Functional Analysis Of OoWRKY40 Gene Of Oxytropis Glabra And Its Interaction With Endophytic Fungi With Endophytic Fungi

As a poisonous grass with a relatively short history of research,the severity of the spread of Oxytropis and its unique toxic substances have brought impacts and losses on the development of grassland ecology and animal husbandry in the western region.On the other hand,because of its good forage characteristics and the medicinal value of anti-tumor activity,Oxytropis has great research prospects.In the early stage,the research team found that Oxytropis showed high tolerance under adversity stress environment,and endophytic fungi isolated from the plant can promote the root growth of the model plant Arabidopsis thaliana,but the stress resistance mechanism,How to promote the growth of Oxytropis is not clear.It has been reported that the WRKY transcription factor family can respond to multiple stress pathways such as organisms and abiotics.Therefore,this study used Oxytropis under the two major transcriptome databases of abiotic stress（drought,salt stress）and symbiosis with endophytic fungi.Screening the differential gene-the transcription factor OoWRKY40,and using it as the research object,through molecular techniques,to explore the unknown function of this gene when it responds to abiotic stress and interacts with the endophytic fungus Alternaria sect.Undifilum.To provide a basis for the later comprehensive understanding and full use of the application value of Oxytropis.The main research contents and results are as follows:（1）After screening the two transcriptome databases of Oxytropis stress and symbiosis with endophytic fungi,the differential gene annotated as comp82399＿c1 was obtained.The primers were designed by Primer 5,and the gene was successfully cloned and named OoWRKY40.The full length of OoWRKY40 is 1177 bp,and the ORF length is 960 bp.Bioinformatics analysis showed that OoWRKY40 protein belongs to theⅡa family of the WRKY family of transcription factors—WRKYGQK-C₂H₂;the phylogenetic tree showed that OoWRKY40 and Chickpea Ca WRKY40 have high homology.（2）The plant binary expression vector p CAMBI1305-OoWRKY40-e GFP was constructed,and the T2 generation transgenic OoWRKY40 Arabidopsis seeds were obtained by the flower soaking method.On the other hand,tobacco subcellular localization experiments showed that OoWRKY40 protein was localized in the nucleus;yeast single hybrid experiments were carried out by constructing yeast recombinant vector p GBKT7-OoWRKY40-BD,and the results showed that OoWRKY40 protein does not have transcriptional activation activity.（3）The results of qRT-PCR showed that the OoWRKY40 gene is involved in the response mechanism of Oxytropis glabra to drought（20%PEG 6000）,salt stress（150 mM Na Cl）,ABA（100μM）,and ETH（100μM）hormone signaling pathways.In order to explore the function of Oxytropis OoWRKY40,in-dish experiments on seed germination rate and root length of wild-type and overexpressing OoWRKY40 Arabidopsis（OEs）were carried out.Under drought treatment（0 mM,200 mM Mannitol,250 mM Mannitol）,compared with the wild type,the overexpression line had a higher seed germination rate and longer roots;salt stress（0 mM,100 mM NaCl,There is no obvious phenomenon in 150 mM Na Cl）.Secondly,the seedlings of Arabidopsis thaliana（OEs）overexpressing OoWRKY40 were treated for 7 days under the condition of 250 mM Mannitol,and the stress genes（PUB22,PUB23,CAMBP,IQM1）and ABA-related genes（NCED,AF1）were detected by qRT-PCR method.The expression level,it was found that the expression levels of these 6 genes showed an upward-regulated trend.The addition of exogenous ABA（0 mM,1μM ABA,3 mM ABA）reduced the germination rate of overexpression lines,inhibited root growth,and delayed plant growth and development.Therefore,it is concluded that OoWRKY40 gene regulates stress-related genes through the ABA signaling pathway to improve the drought tolerance of plants.（4）The results of qRT-PCR showed that OoWRKY40 played a negative regulatory role during the symbiosis period of Oxytropis and the endophytic fungus Alternaria sect.Undifilum;OoWRKY40 participated in the symbiosis of Oxytropis and the endophytic fungus Alternaria sect.Undifilum through the SA and MeJA hormone signaling pathways.During 0-20 days,OoWRKY40 mainly participates in the symbiosis phase through the MeJA signaling pathway;after 20 days,OoWRKY40 mainly participates in this phase through the SA signaling pathway.