Knock-down Of OsLOXs By RNA Interference Leads To Improved Seed Viability In Rice During Storage

Constant innovation of science and technology accelerate the development of modern agriculture,the food production is also gradually increased.However seed security is a global issue and food stuff reserve is the key ring of food security.According to statistics from the Chinese Ministry of Agriculture,annual losses during storage of rice for food and seed accounts for about 8%of total production.Long term maintenance of seed viability is a universal problem.Although optimum packaging materials,microwave treatment,and control of temperature,moisture and storage atmosphere help to slow oxidative deterioration and maintain seed quality,these external methods are costly in terms warehousing and energy.Therefore,taking method of genetic engineering to solve the problem from itself is an economic and effective way to improve seed storability.Four LOX genes in rice seed have been isolated and characterized,including LOX-1(DQ389164),L-2(X64396),LOX-3(Os03g0700400),and r9-LOXl(AB099850).In order to elucidate their functions,our laboratory isolated these genes individually and uncovered their role by investigating over-expression and knock-down lines.The LOX-1 product was involved in tolerance of the rice plant to wounding and brown planthopper attack.Although L-2 was previously cloned from 3-day-old seedlings,knock-down of OsLOX2(corresponding to L-2)slowed the process of seed deterioration.The main role of OsLOX3 was lipid oxidation of rice seeds,negatively affecting seed longevity and production of stale odor.Through using RNAi technology to silence the endogenous OsLOXs gene to cultivate new varieties is significant.In our study,we generated 12 interference families which were validated by PCR analysis to get positive transgenic plants.In our study only 4 interference families(NPF1,NPF2,X03,X06)demonstrated an positive effect on improved seed viability especially NPF1 and it was used as the germplasm for the following research.In the southern-blot hybridization,the result showed that line NPF1 possessed 2 interference copies of OsLOX3.The size of the first one was between 2,799 bp and 1,953 bp,and the second one was between 1,953 bp and 1,515 bp.The RT-PCR analysis of OsLOX3 showed that there was weak expression in other parts of both transgenic and wild-type plants.However,expression of OsLOX3 in all tissues of knock-down plants was significantly lower than in the wild-type.The improved TTC reduction assay showed that the content of TTCH in the interference families was higher than that of the wild type.Using linoleic acid as substrate for determination of LOX activity also exhibited the seeds of interference lines were more vigor than that of wild type.Lipid peroxidation of OsLOX3 knock-down seeds was lower than that of wild-type seeds.Analysis of protein content,starch and amylose content,W001 showed a little change than that of NPF1.Investigating the agronomic traits of the 4 interference families,minor differences in agronomic characteristics between W001 and knock-down lines were detected in two years of assessment.With further breeding and selection these lines may have application in improving the seed viability of stored rice grain.