Cloning And Functional Analyses Of Sub-red (Rs)Candidate Gene In Upland Cotton (Gossypium Hirsutum L.)

Sub-red plant (Rs) of upland cotton is a newly-found genetic marker gene in cotton. The leaf color of Rs cotton falls in between classic red-plant (R1) and normal green-plant cotton, while the petal color is deeper in Rs cotton than R1cotton on anthesis. In addition, compared to R1and normal green plants, Rs cotton possesses higher photosynthetic efficiency. Identifying genes key to the regulation of anthocyanidins synthesisin Rs cotton, may lay the foundation not only for the map-based cloning of Rs gene, but also for high photosynthetic efficiency breeding of cotton.Previously, the Rs gene in cotton was mapped at chromosome7of A genome which is corresponding to R1locus on chromosome16of D genome. Our previous works had indicated that a homologous gene GhPAPID of Arabidopsis R2R3-MYB transcription factor (PAP1) was related the regulation of anthocyanidin biosynthesis in R1cotton, and might bean R1candidate gene. To explore the key anthocyanidin regulatory gene in Rs cotton, we cloned4homologous genes of PAP1from Rs cotton, and determined their functions via sequence analyses, transgenic tobaccos and linkage analysis in a F2population.The main results are as followings:1. Cloning and sequence analyses of PAP1homologous genesAccording the sequences of GhPAPID, we cloned three homologous genes (namely GhPAP1A, GhPAP2A and GhPAP2D) and corresponding cDNAs from Rs and green-plant cotton. Sequence comparison showed that only GhPAP1A had3single nucleotide differences between Rs and green-plant cotton. The multiple-sequence alignment of amino acid sequences indicated that all the4PAP1homologous proteins (GhPAP1A, GhPAP2A, GhPAP1D and GhPAP2D) shared high sequence similarity in the R2R3-MYB domain with homologous proteins from various species, such as cocoa, Arabidopsis and broccoli, etc. suggesting that these4PAP1homologous proteins belonged to the R2R3MYB protein supfamily. Moreover, these MYB proteins contained a conserved C-terminal motif (KPRPR[T/S]), characteristic of the6th subgroup MYB proteins (Sg6).Phylogenetic tree analysis using4cotton PAP1homologous proteins (GhPAP1A, GhPAP2A, GhPAP1D and GhPAP2D) and Arabidopsis secondary metabolism related MYB proteins showed that all the4cotton PAP1homologues were clustered with the Sg6MYB proteins, suggested that the proteins GhPAP1A, GhPAP2A, GhPAP1D and GhPAP2D were homologous to Sg6R2R3-MYBs, and might participated in regulating the biosynthesis and accumulation of anthocyanidins.2. Ectopic expression analyses of PAP1homologous genesIn order to further unveal their biological functions, cotton PAP1homologous genes [GhPAP1A-Rs and GhPAP1A-G (GhPAP1A gnes from Rs and green-plant cotton, respectively), GhPAP2A, GhPAP1D and GhPAP2D] were constructed downstream to CaMV35S promoter and transferred into tobaccos by Agrobacterium-mediated method. It was found that overexpression of all the cotton PAP1homologous genes might activate the expression of anthocyanidin structural genes, and promote the biosynthesis and accumulation of anthocyanidins in transgenic tobaccos. These results indicated that all these cotton PAP1homologous genes encoded functional R2R3-MYB proteins, and could promote anthocyanidin biosynthesis in plants.3. Expression analyses of PAP1homologous genes in Rs cottonTo figure out relationship between PAP1homologous genes and anthocyanidin biosynthesis in Rs cotton, we tested the relative expression levels of GhPAP1A, GhPAP2A, GhPAPID and GhPAP2D in Rs and green-plant cotton by quantitative RT-PCR. Only the expression level of GhPAP1A was significantly higher in Rs cotton than in green-plant cotton, wherea the other three genes sowed no significant difference in expression levels between Rs and green-plant cotton. This result implied that GhPAP1A might play a role in controlling the bio synthesis of anthocyanidins in Rs cotton. 4. The research of GhPAPlA promoterTo relate GhPAPlA to anthocyanidin biosynthesis in Rs cotton, we further cloned the promoter sequence of GhPAPlA(pGhPAPlA, about2.5kb upstream to GhPAPlA ORF) from Rs and green-plant cotton. Compared to green-plant cotton, pGhPAPlA comtained a50bp tandem repeat in Rs cotton. Using a F2population (Rs×green-plant cotton) of148individuals, we detected the linkage relationship between the repeated sequence and Rs gene. It was found that the repeated sequence in pGhPAPlA was cosegregated with Rs, indicating that GhPAPlA is a key regulatory gene of anthocyanidins biosynthesis in Rs cotton.Taken togather, we envisioned that GhPAPlA was a candidate gene of Rs, and the sequence varioation in the promoter region may cause higher expression of GhPAPlA, hence promote the anthocyanidin synthesis and accumulation in Rs cotton.