Function Analysis Of Transcription Factor CpDi19-5 Gene From Wintersweet (Chimonanthus Praecox)

Wintersweet[Chimonanthus praecox(L.)Link]is a unique deciduous shrub of the wax plum family.As an important material of fresh cut flowers and potted landscape,the wintersweet has high ornamental value and economic value.However,with the global warming and frequent extreme weather,the planting of wintersweet is affected by abiotic stress such as high temperature and drought,which becomes more and more prominent.Production practice has shown that the cultivation of crop varieties with strong stress resistance is one of the most economical and effective ways to reduce the effects of stresses such as high temperature and drought,and this goal needs to be realized on the premise of understanding the molecular mechanism of plant resistance.C2H2 type zinc finger transcription factor is one of the largest transcription regulation family in plants.Di19(Drought-Induced)is a kind of small molecular protein of C2H2 type zinc finger transcription factor.Studies show that it extensively participates in resistance to abiotic stress of plants.Related functions have been isolated and identified in a variety of model plants,but there are still few studies on Di19 transcription factors in woody plants such as wintersweet.In previous studies,a CpDi19-5 gene of zinc finger protein transcription factor Di19 family was isolated,which was significantly induced by abiotic stress such as high temperature and drought.It was speculated that CpDi19-5 gene might be an important transcription factor gene involved in abiotic stress response such as high temperature and drought of wintersweet.Therefore,on the basis of previous studies,the induction characteristics of CpDi19-5 gene under abiotic stress were analyzed and verified in this paper,and the plant expression vector was constructed to transform poplar,and abiotic stress tolerance of transgenic poplar plants was analyzed.In addition,the interaction protein of CpDi19-5 was screened by yeast two-hybrid and its regulatory network of protein interaction was verified and analyzed.The main research results are as follows:1.Cloning and activity verification of promoter of CpDi19-5 geneThe study of abiotic stress expression pattern showed that PEG,42℃ and 4℃could induce the up-regulated expression of this gene.The CpDi19-5 gene promoter fragment 2004 bp was cloned from the genomic DNA of wintersweet by PCR method,and was named CpDi19-5pro.Cis-acting element analysis found that it contains basic elements of CAAT-box and TATA-box promoters and multiple light responsive elements such as GA-motif and G-box,etc.In addition,it also contains some functional elements responding to abiotic stress and hormone induction,such as:ABRE,ERE,LTR,CGTCA-motif,and MYB,MYC binding sites.CpDi19-5pro was constructed with the fusion expression vector p CAMBIA1305.1-CpDi19-5Pro containing GUS gene,and transformed into tobacco for transient activity verification.The results showed that CpDi19-5pro had promoter activity.2.Study on the stress resistance function of CpDi19-5 gene in Populus tomentosaThe plant expression vector p CAMBIA1300-CpDi19-5 was constructed by seamless cloning method and transformed into Populus tomentosa by leaf disk method mediated by Agrobacterium tumefaciens.After tissue culture and antibiotic screening,the transgenic populus tomentosa plants were identified by PCR and q RT-PCR.Transgenic poplar seedlings were treated with 42 ℃ high temperature stress and PEG simulated drought stress,respectively.Phenotypic observation and physiological index analysis showed that overexpression of CpDi19-5 gene in populus tomentosa significantly improved the tolerance of transgenic plants to high temperature and drought stress.3.Screening CpDi19-5 interacting proteins by yeast two-hybridizationThe transformed yeast cell vector p GBKT7-CpDi19-5 was constructed,and it was found that CpDi19-5 has self-activated in yeast cells.The optimal concentration gradient was selected to inhibit the self-activation of CpDi19-5 by adding Auridibasidiin A(Ab A)to auxotroph medium SD/-Trp/-Leu.Finally,it was determined that 400 ng/m L Ab A could significantly inhibit the self-activation phenomenon.The library was screened by co-transfer method,and a total of 21 candidate interacting proteins with clear bands were initially screened.BLAST results showed that AQPs(aquaporin)in wintersweet,Nuclear Factor Y(NF-Y)in soybean,ubiquitin-binding enzyme E2,zinc finger protein and other abiotic stress related genes had been studied in other plants.Among them,nuclear factor NF-Y was selected for rotation verification,and the results showed that there was protein interaction with CpDi19-5.