| Wheat is one of the most important food crops in the world.Wheat powdery mildew is caused by Blumeriagraminis f.sp.tritici(Bgt),which is one of the main diseases in major wheat producing areas in the world,seriously affect wheat yield and quality.At present,changes in farming systems and cultivation techniques are difficult to solve the infestation and spread of powdery mildew.Although relying on chemical control has achieved some results in controlling the occurrence and epidemic of powdery mildew,it inevitably causes increased costs and environmental pollution,which is contrary to the concept of green development.Therefore,the first method to control the epidemic of powdery mildew and reduce economic losses is to explore the use of wheat powdery mildew resistance genes and molecular breeding technology to select disease resistant varieties.Aegilops tauschii is a donor of wheat D genome,which is rich in stress and disease resistance genes.Among them,Pm35 has been proved to be an excellent gene with a broad resistance spectrum and no negative effects of agronomic traits.In the early stage,we have mapped Pm35 finely,assembled a physical map containing Gap in the target gene interval,and predicted 3 LRR candidate genes.In order to further clarify the physical map information of the candidate gene's interval,this study intends to use CRISPR-Cas9 enzyme site-directed cleavage to clone a large segment of target DNA directly from crop genomic DNA for the first time using ExoCET technology,thereby confirming the candidate gene's sequence.In addition,Pm35 interaction protein was obtained through yeast library screening,and the molecular mechanism of Pm35 background resistance change from diploid Aegilops to hexaploid wheat was revealed.The specific research results are as follows:(1)In the early stage,on the basis of finely mapping the powdery mildew resistance gene Pm35,we assembled a physical map containing Gap in the target gene interval,and based on the physical map information,used CRISPR-Cas9 nuclease to cut the crude goat grass genome in vitro.Using ExoCET direct cloning technology to successfully capture a 39 kb target fragment from Aegilops genomic DNA,the plasmid was then sequenced using Illumina Hiseq 4000,and sequence assembly was performed through SPAdes to clarify the physical map of the candidate gene region.(2)Through analysis,it was found that Pm35 showed full growth resistance in the background of diploid Aegilops tauschii,but both hexaploid wheat and transgenic wheat synthesized with it as the donor showed resistance at the plant adult stage.In order to reveal the cause of this phenomenon,the full length of Pm35 was used as the bait protein to screen the yeast library to obtain a transcription factor containing the WRKY domain,which showed an adult-stage expression pattern and was induced by pathogens.Therefore,this WRKY gene was identified as the focus of further research.(3)In this study,we used BSMV-VIGS technology and used Aegilops tauschii 2147 as the recipient to obtain WRKY silencing plants.Combining the resistance of previously over expressed and silently expressed plants with Pm35 to further verify the disease resistance of Pm35 and WRKY.Later,the WRKY transcription factor was subcellularly located and found to be localized in the nucleus.(4)Further use yeast two-hybrid,BiFC,Luciferase to fully verify the interaction between Pm35 and WRKY transcription factors.The yeast two-hybrid was used to verify that Pm35 only interacted with WRKY transcription factors on the D genome of wheat,but did not interact with WRKY on the A and B genomes.The reasons for the weakened resistance of Pm35 into hexaploid wheat were tentatively explained.(5)To further study the regulatory mechanism of Pm35,we constructed expression vectors containing the Pm35 CC domain and LRR domain,respectively,and verified the interaction with WRKY by yeast two-hybrid.Finally,it was proved that the CC domain of Pm35 interacted with the WRKY transcription factor. |
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