proanthocyanidins biosynthesis in plants are mainly controlled by three kinds of transcription factors (MYB, bHLH and WD40proteins), which form a transcriptional complex to target promoters of downstream structural gene, resulting in activation of these genes and biosynthesis of anthocyanidins and/or proanthocyanidins.Cotton is the most important fiber crop in the world. This study performed bioinformatic analysis of bHLH protein gene on the basis of the whole genomic sequence of Gossypium raimondii. Furthermore, bHLH protein genes associated with anthocyanins and proanthocyanidins biosynthesis were cloned from upland cotton, and their expressions were analyzed.The main results are as followings:1. Identification of bHLH protein genes in cottonOn the basis of genomic and expression sequences of Gossypium, we totally identified294bHLH protein genes distributed on the13chromosomes of G. raimondii, and each chromosome contained13to43bHLH protein-coding genes.2. Evolutionary classification of bHLH protein in cottonTo analyze comprehensively the characteristics of bHLH protein family in cotton and their relationship with homologous proteins from other plants, we carried phylogenetic tree analysis of281cotton bHLH proteins with172bHLH proteins from Arabidopsis using maximum parsimony and neighbor-joining methods. Accordingly, cotton and Arabidopsis bHLH proteins were classified them37protein subfamilies. Compared with Arabidopsis, the subfamily No. remained unchanged in cotton, wherea the protein protein No. in certain subfamilies increased.3. Bioinformatic and expression analysis of the fifth bHLH subfamilyIn phylogenetic analysis, cotton GobHLH058, GobHLH060, GobHLH106, GobHLH119and GobHLH126and Arabidopsis AtbHLH001, AtbHLH002, AtbHLH012and AtbHLH042classified in the same subfaminly (the fifth group). Multiple sequence alignment indicated that the N-terminal of bHLH structure domain had three highly conserved domains (BOX11, BOX14and BOX33), in addition to the highly conserved bHLH domain. Quantitative RT-PCR analyses showed that the expression level of GobHLH106and GobHLH119increased significantly in the brown fiber and red leaves, accumulating high levels of anthocyanins and proanthocyanidins, respectively. This result indicated that GobHLH106and GobHLH119might be involved in regulation of anthocyanin and proanthocyanidin biosyntheses in cotton.4. Cloning, bioinformatic and expression analysis of GhbHLH106and GhbHLH119in upland cottonHomologous genes of GobHLH106and GobHLH119were cloned from G. hirsutum, G. arboretum and G. raimondii. It was found that G. hirsutum harbored two copies of GhbHLH106and GhbHLH119genes from A and D genome, named GhbHLH106A/D and GhbHLH119A/D, respectively. Sequence comparison indicated that the gene structure of GhbHLH106and GhbHLH119were highly conserved in G. hirsutum, G. arboretum and G. raimondii. Phylogenetic analysis showed that the two copies of GhbHLH106and GhbHLH119genes in G. hirsutum were respectively orthologous to GabHLH106and GabHLH119from G.arboretum (AA) and GrbHLH106or GrbHLH119from G. raimondii (DD). Quantitative RT-PCR showed that the expression levels of GhbHLH106A/D and GhbHLH119A/D all increased significantly in the brown fiber and red leaves. These results suggested that bHLH protein genes from diploid ancestors might largely remain in allotetraploid cotton.5. Transient expression in tobaccoTo determine the functions of GhbHLH106and GhbHLH119in regulating anthocyanin biosynthesis, we cloned the cDNAs sequences of GhbHLH106A and GhbHLH119D from upland cotton, and analyzed their functions to activate anthocyanin structural genes by transient expression in tobacco.Dual luciferase assay indicated that GhbHLH106and GhbHLH119, in combination with a cotton R2R3-MYB gene GhPAP1-D2, could both activate a Arabidopsis DFR promoter.This result suggested that GhbHLH106and GhbHLH119might interact with GhPAP1-D2and positively regulate the expression of anthocyanin structural genes.