Identification And Functional Characterization Of Several Disease Resistance-Related Rice Insertion Mutants Induced By Retrotansposon Tos17

Rice is one of the most important cereal crops to feed more than half of the population in the world,so research about its disease resistance has became a focus.With the completion of genome sequencing of rice as a model for monocot plants and cereal crops,the next important challenge is to study the biological functions of genes.Retrotransposon Tos 7-induced mutants of rice are very useful tools in studying the mutation and functional analysis of genes.In order to research the molecular mechanisms of disease resistance in rice,we introduced a number of rice Tosl7 insertion lines from NIAS(Japan),in which the Tosl7 disrupted genes possibly underlying disease resistance.After the seed propagation,the homozygous Tosl7 insertion mutant lines were identified with PCR and finally the CLAT 3-15,NBS 6-2,RLK 7-10,bZIP1-13,bZIP1-16 and bZIP19-1 were screened for further study.Of these genes,NBS encodes protein that containing nucleotide binding domain,RLK encodes receptor kinase-like protein,and CLAT is involved in vesicle formation and transportation in plant cells,while bZIP is a disease related transcription factor,which are suggested to related to disease resistance in rice.The coding region of CLAT,NBS,RLK and bZIP genes were amplified by PCR and cloned into the Gateway enter vector pENTR-D-TOPO,the transient GFP fusion expression vector p-NBS-GFP,p-CLAT-GFP,p-RLK-GFP and p-bZIP-GFP were constructed subsequently through LR reaction.The subcellular localization experiments indicated that CLAT and NBS mainly located in the membrane,RLK and bZIP located both in the membrane and the nucleus.To investigate the disease resistance of mutant and functional characterization of genes,the selected homozygous mutants CLAT 3-15,NBS 6-2,RLK 7-10,bZIP1-13,bZIP1-16 and bZIP19-1 were treated by BTH in different time with wild type Nipponbare as control.The dynamic expression of plant defense genes PBZ1 and Act1 was analysised.The results show that expression of PBZ1 can be induced by BTH,and its expression increased with treatment time gradually in RLK 7-10,bZIP1-13 and bZIP1-16.While in the mutant CLAT 3-15,NBS 6-2 and bZIP19-1,the expression of PBZ1 was not induced by BTH.The expression of Act1 results show that Act1 is constitutively expressed,not induced by BTH.The mutant lines resistance to infection against Xanthomonas oryzae pv.oryzae(Xoo)was evaluated and data was analysised with SPSS,the result showed that there were significant differences between homozygous mutant CLAT 3-15,NBS 6-2,RLK 7-10,bZIP1-13,bZIP1-16,bZIP19-1 and wild-type.In addition,we detected the enzymatic activity of PAL and LOX which are involved in the defense response signaling pathways after the treatments of BTH in different times.We found that PAL enzyme involved in disease resistance response in Tosl7 insertion mutants.Especially in RLK 7-10,bZIP19-1,PAL activity reached a continuous high level when induced by BTH.The enzymatic activity of the LOX illustrated that BTH may induce an enhancement of LOX activity in the mutants bZIP1-13,bZIP1-16 and bZIP19-1,while in the CLAT 3-15,NBS 6-2 and RLK 7-10,LOX activity was possibly postponed upon the induction of BTH,and then the rice disease resistance might delayed.