Cloning And Functional Analysis Of Lignin Biosynthesis Related MYBs Genes From Citrus Maxima Juice Sacs

Citrus maxima(Burm.)Merr.'Hongroumiyou' is a bud mutant cultivars from‘Guanximiyou'.Juice sacs granulation which occurred in the later stage of fruit ripening and postharvest storage influeced the fruit quality seriously.To provide a new reference for the study of juice sacs granulation mechanism,the genetic characteristics and transcription regulation of MYBs involed in lignin biosynthesis in juice sacs from‘Hongroumiyou'were studied.The main results were as follows:1.A total of 86 differentially expressed Citrus maxima MYBs genes sequences were screened from RNA-seq database.According to phylogenetic analysis by MEGA5.2,Citrus sinensis genome-wide analysis and sequences alignment analysis with Citrus sinensis and Arabidopsis thalian,17 sequences were picked from the 86 MYBs.Finally,sixteen Citrus maxima MYBs genes encoding region sequences from'Hongroumiyou' including CmMYB58-2,CmMYB330,CmMYB85-1,CmMYB85-2,CmMYB20,CmMYB58-1,CmMYB61,CmMYB55,CmMYB86,CmMYB103,CmMYB46-1,CmMYB46-2,CmMYB77-1,CmMYB77-2,CmMYB52-1,CmMYB52-2 were cloned respectively.2.Bioinformatics softwares employed on CmMYBs sequences analzying.The results showed that 16 CmMYBs were unstable,hydrophilic and non-secretory proteins,which contained a plurality of different kinase phosphorylation sites.They were located in the nucleus.Protein secondary and tertiary structure analysis,domain prediction and amino acid sequences alignment results showed that the existence of ?-helix and ?-turn made CmMYBs form the helix-turn-helix structure.CmMYB58-1 was 1R MYB containing a MYB domain,the other CmMYBs were typical R2R3 MYB,containing 2 MYB domains.According to phylogenetic tree analysis,CmMYB58-2 belonged to the R2R3 MYB 3 subgroups,CmMYB330 belonged to 4 subgroups,CmMYB61,CmMYB55 and CmMYB86 belonged to 13 subgroups,CmMYB77-1 and CmMYB77-2 belonged to 22 subgroups,CmMYB52-1 and CmMYB52-2 belonged to 21 subgroups.CmMYBs were similar to MYBs from the corresponding Arabidopsis,Citrus sinensis,Populus trichocarpa,Pinus taeda.The results suggested that CmMYBs should be involved in the regulation of lignin metabolism genes.3.CmMYBs qPCR results indicated that the expression level of CmMYBs showed different changing tendency along with the advancement of juice sacs development in 'Hongroumiyou'.Most of the expression level of CmMYB46-1 was very low and less than 0.1 during the whole juice sacs development periods,the expression level of CmMYB55 in the parts of juice sacs near the core and be far away from the core was the highest on 161 DAF respectively,while the expression level of CmMYB77-l and CmMYB77-2 in the parts of juice sacs near the core and be far away from the core were the lowest on 161 DAF respectively.With the advancement of juice sacs development in 'Hongroumiyou',juice sacs granulation degree was gradually increased,the expression level of CmMYB58-2,CmMYB330,CmMYB85-1,CmMYB85-2,CmMYB20,CmMYB61,CmMYB86,CmMYB103,CmMYB46-2,CmMYB52-1 and CmMYB52-2 in the part of juice sacs near the core were increased in general,and then declined in the later stage of juice sacs development.According to the expression pattern of CmMYBs in the development of juice sacs,CmMYBs genes may be involved in the juice sacs granulation process,such as CmMYB58-2.4.Results of CmMYBs subcellular localization analysis showed that CmMYB330,CmMYB85-1,CmMYB85-2,CmMYB20,CmMYB58-1,CmMYB61,CmMYB55,CmMYB86,CmMYB103,CmMYB46-1,CmMYB46-2,CmMYB52-1 and CmMYB52-2 were nuclear localization proteins.Results of CmMYBs transcriptional activation activity analysis showed that CmMYB330 and CmMYB46-2 were no transcriptional activation activity,CmMYB58-2,CmMYB85-1,CmMYB85-2,CmMYB20,CmMYB58-1,CmMYB61,CmMYB55,CmMYB86,CmMYB103,CmMYB46-1,CmMYB52-1 and CmMYB52-2 had transcriptional activation activity in Y2H Gold yeast.The transcriptional activation activity region of CmMYB20,CmMYB55 and CmMYB86 were at their C-terminal.5.Regulatory sequence of the 5' terminal in 'Hongroumiyou' lignin biosynthesis genes including proCmC4H,proCm4CL,proCmC3H,proCmCCoAoMT1,proCmCAD1,proCmF5H and proCmCOMT1 were cloned by analysis of 'Hongroumiyou' lignin biosynthesis genes that had been cloned and Citrus sinensis genome sequences.They were predicted to contain a number of different types of MYB binding sites elements,such as AC-I,AC-?,MBS,MBSI,MRE and so on.They also contained many different types of hormone response elements,such as ethylene,auxin,gibberellin,salicylic acid,methyl jasmonate.6.Point-to-point yeast one hybrid analysis results of the interaction between the regulatory sequence of the 5' terminal of CmC4H,Cm4CL,CmC3H,CmCCoAoMT1,CmCAD1,CmF5H,CmCOMT1 and CmMYB58-2,CmMYB330,CmMYB85-1,CmMYB85-2,CmMYB20,CmMYB58-1,CmMYB61,CmMYB55,CmMYB103,CmMYB46-2,CmMYB52-1,CmMYB52-2,respectively indicated that the lowest concentration of AbA resistance for the bait yeast strains of the regulatory sequence of the 5' terminal of CmC4H,CmCCoAoMTl and CmCOMT1 could not be determined,and subsequent yeast one hybrid analysis only carried on the regulatory sequence of the 5' terminal of Cm4CL,CmC3H,CmCAD1 and CmF5H.This study demonstrated that CmMYB58-2 bound to the regulatory sequence of the 5' terminal of CmC3H and then involved in transcriptional regulation of Citrus maxima juice sacs lignin biosynthesis.