Bioinformatics Analysis Of Banana TGA Transcription Factor Family And Function Analysis On The Disease Resistance Of MaTGA8

Banana wilt caused by Fusarium oxysporum f.sp.Cubense(Foc)is one of the main diseases of bananas in my country,which seriously affects the yield and quality of bananas.Therefore,defense genes of banana wilt were excavated and their molecular mechanism of the reaction is explored of great significance for the prevention and treatment of fusarium wilt.TGA is a member of the D subfamily of the bZIP(basic region-leucine zippers,bZIP)family.It is a type of transcription factor first identified in plants and is the main regulator of many plant’s development and physiological processes,including morphogenesis,seed formation,in response to abiotic and biotic stress,maintains normal growth of plant.However,the functional role of TGA in the resistance of bananas to fusarium wilt is still unclear.In order to explore the function of banana TGA,this study used Arabidopsis TGA transcription factor family member proteins as the query sequence.We conducted blast search for the TGA transcription factor family in the banana genome database,and performed bioinformatics analysis as well as RT-qPCR analysis on the family members.The MaTGA8 gene was cloned and subjected to bioinformatics analysis and subcellular localization experiments;Banana susceptible and resistant varieties as materials,the salicylic acid(SA)was used to bananas at the seedling stage for abiotic stress,then analysed the MaTGA8 gene and expression level of the downstream target gene MaNPR1 family,further used yeast two-hybrid experiment to determine the interaction relationship between MaTGA8 and MaNPR1;Then MaTGA8 was transformed into Arabidopsis,the transgenic Arabidopsis was selected and treated with Foc TR4,observed the disease resistance,and detected the abundance of Foc TR4 and the differential expression of MaTGA8 in vivo.This study provides candidate genes for application of MaTGA to molecular breeding of banana with resistance to wilt disease.The main results of this research are as follows:A total of 9 family members were identified,named MaTGA1～MaTGA9.TGA family proteins of banana was rich in acidic amino acids,and most of the proteins are alpha helices;the subcellular location is mainly in the nucleus.The phylogenetic tree,nine MaTGA transcription factor members could be classified into two groups,ClassⅠand ClassⅡ.The distribution of gene structure and functional domains also showed a high degree of consistency.The RT-qPCR analysis showed that MaTGA2,MaTGA3,and MaTGA8 were significantly down-regulated both in Williams(susceptible)and Nantianhuang(disease-resistant)after Foc infection.and the gene espression of MaTGA1、MaTGA6、MaTGA7 and MaTGA9 declined first and then rose,however MaTGA4 and MaTGA5 were only up-regulated in William,indicating that MaTGA2,MaTGA3,and MaTGA8 play important biological functions in banana resistance to wilt.The MaTGA8 gene was cloned with a size of 1675 bp,in which the open reading frame length is 1515 bp.The amino acid composition,instability coefficient,fatty acid coefficient and hydrophobicity coefficient of the gene were analyzed by software,and the results showed that MaTGA8 is unstable fat-soluble hydrophilic acidic protein,and contains more cis-acting elements such as light response elements and hormone response elements,endosperm expression elements,and seed specificity.Through the analysis of MaTGA8 protein network interaction,it is found that this gene interacts with multiple MaNPR1 and mainly participates in the SA signal transduction pathway;under the treatment of the different concentrations of SA,banana MaTGA8 and MaNPR1 family genes have different expressions in response to SA.We were speculated that MaTGA8 may interact with MaNPR3,MaNPR4,and MaNPR11.The yeast two-hybrid bait vector pGBKT7-MaTGA8 was constructed,and transformed into yeast strain Y2HGold to carriy out self-activation and toxicity tests on SD/-Trp medium.The results showed that the pGBKT7-TGA8 plasmid was non-toxic and had a certain degree in the case of two reporter genes.However,the degree of self-activation is not serious.The yeast hybrid prey vectors pGADT7-NPR3,pGADT7-NPR4,and pGADT7-NPR11 were constructed and verified by transforming the yeast strain Y2HGold(containing the pGBKT7-MaTGA8 plasmid).The results showed that MaTGA8 had weak interaction with MaNPR4 and strong interaction with MaNPR11.The pCAMIB1302-MaTGA8 expression vector containing the GFP protein was successfully constructed,and subcellular localization imaging of onion epidermal cells revealed that the MaTGA8 transcription factor was located in the nucleus.MaTGA8was genetically transformed into Arabidopsis by Agrobacterium tumefaciens infection.The selecting of specific fragments showed that the positive rate of plants was as high as 96%.The expression of MaTGA8 in transgenic Arabidopsis was significantly higher than that of the wild type.Compared with the uninfected Foc TR4 wild type,the Arabidopsis plant infected with Foc TR4 is smaller.And The transgenic Arabidopsis shows better resistance than the wild type,and the chlorophyll content is not significantly different from that of the untreated wild type.At 96 h,the Foc TR4abundances in transgenic and wild-type Arabidopsis were 1.55×10~6copies/ml and1.66×10~6copies/ml,respectively.The expression of MaTGA8 in transgenic Arabidopsis showed an upward trend,but it was generally lower than that of the untreated gene.It shows that MaTGA8 has anti-disease effect on Foc TR4.In summary,the banana MaTGA8 gene may be involved in the regulation of banana fusarium wilt resistance through interaction with MaNPR1.