Screening And Cloning Of The OsXCP2 And OsMLP Gene For Interaction Between Rice And Hirschmanniella Mucronata

Rice is one of the significant and widely cultivated staple grain crops planted in the world,but rice yield is restricted by many kinds of pathogens.Among of them,rice parasitic nematodes,one of the most important rice diseases,causing the world-wild rice yield reduced by 10 %-25 %,economic losses topped to 157 billion dollar in annual.As the most economically pathogen of rice,Meloidogyne graminicola and Hirschmanniella spp.embracing different parasitic life style play a crucial role in demonstrating the interaction between rice and its parasitic nematodes.In the previous experiment,an excellent nematode resistance germplasm R51,was screened from 560 rice germplasms,and the identification results of many years and many methods have proved that the resistance is credible.We contrasted the RNA-seq results of nematode-infected or non-infected two rice germplasms systematically,acquiring two gene OsXCP2 and OsMLP,embracing significantly differential expression,and so far no any researches elaborated their functions in the interactions between rice and its sedentary parasitic nematodes.This paper analyzed the RNA-seq results of resistant and susceptible rice root infected by H.mucronata,and then the coding sequences of OsXCP2 and OsMLP were cloned into the plant overexpression vector pCAMBIA1302 and the plant RNA interference vector pB7GWIWG2(II).For studying the genes' function in the interaction of rice and plant-parasitic nematodes,the recombinant plasmids were transformed into Nicotiana benthamiana and Nipponbare via Agrobacterium GV3101 or EHA105.1.Analyzing the RNA-seq results of nematode-infected rice R51,R155 and non-infected rice R51,R155.When RPKM >1,there were about 24 thousand genes expressing in the samples,about 60% of the rice genome encoding genes.Executing expressional differential analysis,Gene Ontology and KEGG analysis to R51 vs.R155,RN51 vs.R51,RN155 vs.R155,RN 51 vs.RN155.The results indicated that after rice infected by H.mucronata,genes involved in phenylpropanoid biosynthesis,phenylalanine metabolism,alpha-Linolenic acid metabolism,biosynthesis of secondary metabolites and diterpenoid biosynthesis were significantly up-regulated.In rice RN51 and RN155,genes coding beta-1,3-glucosidase,and chitinase(CHI1,CHI2)were up regulated too.Moreover,the changes of their expression quantities in RN155 root tissue were more than that in RN51 root tissue.Rice could resist H.mucronata infections via regulating the enzymes related to synthetase lignin and wax and gene coding xylanase were repressed,but the date presentation that expansin was regulated up,which could change cell-wall tenacity and intensity.Jasmonic acid(JA)and brassinolide(Br)signal transduction pathways were used to control resistantly genetic expression,and to some extent,some genes related to ethylene(ET)signal transduction pathway were suppressed.Rice regulated WRKY18 and WRKY60 up,which could make rice root tissue embrace a low sensitivity to abscisic acid(ABA),but because of interaction between rice and H.mucronata,antithetic WRKY40 was up-regulated,and two ABA hydroxylases were down-regulated.Only PR1,PR12,PR13 and PRB were significantly expression in rice R51 and R155 infected by migratory nematodes.2.The coding sequences of gene OsXCP2 and OsMLP were cloned into TA vector pEASY-T3,acquiring recombinant T3:destination gene.And then by specific primes containing the sites of restriction enzymes Bgl II and SpeI the open reading frame were connected into plant overexpression vector pCAMBIA1302(GFP,his-tag).Blastn was used to found their specific sequence information as the RNAi target sequences,and they were recombinated into plant RNA inference vectors via Gateway technology.Finally,these recombinant plasmids were transformed into agrobacterium GV 3101 and EHA 105.3.Subcellular localization experiments of rice OsXCP2 and OsMLP genes were carried out via GFP tag,and the results showed that they were all located in cell wall or cell gap.The recombinant plasmids were transformed into Nicotiana benthamiana and rice Nipponbare,destination sequences recombinating into plant genomes.But RNAi recombinants only were transformed into rice.Then PCR and PAR/bar test papers were used to verify experiment success.Finally,we acquired one OsXCP2 and three OsMLP over-expressed tobaccos,twenty-four and eighteen transgenic rices,twenty-seven and twenty-six RNA interference rices.qRT-PCR indicated that OsXCP2 and OsMLP embraced 13 copies and 8 copies in transgenic tobaccos,7 copies and 13 copies in transgenic rice.Real-time quantitative PCR showed that they expressed 13.29 and 6.6 times in transgenic tobaccos than in wild type,and all of them were significantly differential expression in transgenic rice.