| Rice blast seriously devastates rice growth and affects rice yield and quality worldwide.Dissecting mechanisms of rice disease resistance and exploiting novel rice resistance genes without penalties of growth is a significant approach for crop breeding improvement.Pi63,a broad-spectrum rice blast resistance gene,exhibits durable field resistance and does not affect rice yield and quality.Previous studies have shown that the expression of Pi63 gene is induced by rice blast infection,showing obvious temporal and spatial expression specificity,and Pi63-mediated resistance is positively related to the expression of this gene.As a key element of gene expression regulation,the promoter is particularly critical in the process of plant resistance response.The results of bioinformatics analysis showed that the Pi63 promoter sequence was abundant in plant disease resistance response regulatory elements.According to the distribution of regulatory elements,4 deletion fragments of Pi63 promoter,named P0-P3,were constructed successfully.This study intends to construct a Pi63 yeast one-hybrid library,screen the trans-acting factors that interact with the rice blast resistance gene Pi63 promoter sequence using yeast one-hybrid technology,and validate the screened proteins to lay the foundation for elucidating the regulation mechanism of Pi63 promoter and studying the balance mechanism between crop immunity and yield.The main results of this research are as follows:(1)Rice leaves were inoculated in vivo with Magnaporthe grisea,and mixed samples was used to construct a yeast one-hybrid library.The library titer was 2.15×10~9cfu/mL,and the average inserted fragment of the library was 500～2000 bp.After repeated subculture,recombinant plasmids still existed stably in the bacteria,indicating that the yeast one-hybrid library was successfully constructed and it could be used for later library screening.The 4deletion fragment sequences of Pi63 promoter were constructed into pAbAi bait vector,the recombinant plasmids were transformed into yeast competent cells,and 4 yeast one-hybrid bait strains were obtained after positive identification.(2)The above 4 bait strains were used to screen the yeast one-hybrid library,and a total of25 proteins were screened with possible interactions with the regulatory elements of Pi63promoter sequence.In the point-to-point response verification experiment,the proteins No.1,4,5 and 12 showed strong interaction with the corresponding promoter fragments.The No.1protein which is WIH1-like protein and the No.5 protein which is metallothionein-like protein are both cysteine-rich protein family and play the role of the plant immune response.No.4protein is an undefined protein.No.12 protein is an aldehyde oxygen dehydrogenase family protein and participates in a variety of plant signal pathways.In addition,it also showed that these proteins interact with the regulatory elements on the Pi63 promoter were mainly located in the sequence of P0 to P1,which was consistent with the expected localization of regulatory elements.(3)The No.1,4,and 5 proteins from the yeast one-hybrid point-to-point verification were successfully constructed into the protein expression vector pGEX-6p-1,and the soluble fusion protein was successfully obtained by induction.The EMSA experiment was carried out with three probe sequences covering the Pi63 promoter,and the interaction between protein 1 and protein 4 and regulatory elements was successfully verified.The No.1 protein screened can bind to the GCCCORE element,CATATGGMSAUR element,and BIHD1OS element in the upstream region of the Pi63 promoter,and participate in the JA-mediated defense response process.Although the No.4 protein molecule has not been studied yet,the experimental results show that it can combine with GCCCORE element,ASF1MOTIFCAMV element,CGCGBOXAT element and BIHD1OS element. |
