Identification And Functional Analysis Of R2R3-MYB Gene Family Membersand CiCesAs From Caragana Intermedia

Caragana intermediais a perennial shrub of Caragana,Leguminous.lt is drought andsaline-alkalitolerant,and easier to survive in extremeconditionsbecause of the well-developed roots.C.intermedia played an important role in maintaining a good ecological environment.R2R3-MYB transcription factors family regulated numerous cellular and molecular mechanisms,such as plant development,primary and secondary metabolism,cell differentiation,and stress response.CesA regulates the cellulose synthesis,and responds to abiotic and biotic stress.At present,there are few studies on R2R3-MYB gene and CesA gene in C.intermedia.In this study,CiMYB gene family was isolated and identified fromdrought treated transcriptome of C.intermedia,functional analysis of threeCiMYBs were carried out;furthermore,CiCesAs were cloned,and functions were analyzed:1.Totally 40 CiMYBs withcomplete R2R3-MYB domin were obtained from the drought treatecd transcriptome of C.intermedia,and 29 CiMYBshave complete open reading frames(ORFs);the ORF length is between 700 and 1200bp,the number of encoded amino acids ranged from 200 to 400.Among them,30 CiMYBs were clustered into 14 different subgroups of R2R3-MYBof Arabidopsisthaliana;the remaining 10 CiMYBs belong to the un-grouped R2R3-MYB,just like their homologous AtMYBs.2.Expession pattern analysis showed thatthe majority of CiMYB genes responded to drought,ABA,NaCl,cold and UV-B stresses.The expressions of CiMYBs have certain tissue specificity,and higher expression level is found in stem and leaf.3.All of CiMYBs localized in nucleiaccording to prediction bysubcellular localization software,NLS was found in 12 CiMYBs;furthmore,the localization assays revealed that CiMYB 15,CiMYB74 and CiMYB 116 localized in the nuclei.4.The expression of CiMYB15 was induced by UV-B;the main cis-elements of CiMYB15 promoter includesabioticstress responded elements(G-box,P-box,GT1-motif and MBS),biotic stress responded elements(BOX-W1 and EIER),and MYB binding sites of flavonoids synthase regulatory genes.Total flavonoids content of CiMYB15 overexpression plants was higher than that of wild type Arabidopsis;in addtion,the expression level of AtCHS was increased in transgenic Arabidopsis.5.The predicted cis-elements of CiMYB74 promoter are mainly CGTCA-motif,TGACG-motif,CE3,ARE,and Circadian-related.CiMYB74 overexpression lines were more sensitiveto salt stress during seeds germination,and the transcript level of SOS1?SOS2?SOS3and HKT was decreased slightly in transgenic Arabidopsis.6.CiMYB116 promoter contains some stress responded elements,such as HSE(heat shock element),TCA-element,TC-rich repeat and circadian element;Overexpressionof CiMYB116 gene reduced the sensitivity of transgenic plants to ABA at seed germination stage.7.CiCesA8 expressed widely in different tissuesofC.intermedia,the expression of CiCesA8 was inhibited by drought,ABA and BR stresses.CiCesA8overexpression linesexhibitedhypersensitivityto ABA at seed germination stage,and drought tolerance of transgenicArabidopsis was reduced;ABA-and stress-related genes were down-regulated in the overexpression lines.Complementation of CiCesA8 rescued the dwarf phenotype of its Arabidopsis homologuemutantlew2-1.