Functional Analysis Of ZmHAK1 Promoter And Interaction With Transcription Factors ZmRAP2.11 And ZmARF2 In Maize

The gene of the high-affinity potassium transporter(ZmHAK)family plays a key role in the uptake and transport of potassium nutrition in maize(Zea mays L.).Among them,ZmHAK1 can increase the potassium uptake capacity and plant stress resistance in maize under a low amount of potassium stress.This gene is regulated by promoters and transcription factors.The promoter and regulatory elements on the promoter of the ZmHAK1 gene play a crucial role in the regulation of the ZmHAK1 gene.Therefore,analyzing the structure of the promoter and the function of the transcription factor can provide a theoretical basis for improving the plant resistance through genetic engineering,which has an important significance in the scientific research process.In this study,the promoter of maize ZmHAK1 gene was analyzed and the functions of transcription factors ZmRAP2.11 and ZmARF2 were explored.The main findings are as follows:1.The 2246 bp fragment of ZmHAK1 gene promoter,obtained from the genome DNA of the maize leaves by TA-cloning method,which was named PZmHAK1:pMD-18 T.A number of putative cis-acting elements of ZmHAK1 gene promoter sequence were discovered using PLANTCARE database,containing the core elements TATA-box,CAAT-box,abiotic stresses(drought,stress),hormones(methyl jasmonate,auxin,abscisic acid).2.The 35 S promoter was replaced by the ZmHAK1 promoter in the plant binary expression vector pCAMBIA1301,which was fused with the GUS reporter gene.Then a series of 5' deletion expression vectors were constructed and transformed by Agrobacterium transient transformation in tobaccos.GUS tissue histochemical staining results indicated that the full-length promoter was expressed in roots,stems and leaves of plants;GUS quantitative results showed that ZmHAK1 gene promoter was induced by abiotic stress(low potassium,high temperature,low temperature,salt,drought,darkness)and hormones(IAA,ABA,MeJA,SA).3.The ZmHAK1 gene promoter was transferred into the maize inbred line by pollen-tube pathway,and the positive seedlings were successfully screened by hygromycin screening and PCR identification.4.The transcription factors ZmRAP2.11 and ZmARF2 gene were successfully cloned from the cDNA of the maize leaves.Bioinformatics analysis of these two transcription factors showed that the transcription factor ZmRAP2.11 was a member of the AP2/ERF family,and ZmARF2 belonged to the auxin response factor family.The results of subcellular localization showed that the transcription factor ZmRAP2.11 was mainly located in the nucleus,and the transcription factor ZmARF2 was mainly located in the nucleus and membrane.5.Yeast one-hybrid experiments showed that the ZmHAK1 promoter interacted with the transcription factors ZmRAP2.11 and ZmARF2.The promoter was divided into six segments to analyse the interaction between promoter and transcription factors.The results indicated that the transcription factors ZmRAP2.11 interacted with the third and sixth segments of the ZmHAK1 promoter.And the transcription factor ZmARF2 had interaction with the first and third segments of the ZmHAK1 promoter.