Identification Of Interaction Partners Of MYB55 From Brassica Napus And Arabidopsis Thaliana And Functional Charcterization Of BnKNAT7

Rapeseed（Brassica napus）is the largest oil crop in China,however,the yield and quality of rapeseed are seriously threatened by multiple biotic or abiotic stresses,such as the poor resistance to lodging and Sclerotinia stem rot,which seriously damage the agricultural production safety.Therefore,creating and breeding novel germplasms and rapeseed cultivars with optimal morphology,lodging resistance,disease resistance,high yield and high quality has important significance.MYB transcription factors（v-myb avian myeloblastosis viral oncogene homologs）,a large family in plants,has a variety of functions in which involved almost all aspects of plant growth,development and metabolisms.For example,MYBs are crucial in regulating the biosynthesis of plant secondary cell walls.The previous studies of our group showed that the number of branches,siliques,grains,yield,etc.in Bn MYB55-overexpressing transgenic rapeseed increased significantly,while the content of lignin,cellulose,pectin and cell density in the stem also increased significantly,behaving as a positive regulator of lodging resistance,S.sclerotiorum resistance,yield,etc.of rapeseed.However,its molecular ineraction mechanism in functional regulation is completely unknown.In this study,the interaction partner factors of MYB55 in Arabidopsis thaliana and B.napus were identified and verified by Yeast Two-Hybrid（Y2H）and Bimolecular Fluorescence Complementation（Bi FC）point-to-point identification.Then,the physiological function of Bn KNAT7 with strong interaction signal in the partner factors of MYB55 was functionally analyzed.In addition,the primary identification of the downstream target genes of At MYB55 was carried out by Yeast One-hybrid（Y1H）and Luciferase Complementation Assay/Dual-luciferase Reporter System（LUC）.The specific findings are as follows:1.MYB55 could interact with 8 transcription factors and 2 MPK kinases in A.thaliana and B.napusBased on the previous Bn MYB55 research result of our group on agronomic,physiological,biochemical level,tissue anatomy,molecular biology,transcriptomic and metabolomics levels,this study first bioinformatically predicted possible interacting partners of MYB55,and then determined 26 candidate partner factors of MYB55,and carried out point-to-point identification and verification using Y2H and Bi FC methods for the genes from both A.thaliana and B.napus.The coding regions of MYB55 and candidate partner factors were cloned,inserted into the corresponding platform vectors of Y2H and Bi FC to construct several batches of expression vectors.The p GBKT7-MYB55 bait vector was converted to Y2HGold for self-activation verification,and the results showed that both At MYB55 and Bn MYB55have self-activating activity.The interaction proteins of At MYB55 and Bn MYB55 were then screened by 0.5 m M 3-AT DO Supplement-Ade/-His/-Leu/-Trp（QDO）medium by3-AT concentration screening test.Finally,10 interaction partner factors were obtained in both A.thaliana and B.napus.They are MYB transcription factors MYB46,MYB43,MYB86 and MYB55 itself,which are involved in the synthesis of secondary cell walls,KNAT3,KNAT4,KNAT5 and KNAT7 of KNOXII subfamily members involved in regulating secondary cell wall trait,and MPK1 and MPK6 in the MPK cascading signaling pathway.Among them,MYB55 showed strong interaction with MYB55,MYB46,MYB86,KNAT3,KNAT4,KNAT5,KNAT7 in both Y2H and Bi FC experiments,showed stronger fluorescence signals in Bi FC while weak interaction in Y2H with MPK1 and MPK6,and weack interaction with MYB43 in both Y2H and Bi FC experiments.2.Bn KNAT7 negatively regulates cell wall lignification interfasicular fibers,S.sclerotiorum resistance and drought toleranceSince the interaction signals between MYB55 and KNAT7 were the strongest in both Y2H and Bi FC methods,Bn KNAT7-3,a representative member of the Bn KNAT7 family,was selected for cloning,subcellular localization experiment,expression characteristic studies and transgene functional studies.Through homologous alignment and evolutionary study of At KNAT7 and Bn KNAT7 family,it was found that there are 5 corresponding genes of At KNAT7 in rapeseed forming a small family:Bn KNAT7-1～Bn KNAT7-5.They encode proteins of291-295 amino acids,with hydrophobic amino acids accounting for the highest proportions（37.06%-37.45%）.More than 50%of their secondary structure is composed ofα-helix,and the serine phosphorylation site accounts for the largest proportion（50%-55%）of phosphorylations.Their subcellular localization is predicted to be in the nucleus,which is supported by the results of Bn KNAT7-3 subcellular localization experiment.q RT-PCR results showed that Bn KNAT7-1,Bn KNAT7-3 and Bn KNAT7-4 were dominantly expressed in seeds and stems,Bn KNAT7-2 was dominantly expressed in bud,and Bn KNAT7-5 was expressed specifically in stems,implying that Bn KNAT7 family was involved in the regulation of secondary wall biosynthesis in multiple tissue types of B.napus.The coding region of Bn KNAT7-3 was cloned to construct the 35S::Bn KNAT7-3overexpression vector,which was transformed into A.thaliana WT and knat7 mutant respectively.Through multi-generation screening,multiple positive transformation plants were obtained.The frozen sections of the lower-part inflorescence stems of 6th week transgenic Arabidopsis plants were prepared,and spontaneous fluorescence of lignin in xylem was observed.The results showed that the xylem vessel molecules of knat7 plant collapsed inward,the vessel cell wall became significantly thinner,and the interfasicular fiber wall became significantly thicker.However,the vessel molecules of overexpressing transgenic plants were normal,while the interfasicular fiber wall was a little thinner.The compensation transgenic plants were similar to the wild type in terms of vessel molecular morphology,vessel cell wall thickness and interfasicular fiber wall thickness.In Phloroglucinol-HCl and M（?）ule staining,it was found that the xylem coloring of knat7was darker,the xylem coloring of overexpressing transgenic plants was lighter,and the phenotype of the compensation transgenic plants was not significantly different from the wild type.The results of Sclerotinia sclerotiorum inoculation identification test of detached leaves of Bn KNAT7 transgenic Arabidopsis,WT and knat7 mutant plants showed that,compared with the wild type,after 24 h of inoculation the leaf lesion size of knat7 plants decreased by 36.7%,the Bn KNAT7-3 compensation transgenic plants decreased by 18.7%,and there was no significant difference in Bn KNAT7-3 overexpressing plants.The results of drought tolerance identitification indicated that after 400 m M mannitol treatment,the root length of knat7 was 55%longer than the WT,and the root length of Bn KNAT7-3compensation transgenic plants was 90.6%of that of the WT,and there was no significant difference in the 35S::Bn KNAT7 everexpressing plants.In summary,in the knat7 mutant,the interfasicular fiber wall is significantly thicker,the resistance to S.sclerotiorum and the drought tolerance are significantly stronger,indicating that KNAT7 negatively regulates the biosynthesis of secondary wall in interfasicular fibers and,perhaps,resistance to S.sclerotiorum and drought tolerance.3.Preliminary study on the downstream target genes of At MYB55The research on Bn MYB55 overexpressing transgenic rapeseed in our group,DEGs are enriched in phenylpropanoid pathway,flavonoid pathway,disease resistance pathway,hormone pathways,etc.In this study,promoter elements of At MYB55 candidate target genes were predictely bioinformatically,and 9 DEGs（At PAL2,At PAL4,At WRKY75,At SAUR22,At IAA9,At WRKY33,Atb ZIP25,At KNAT7 and At KNAT4）were selected as candidate target genes of At MYB55 to carry out LUC and Y1H point-to-point identification experiments in A.thaliana.The coding region of At MYB55 and the promoter sequences of candidate target genes were cloned,and batches of LUC and Y1H vectors were constructed.The results of LUC experiments showed that there was no significant difference between the LUC/REN values of the co-injection of At MYB55 and Ben’s tobacco with 9 candidate target genes and the LUC/REN values of co-injection of 62SK and the candidate target genes.In Y1H experiments,the GAL4 AD-At MYB55 recombinant plasmid and the p GADT7 empty carrier plasmid were transferred into a competent yeast cells prepared by Y1H strains containing the candidate target gene promoter,respectively,and the yeast strains co-transformed with At PAL2,At PAL4,At WRKY75,At SAUR22 and At IAA9 did not grow on SD/-Leu/Ab A⁵⁰ solid medium.Yeast strains co-transformed with Atb ZIP25,At WRKY33 and At KNAT7,respectively,could not grow on SD/-Leu/Ab A¹⁰⁰ solid medium,and yeast strains co-transformed with At KNAT4 could not grow on SD/-Leu/Ab A¹⁵⁰ solid medium,but they could grow normally on SD/-Leu solid medium.Strains transformed with the p GADT7 empty carrier plasmid grew normally on both SD/-Leu and SD/-Leu solid media containing different concentrations of Ab A.In summary,At MYB55 can not bind to the promoter fragments of At KNAT4,At KNAT7,At PAL2,At PAL4,At IAA9,At SAUR22,Atb ZIP25,At WRKY33 and At WRKY75,which may not be direct target genes of At MYB55.