Study On Function And Mechanism Of24nt-siRNA During Superior And Inferior Rice Grain-filling

As the most abundant small interfering RNAs in rice grains，24nt-siRNAs showsa regularity distribution in superior and inferior spikelet during the period of ricegrain-filling: The amounts of24nt-siRNA declined with the process of grain filling in bothsuperior and inferior grains, but24nt-siRNA in inferior grains was much higher than that ofsuperior grains in each period. So it is important to study the influence of24nt-siRNAexpression alteration in spikelet on the rice grain filling.By bisulfate sequencing, we detected the cytosine methylation status of one DNA regionon the12th genome in rice Xinfeng2, and we also detected the cleavage of24nt-siRNA ontarget gene by5’ RACE amplication. With method of simple RNAi construction, weconstructed three kinds of rice endosperm-specificity interfering vectors: DCL3ai, polIViand RDR2i, got three kinds of RNAi transgenic rice T0generation and detected theexpression quantity of target gene and the spikelets’ phenotype of positive transgenic rice.The mainly results are as follows:1. The result of bisulfate sequencing showed that the DNA methylation levels in superiorwas higher than that in inferior. The rate of two categories CHG, CHH accounted for72.5%in target gene cytosines, and the level of CHH methylation were significant difference(P<0.05) in superior and inferior. The levels of CG methylation arrived at98%, but therewas no difference between superior and inferior. The RT-PCR validation showed that24nt-siRNA expression in inferior grains was higher than that of superior. It was concludedthat the different methylation levels between superior and inferior grains was due to CHGand CHH methylation, and the abundance of24nt-siRNA might be the result of low levelsof cytosine methylation in inferior.2. Choosing nine target genes for cleavage verification of5’ RACE from the predicationconsequence of siRNA target of superior and inferior in Xinfeng2, we got TA clones ofthree target genes after gene amplification and TA cloning, and only the result of geneLOC_Os06g19990is right by analyzing the sequencing result. We together collected13siRNAs near the LOC_Os06g19990adaptor of5’ RACE according to the sequence of LOC_Os06g19990and the siRNA sequencing. After analizing the13siRNAs, onlysiR478391in24nt length match with target sequence completely, and its TPM is relativelyhigh, the9th and10th base from5’ end of siR478391corresponds to the cleavage site,therefore siR478391has the most possibility to cleaved the target gene LOC_Os06g19990.3. We constructed three kinds of rice RNAi expression vectors: DCL3ai, polIVi andRDR2i by the ways of simple RNAi construction, which are activated by riceendosperm-specificity promoter Gt13a, and we got three kinds of transgenic rice T0generation by agrobacterium-mediated transformation. After treating the leaves of RNAitransgenic rice with GUS staining, we found there were ten transgenic rices being blue at thecut edge of leaves. The result of qRT-PCR showed that the relative expression quantity ofthe target gene decreased to18%~55%on the level of mRNA among10RNAi transgenicseeds with significant difference (P <0.05), suggesting that the RNAi vector had alreadyexpressed in rice plants and the24nt-siRNA accumulation reduced to some degree. Aftermeasuring seed’ phenotype, we found the single kernel weight of ten transgenic ricesdecreased significantly (P <0.05), the size of transgenic rice kernel was smaller than that ofwild type rice, both length and width changed significantly (P <0.05), which indicated thatthe chang of24nt-siRNA accumulation had a certain influence on kernel’s size and singlekernel’ weight.