Functional Analysis Of A Defense-related Gene Pstpk1 Associated With Pid3 In Rice And Application Of Bsr-d1 Resistance Site In Breeding

?.Functional Analysis of a Defense-related Gene Pstpk1 Associated with Pid3 in RiceRice is one of the most important crops in China.However,rice blast restricts the rice yield severly.Currently,developing resistance genes agaist rice blast and applicating it in rice breeding are important for breeding resistant varieties.,We previously cloned a resistance gene,Pid3,in rice Digu that enhance the resistance on rice blast.The Pid3 encodes a typical CC-NBS-LRR protein.It induced specifically resistance to the race Zhong10-8-14.However,the molecular mechanism of Pid3-mediated ETI pathway has not yet been elucidated.To explore the molecular mechanism of the Pid3-mediated ETI pathway,we extracted the total protein after treating the rice Pid3-TP309 with incompatible strains and compatible strains and and screened candidate proteins that specifically interact with PID3 in resistance reaction mediated by Pid3 using GST pull-down technology.On the basis of the above results,one of the candidate proteins was using forstudying its function.The protein is a serine/threonine protein kinase(Serine/threonine protein kinase)which has not been reported in the resistant researches on rice blast.We named it Pstpk1(Pid3 interaction serine/threonine protein kinase 1).In order to explore the function of Pstpk1 in Pid3-mediated ETI,we overexpressed,knocked out or interfered the Pstpk1 in the Pid3-TP309 and TP309 backgrounds,respectively.And then,identified their resistance on rice blast.;studied subcellular localization of the PSTPK1;tried to verify the interaction between PSTPK1 and PID3 using yeast two-hybrid(Y2H)and bimolecular fluorescence complementary(Bi FC).The main results as follows:1.In order to investigate whether Pstpk1 participates in Pid3-mediated ETI,knockout and overexpression lines of the Pstpk1 gene in Pid3-TP309 background were obtained,respectively.The blast resistance of the Pid3-TP309 with knockout Pstpk1 is similar to that of Pid3-TP309,whereas the lines overexpressing Pstpk1 are more suspectible to rice blast than Pid3-TP309,suggesting that g Pstpk1 might play a negative role in Pid3-mediated resistance on rice blast.2.In order to explore whether Pstpk1 regulates the PTI(Pattern-Triggered Immunity),we got the transgentic lines with kockouting or overexpressing the Pstpk1 gene in the TP309 background.After inoculating Zhong10-8-14,we find that kockouting or overexpressing the Pstpk1 gene does not affect the resistance to rice blast,suggesting that Pstpk1 is not involved in the pathway of the basic immunization.3.The result of subcellular localization showed thatthe PSTPK1-GFP fusion protein is mainly in the nucleus,though localized in both the nucleus and the cytoplasm.4.We verify the interaction between PID3 and PSTPK1 using Bi FC and Y2 H.The results showed that there was no interaction between PSTPK1 and PID3 in tobacco and yeast systems,respecitively.However,PSTPK1 can be interacted with PID3 using GST pull-down,indicated that the interaction between PSTPK1 and PID3 depends on the induction of Magnaporthe oryzae fungi.?.Application of bsr-d1 resistance site in breedingIn this study,a broad-spectrum resistance site,bsr-d1,was used to improve the resistance of Shuhui 527.We identified resistance to rice blast in the improved lines and examined the recovery rate and agronomic traits.We found that the improved lines had increased resistance to rice blast,and there was no significant difference in main agronomic traits with Shuhui 527.The above results showed that the broad-spectrum resistance site bsr-d1 significantly increased the resistance of Shuhui 527,and did not affect the main agronomic traits,it has important breeding and utilization value.The main findings are as follows:1.Using the Bsr-d1 gene mutation site,the corresponding molecular marker Bsr-d1-d CAPS was designed.After the detection,it was found that the marker has a good polymorphism between the donor parent and the recipient parent,and could be used for molecular assisted screening.2.Through the continuous return of parental Shuhui 527 and improved lines,combined with molecular marker-assisted selection,we obtained three disease-resistant improved lines.The results of molecular marker detection showed that the bsr-d1 in the improved line of Shuhui 527 was homozygous.3.To identify the resistance of the improved strains,the pathogenic strains of rice blast were used to infect the improved strains of Shuhui 527,Shuhui 527,Lijiang,and Digu.The results showed that the resistance levels of the three bsr-d1 improved strains of Shuhui 527 were significantly increased.4.Using the genetic polymorphism marker between Digu and Shuhui 527,the background recovery rates of the three improved lines were analyzed,and the background recovery rate of the improved lines was more than 94%.5.The SPSS statistica20 was used to investigate the main agronomic traits of improved lines,including plant height,effective panicles,panicle length,seed setting rate,and 1000-grain weight.The test results showed that there was no significant change in effective panicles and 1000-grain weight,and there were significant differences in other traits.