Identification Of BBX Gene Family In Cotton And Its Regulatory Role In Proanthocyanidin Synthesis

Based on previous studies,the BBX gene family was screened from upland cotton by bioinformatics tools in this paper,and GhBBX27 and GhBBX33 were identified as candidate genes that could positively regulate proanthocyanidin synthesis,and their functions were preliminary analyzed..Main experimental results as follows:(1)63 Gh BBX genes were screened from upland cotton,and were divided into five structural groups based on the B-Box and CCT domains,which contained 11,7,6,31,and8 BBX proteins,respectively.The motifs of the two B-box conserved domains are C-X2-C-X4-A-X3-C-X2-D-X4-C-X2-CD and C-X7-C-X2-CD-X3-H.(2)Gh＿A06G1830.1 and Gh＿D02G0969.1 were cloned from brown cotton fibers,and were named GhBBX27 and GhBBX33,respectively.The full-length c DNA of GhBBX27 is753 bp,which encodes 250 amino acids,and the isoelectric point is 6.19.The full-length c DNA of GhBBX33 is 864 bp,which encodes 287 amino acids with an isoelectric point of5.92.The protein structure prediction showed that the sondary structures of the GhBBX27 and GhBBX33 proteins were mainly random coils,while the tertiary structure of the GhBBX27 protein was similar to that of the Arabidopsis At BBX21 protein.(3)The prokaryotic expression vectors p ET32a-GhBBX27 and p ET32a-GhBBX33 were successfully constructed,and were introduced into BL21 strain for protein expression.The result indicated that the target bands of 67.7 k Da and 71.2 k Da were obtained in the supernatant after induction.(4)Successful transformation of wild-type Arabidopsis thaliana using Agrobacterium-mediated method,DMACA staining of transgenic Arabidopsis seed coat is darker than wild type,and the hypocotyls of transgenic Arabidopsis seedlings are shorter than wild type Arabidopsis.(5)The brown cotton was successfully transformed cotton by the pollen tube channel method.The expression levels of transgenic brown cotton GhBBX27 and GhBBX33 increased compared to normal brown cotton,and the content of proanthocyanidins also increased.Speculate that GhBBX27 and GhBBX33 can promote the accumulation of proanthocyanidins.