| Lilium regale Wilson is a unique wild lily species in C hina,which has the characteristics of drought resistance,salt resistance and strong disease resistance.In addition to its ornamental value,it is an important resource for disease-resistant breeding of modern lily.Due to factors such as geological disasters,man-made excavations,and land reclamation,the distribution area and quantity of L.regale Wilson’s wild resources have decreased significantly,and it has become an endangered species.Therefore,there is an urgent need to strengthen the research on the molecular mechanism of pathogen resistance of L.regale Wilson,and to provide a theoretical basis for disease resistance breeding of lily.In the previous analysis of the transcriptome of L.regale Wilson,c DN As of several WRK Y transcription factors that have responded to exogenous Me JA and Fusarium oxysporum infections have been obtained.In this project,one of the WRK Y transcription factor genes(Lr WRK Y3)was selected for functional analysis.An LrWRKY3 transcription factor with an Open Reading Frame(ORF)of 537 bp was cloned using the c DNA of L.regale Wilson as a template,and the protein encoded by it contained a highly conserved ’WRKYGQK’ heptapeptide sequence.The Lr WRK Y3 subcellular localization fusion protein expression vector was constructed and transiently expressed in onion epidermal cells to analyze the localization of LrWRKY3 in the cells.A prokaryotic expression vector was constructed for heterologous expression in E.coli,and the recombinant LrWRKY3 protein was obtained for EMSA experiments to verify the specific binding site of LrWRKY3.The yeast one-hybrid experiment was used to verify whether the transcription factor has transactivation.The LrWRKY3 plant overexpression vector was constructed to obtain transgenic tobacco(Nicotiana tabacum)lines and study their functions.Finally,the yeast two-hybrid system was used to screen the proteins interacting with LrWRKY3 transcription factor from the c DNA library of L.regale Wilson.The research work and main results obtained in this thesis are as follows:1.Construct p BIN m-gfp5-ER-Lr WRK Y3 subcellular localization vector and transform it into Agrobacterium tumefaciens EHA105.Onion epidermal cells were infected with a positive monoclonal bacterial solution,and then cultured for two days.LrWRKY3-GFP fusion protein was observed in the nucleus by laser confocal microscopy.2 、 A prokaryotic expression vector containing p ET-32(a)-Lr WRK Y3 was constructed and transformed into E.coli BL21(DE3)for heterologous expression.Since the recombinant protein is an inclusion body,the active soluble protein HisLrWRKY3 was obtained through urea denaturation,purification of the protein through a N i-NTA column,renaturation of a dialysis bag,and concentration.The recombinant protein was used in the EMSA experiment.The results showed that the recombinant protein could bind to the probe containing the W-box fragment,and the LrWRKY3 transcription factor had a specific binding site W-box.The Lr PR10 promoter obtained earlier had a W-box to construct a bait vector p Ab Ai-PLr PR10 and a prey vector p GADT7 AD-LrWRKY3 for yeast one-hybrid experiments.The results showed that LrWRKY3 could interact with PLr PR10 and had transactivation.3、A plant overexpression vector p C AMBIA2300S-LrWRKY3 was constructed and introduced into tobacco,and T2 generation transgenic tobacco was obtained after cultivation.q RT-PCR showed that LrWRKY3 was stably expressed in transgenic tobacco lines,and leaf and plant inoculation experiments showed that transgenic tobacco was highly resistant to F.oxysporum.And q RT-PCR was used to detect the high expression of genes involved in jasmonic acid(JA)synthesis and disease resistance in transgenic tobacco at the transcription level.4、Using L.regale Wilson root as material,a c DNA library that meets the requirements of the screening library was constructed,and a p GBKT7-LrWRKY3 bait vector was constructed.The library plasmid p GADT7-c DNA was transferred into AH109 yeast strain containing the bait plasmid,and spread on SD-Trp/-Leu/-His/-Ade plates to screen for interacting proteins.A total of 45 proteins that may interact with the LrWRKY3 transcription factor were preliminarily screened.Including proteins related to plant DNA replication,transcription,protein translation,plant growth and development,photosynthesis,plant stress resistance and plant energy metabolism.Provide basic information for the further study of the function of LrWRKY3.The nuclear-localized Lr WRK Y3 protein specifically binds to the cis-acting element W-box,and the LrWRKY3 transcription factor has transcriptional activity.And through the JA-dependent pathway,it participates in the defense of L.regale Wilson and activates the transcription of a series of disease-resistance-related genes,thereby improving the resistance to F.oxysporum.This research will lay the foundation for further revealing the disease resistance mechanism of Lilium chinense in the future. |
