Functional Research Of MeMYB2 Transcription Factor In Cassava

Cassava（Manihot esculenta Crantz）is a tropical crop which mainly grown in guangzhou guangxi hainan yunnan fujian and other provinces.Cassava has the characteristics of tolerance to poor and drought by root system developed.It has a unique response mechanism to the indirect drought and low temperature in the tropical.Therefore,the response mechanism of tropical crops was analyzed under cold and drought stress that could improve the yield and quality of tropical crops in China.In the previous of study,the MeMYB2 gene was screen in drought and low temperature stress by high-throughput sequence and transcriptome analysis.The expression of MeMYB2 gene under different stress treatments by quantitative real time PCR in cassava.The subcellular localization of MeMYB2 gene was determined by GFP fusion expression technique.Fragment cloned MeMYB2 gene to determine the specific location of the self-activating activity of MeMYB2 gene.Through yeast two hybrid technique and bimolecular fluorescence complementary technology,the interaction protein upstream of MeMYB2 was screened and the relationship of the interaction protein was determined.Screening of MeMYB2 regulated downstream target genes by yeast one hybrid.The results lay the foundation for elucidating the molecular mechanism and the function of MeMYB2.The main results are as follows:The results of Real-time PCR showed that MeMYB2 may not respond to ABA stress response mechainsm.MeMYB2 may be involved in drought stress response,and the expression of MeMYB2 gene is down-regulated.The water loss rate of the leaves was determined by MeMYB2-RNAi transgenic cassava and wild cassava within twelve hours in vitro.MeMYB2 gene was a negative regulator under the drought stress in cassava.Subcellular localization of MeMYB2 showed that MeMYB2 gene was located in the nucleus.The subcellular localization vector 35S: MeMYB2: GFP was constructed using the molecular cloning method.The transformation into tobacco leaves was carried out by agrobacterium-mediated genetic transformation,and the GFP fluorescence signal was detected under a laser confocal microscope to confirm that the MeMYB2 was localized in the nucleus.The specific location of the active domain of MeMYB2 self-activation was determined,and the results showed that the transcription activating domain of MeMYB2 was between267aa-247 aa at the C-terminal of the coding region of the protein.The interaction protein of MeMYB2 was identified by yeast two hybrid technology,and seven interaction proteins were screened by mating method.Manes.15G115100.1（AD22）is a member of the photoregulatory protein family.NPH3 protein can positively regulate the light regulatory signaling pathway of the light regulator pathway,and improve the tolerance under strong light.Manes.05G142000.1（AD26）belongs to copper ion binding protein.Under abiotic stress,it can regulater the homeostasis of copper in plant,thereby improving the antioxidant effect of plant and enhancing the resistance of plants to pathogenic bacteria.Bimolecule fluorescence complementation of MeMYB2 candidate interacting proteins.The results showed that fluorescent signals were present in tobacco leaves co-injected with YC-MeMYB2⁷⁶⁰ and YN-Me NPH3,YC-MeMYB2⁷⁶⁰ and YN-Me Pet B with YN-MeMYB2⁷⁶⁰ and YC-Me Pet B,which were located in cytoplasm and cell membrane respectively.It shows that MeMYB2,Me NPH3 and Me Pet B can interact directly in tobacco leaves.Screening of MeMYB2 promoter binding 33 genes were screened as candidate target genes by yeast one-hybrid identification.Manes.14G021900.1 was HMA（Heavy metal associated）heavy metal binding protein,containing of zinc finger domain in abiotic stress response.It was speculated that MeMYB2 promoter could interact with HMA protein in abiotic stress response mechanism of cassava.In conclusion,these results would provide a theoretical basis for the further studying the molecular mechanism response to abiotic stress and developing of molecular breeding of tropical crops in cassava.