Screening Of Rice Lipoxygenase 3(LOX3) Interaction Protein And Proteomic Analysis Results Verification

Rice is one of the most important food crops in the world,but it is easy to occur metamorphism during the period of preservation,resulted in a large number of food loss each year.So the rice storage ability problems has become one of the main challenges for rice breeders.Lipoxygenase 3(LOX3)is the key enzyme during rice seed fat oxidation process based on previous research.The results indicated that the lack of LOX3 can improve storage resistance of rice seed,but its molecular mechanism is still unclear.In this study,we analyzed the metabolic pathway of LOX3,and clarified that how LOX3 regulated the upstream and downstream genes to affect the storability of rice withthe differential protein expression profile in the previous research.Meanwhile,the proteins interactingy with LOX3 were screened with the yeast two hybrid system,and furtherly verified by GST pull-down technology.At last,the pathway of LOX3 and the molecular mechanism of LOX3 regulation in rice storage ability were identified.The mainly results were as follows:1.The pGBKT7-LOX3 yeast two hybrid bait carrier was contructed.The self-activation experiments indicated that there was no self activation in pGBKT7-LOX3.So we make the hybridization between bait vector and cDNA library of rice.The interaction proteins were screened by four elements deletion of culture medium filtering.The pGADT7 carrier for converting screen was contructed.Finally two proteins interacting with LOX3 were screened and their corresponding accession number of genes were:Os02g0758000,Os09g0438700,respectively.2.The label pMAL-c5X-LOX3 prokaryotic expression vector with pMAL,the GST label pGEX-6p-1-Os09g0438700 and pGEX-6p-1-Os02g0758000 prokaryotic expression vector were contructed and inserted into BL21 strain.After being induced by SDS-PAGE analysis,the pMAL-LOX3,GST-Os09g0438700 and GST-Os02g0758000 fusion proteins were confirmed.3.Six differentially expressed genes were screened with the seeds of anti-LOX3 in rice before and after artificial aging(RH88,42?).The six genes accession number were Os01g0348600,Os11g0605500,Os06g0204400,Os02g0116500,Os02g0116500,Os09g05 70300 and Os02g0676000 respectively.These genes expression level were analyzed by qRT-PCR.The results indicated that the expression level of those genes was significantly different before and after artificial aging(RH88,42?).The results were consistent with the results of proteomics.