Genome-wide Identification And Function Analysis Of The BHLH Gene Family And The TIFY Gene Family In Chinese Jujube

Chinese jujube(Ziziphus jujuba Mill.)is a species with high economic value in the family Rhamnaceae and is also one of the most representative national fruit trees in China.Jujube trees have strong resistance to stress,and their fruits are rich in nutrition.The bHLH(basic helix-loop-helix)gene family is one of the largest transcription factor families in plants,and the TIFY family is widely found in terrestrial plants.The two kinds of transcription factors are not only essential for the normal growth and development of plants,but also participate in the response process of plants to a variety of adversity.In addition,some bHLH family members can interact with TIFY family members,especially the JAZ proteins,to participate in the regulation of jasmonic acid pathway on plant growth and development,cold resistance and pathogen resistance.However,the bHLH family and TIFY family in Chinese jujube have not been reported before and the mechanism of their interactions is still unclear.Therefore,in this study the number,classification,gene structure and expression of jujube bHLH family and TIFY family genes were systematically analyzed,and the candidate genes related to jujube flower,fruit development and stress resistance were screened out.Meantime,the expressions of ZjJAZs in ZjTIFY family changed signifanctly in JWB-infected leaves.In addition,the interaction between ZjMYC2 and ZjJAZs was verified by yeast two-hybrid(Y2H)screening,and the core signaling factors of jasmonic acid pathway in jujube was preliminarily identified,which paved the way for further research on the jasmonic acid pathway regulating jujube-phytoplasma interaction.The main results are as follows:1.Ninety-two bHLH genes in jujube genome were identified,and they were divided into 16 classes by constructing phylogenetic trees,analyzing the conservative domains and comparing homologous proteins.The conservative domains in each class were relatively conservative,and 10 atypical bHLH genes were also found.The number of amino acid residues of the bHLH proteins ranged from 94 aa～891 aa,and the molecular weight ranged from 10.6 KD～78.7 KD.These proteins with isoelectric points less than 7 accounted for 66.3%of the total,and most of them were weakly acidic.Based on their physical and chemical properties,the family members should behave different characteristics.2.Of the 92 ZjbHLHs,70 genes were mapped to 12 pseudochromosomes in the jujube genome,17 genes were located on 12 scaffolds.ZjbHLHs were not evenly distributed across the 12 chromosomes,with the most distributed on Chr.1(9),-4(7),-6(7),-8(9)and-9(9).In addition,there are some genes distributed in clusters on the chromosome,forming tandem repeat genes(ZjbHLH27 and-53;ZjbHLH38,-40 and-41;ZjbHLH64 and-37;ZjbHLH17,-20 and-21).3.The interactions among ZjbHLHs were predicted by STRING,laying the groundwork for the discovery of functionally related genes.4.Thirty-seven bHLH genes were selected for tissue-specific expression analysis in jujube and wild jujube.The results showed that the expression patterns of most ZjbHLHs were similar in jujube and wild jujube.Some genes were mainly expressed in vegetative organs(ZjbHLH1,-2-3,-4,-11,-63,-65,-81,-83,and-87)or reproductive organs(ZjbHLH60).And it was also found that ZjbHLH62 was stably expressed in various organs of both jujube and wild jujube.These results showed that most of the ZjbHLHs had diverse tissue-specific expression patterns,indicating that they play multiple roles in various organs.5.RT-qPCR was used to further analyze the expression of ZjbHLH genes in flowers and fruits at different development stages and leaves at different diseased degrees of JWB.Some key candidate genes related to flower and fruit development and stress resistance were identified.These results lay a foundation for further function verification.6.A total of 15 ZjTIFYs have been identified,and the length of the open reading frame varies greatly,ranged from 444 bp to 1266 bp,the size of the coding protein ranged from 147 aa～421 aa,and the molecular weight ranged from 37.25 KD～105.28 KD.The isoelectric points were very similar,with an average value of 5.11,all of which were weak acidic proteins.The family is divided into four subclasses.Different class can be distinguished by their different conservative domains,and the TIFY domain is the conservative one.Seven ZjJAZs were identified.7.The expression of 7 ZjJAZs in the leaves at different diseased degrees of JWB was analyzed.The expressions of ZjJAZl,ZjJAZ6 and ZjJAZ7 were enhanced and ZjJAZ2,ZjJAZ3.ZjJAZ4 and ZjJAZ5 were decreased in the diseased leaves,indicating that ZjJAZs were indeed involved in the jujube-phytoplasma interaction.8.The protein interaction of ZjbHLH9,ZjbHLH10 and ZjJAZs were analyzed by yeast two-hybrid(Y2H)screening,and the core signaling factors of jujube jasmonic acid pathway in response to phytoplasma infection were preliminarily identified.In conclusion,based on the genomic search the members of jujube bHLH family and TIFY family were identified.Some key candidate bHLH genes related to jujube flower and fruit development and stress resistance were screened out.In addition,the cruial ZjJAZs in response to phytoplasma infection were analyzed and the interaction of ZjMYC and ZjJAZ proteins in jujube jasmonic acid pathway was implemented,which provide clues and basis for further revealing the molecular mechanism of jujube-phytoplasma interaction.