Isolation And Functional Identification Of The Low Phosphorus Stress-related Transcription Factor Gene FtWRKY29 From Tartary Buckwheat

Tartary buckwheat(Fagopyrum tataricum)is an annual dicotyledonous herb of the family Polygonaceae buckwheat,is a traditional small grain in China.Tartary buckwheat with drought,salt,cold,barren and UV-resistant and other excellent cultivation characteristics and production traits,in the alpine mountains of the Asian and European continents are more planted.In recent years,tartary buckwheat abiotic stress-related research has mainly focused on the function and role of plant flavonoids and transcription factors in plant resistance,with little focus on plant nutrient stress has been reported.The WRKY gene family is one of the largest families of transcription factors in higher plants and extensively regulates the plant response to a variety of key stressors,among which the WRKY transcription factors involved in regulating phosphorus stress have recently received attention.This study to Sichuan Liang Shan State cultivated buckwheat species "Xiqiao 2" as experimental material,based on tartary buckwheat genomic and transcriptomic data,screened and cloned a gene encoding a class II WRKY transcription factor FtWRKY29.Further characterization of the transcriptional activation activity of its encoded protein FtWRKY29 using transcriptional activation assays and verification of the binding capacity of FtWRKY29 and W-box cis-acting elements using yeast single hybridization and tobacco transient cotransfection.Transgenic Arabidopsis was used to analyze its effects on plants under low phosphorus stress,and the activation of the phosphotransfer protein PHT1;1 gene promoter by FtWRKY29 was analyzed by yeast single-hybrid and tobacco transient cotransfection assays.Identification of MPKs members interacting with them by yeast two-hybrid.The present study on the fine molecular identification of FtWRKY29,a preliminary elucidation of the buckwheat transcription factor FtWRKY29 in Arabidopsis in response to low phosphorus stress molecular mechanism to deepen the understanding of tartary buckwheat resistance to regulatory mechanisms,for the future use of complementary molecular breeding techniques to improve tartary buckwheat resistance,increase yield provides theoretical support.The main findings are as follows:1.Bioinformatics analysis of the molecular characteristics of the tartary buckwheat FtWRKY29 transcription factor showed that FtWRKY29 contains two exons and an intron,and the deduced FtWRKY29 protein has a WRKY structural domain and a C2H2 zinc finger structure,belonging to the class II WRKY transcription factor.Expression profiling showed that the expression of FtWRKY29 in tartary buckwheat was tissue-specific and highest in tartary buckwheat roots;its expression was strongly upregulated under low phosphorus stress(6 h,P<0.01;12 h,P<0.05).2.Construction of the p Bridge-FtWRKY29 recombinant plasmid and identification of its transcriptional activation by yeast single hybridization showed that FtWRKY29 could activate reporter gene expression and show β-galactosidase activity in yeast strain AH109,indicating that FtWRKY29 has transcriptional activation activity alone.Construction of the p CHF3-FtWRKY29-YFP recombinant plasmid and stable transformation of Arabidopsis plants showed that the expression of the FtWRKY29-YFP fusion protein overlapped with the DAPI staining results and that the gene was localized to the cytosol.3.Analysis of the binding capacity of FtWRKY29 and W-box cis-acting elements using yeast single hybridization showed that FtWRKY29 specifically recognizes and binds synthetic W-box elements and activates expression of the reporter gene HIS3 in yeast;no interaction with the mutant m W-box.Further transient cotransfection experiments in tobacco using transgenic techniques showed that FtWRKY29 enhances GUS gene expression in plant cells through intercropping with the W-box and basal promoter 35 S mini.4.The p CHF3-FtWRKY29-YFP plant expression vector was constructed and transfected with the Agrobacterium GV3101 by floral dip method,respectively,and the positive strain T3 was screened.Phenotypic analysis of Arabidopsis plants showed that overexpression of FtWRKY29 could affect the morphological establishment of Arabidopsis seedlings and increase the extension of Arabidopsis root system compared with the control;under low phosphorus culture conditions,the phosphorus content of transgenic Arabidopsis was significantly higher than that of wild-type plants(P<0.05).Anthocyanin accumulation was significantly lower than that of the control(P<0.05),indicating that overexpression of FtWRKY29 could increase Pi uptake in Arabidopsis seedlings and relieve the induction of anthocyanin synthesis by low Pi stress.5.The Arabidopsis PHT1;1 gene was selected and its 2000 bp promoter sequence was cloned,which showed that the upstream promoter region of PHT1;1 contained three W-box components.The interaction of FtWRKY29 with Pro PHT1;1 was identified using yeast single-hybrid and transient cotransfection assays of tobacco leaves,which showed that FtWRKY29 could interact with Pro PHT1;1 in yeast cells and plants,respectively,initiating the expression of reporter genes HIS3 and GUS and showing activity.6.This study performed functional complementation experiments in the Arabidopsis wrky75 mutant.The results showed that wrky75::FtWRKY29 transgenic Arabidopsis strains had longer root lengths in low-Pi-stressed MS,and that overexpression of FtWRKY29 effectively restored the Pi uptake impairment caused by wrky75 deletion.7.Phosphorylation and dephosphorylation are important modalities for regulatin g the biological function of WRKY,and in this study,we screened and cloned pote ntially interoperable Arabidopsis AtMPK3 and AtMPK6 genes for FtWRKY29 and co nstructed yeast two-hybrid vectors for p GADT7-FtWRKY29 and p GBKT7-AtMPK3/AtMPK6/FtMPK3/FtMPK18/FtMPK38/FtMPK45.Positive yeast transformants were iden tified by SD-Trp/Leu and SD-Trp/Leu/His/Ade defective plate screens,which showe d that FtWRKY29 interacts with Arabidopsis AtMPK3 and AtMPK6,FtMPK3,FtM PK18,FtMPK38,and FtMPK45 to activate expression of reporter genes ADE2 and HIS3.In summary,FtWRKY29 is a class II WRKY transcription factor with transcriptional activation function,the expression of this gene in buckwheat has tissue specificity and is induced by low phosphorus stress;FtWRKY29 may sense low phosphorus signals through the mitogen-activated protein kinase cascade signaling system,the mitogen-activated protein kinase cascade signaling system regulates FtWRKY29 transcription.Overexpression of FtWRKY29 in Arabidopsis can change the root length of Arabidopsis,improve phosphorus uptake in Arabidopsis through interactions with the phosphotransfer protein gene PHT1;1 promoter W-box;enhance tolerance to low phosphorus stress.