Mechanism Analysis Of Powdery Mildew Resistance Gene Pm35 In Aegilops Tauschii

Wheat powdery mildew is an airborne disease caused by Blumeria graminis f.sp.tritici,which seriously threatens the quality and yield of wheat.The discovery and utilization of powdery mildew resistance genes in wheat and its relatives is of great significance for broadening the genetic basis of wheat powdery mildew resistance and serving actual disease resistance breeding.As a donor of the wheat D genome,Aegilops is rich in disease resistance,stress resistance and other excellent genes,and it is easier to transfer to wheat.The powdery mildew resistance gene Pm35 in Aegilops can significantly improve the powdery mildew resistance of wheat and has good utilization value.The wheat powdery mildew strain E09 was used to infect Aegilops crassicarpa 2147,the extracted c DNA was used to construct a yeast library,and the protein WRKY-IR interacting with Pm35-R was obtained by yeast two-hybrid screening.Further research found that Pm35 specifically interacts with the transcription factor WRKY-IR（D）from the D genome,but not with the two homologous transcription factors on the A and B genomes.This study uses Co-IP,subcellular localization,genetic modification and other methods to study its interaction mechanism.The specific research results are as follows:1.This study uses subcellular localization experiments to locate both Pm35 and WRKY-IR（D）in the nucleus,which is consistent with the predicted results,indicating that Pm35 and WRKY-IR（D）recognize each other in the cell and exercise disease resistance.And find the CC domain of Pm35 to guide Pm35 into the nuclear region.2.Through Co-IP experiment,verify that Pm35 and WRKY-IR（D）actually have protein interaction.3.Using transgenic technology to transform Fielder,using ordinary PCR technology to initially determine the transgenic positive seedlings,using RT-q PCR technology to identify,transgenic T₀ generation plants transformed WRKY-IR（D）segment expression level is the control 33-39 times,the result showed that the WRKY-IR（D）segment used for RNAi was successfully expressed;the transgenic positive vaccine was successfully obtained,and the disease resistance was identified.The result was no significant difference in disease resistance compared with the control Fielder.We It is believed that WRKY-IR（D）alone in common wheat does not have disease resistance;4.Due to the high homology of WRKY from A,B,and D,RT-q PCR technology identification results showed that the expression of WRKY in A and B genomes were all down-regulated to varying degrees,indicating that WRKY-IR（D）-RNAi transgenic plants successfully interfered WRKY-IR（D）,and the interference efficiency has a certain specificity.The above research results lay the foundation for further hybridization with Pm35 containing wheat to study disease resistance.5.Pm35 transgenic seedlings and SN7064 as a background crossed with disease-resistant parents and backcrossed progeny BC₃F₄multiple times while ensuring disease resistance,the yield-related traits such as plant height,ear length,spikelet number,ear kernel number and1000-grain weight were compared with the control JW1 compared with the parent SN7064,the difference is not significant,indicating that Pm35 can be transferred to common wheat for transfer use.