| Rice grassy stunt virus(RGSV),a species of TenuivIrus,is spread by Brown Phanthopper.It has broken out in southeast areas of Asia for many years,since it was confirmed in 1963 in the Philippines.The recent large damages occurred in Vietnam’s Mekong Delta region in the year of 2006-2009,resulted in 700000 tons of rice losses and suspension of exports.To China,it occurs in some southeast provinces.The obvious symptoms such as multi-tillering,dwarf,irregular spots in leaves and low fruiting rate in RGSV-infected rice plants.As a result,RGSV brings serious damage to rice planting in China and southeast areas of Asia.DNA methylation is an important method of epigenetic and also an important way to regulate genes in plants.It is closed related to the life processes of plants,such as plant developing,resistance,foreign genes defense and silencing,genome maintenance and transposons suppression.So,the phenotype of the plants will be different when the methylation is influenced.And the study of function analysis on these related genes of methylation will be significant.The experiment of a rice mutant which showed the reduction of DNA methylation,resulted in dwarf,delayed heading or vanished thoroughly.Most of these symptoms are similar to the 5-azacytidine treated rice,DNA methylation inhibited rice,and RGSV infected rice.The early related research based on the DNA methylation high-throughput genomic sequencing data,methylation level and the transcriptome data of RGSV-infected rice.This preliminary analysis showed the DNA methylation level decreased and the related gene expression level changed significantly in RGSV-infected rice.This study mainly aims at function analysis on some methylation related genes which change gene expression and methylation level significantly in RGSV-infected rice,for the further study on the pathogenic mechanism of RGSV infected plants.Based on the early period of the related research in the laboratory,this study selects a group of methylation decreased and related genes whose gene expressions significant change after infected RGSV.Through further analyzing and screening,we found out three related genes:OsMET04,OsMET07 and OsMET12.These three genes change gene expressions significantly,and they are directly or indirectly related to tillering in NCBI putative symptoms.This study starts with further validation of RGSV-infected rice in gene expression changes of this three genes by qPCR,and then construct the over expression and the RNA interference vectors.In this study,a kind of agrobacteria mediated rice genetic transformation method is optimized.This method,compare with the original method,increases the amount of rice calli,develops an effective antifungal method and invents an degerming liquid which decreases 50%-70%fungi and agrobacteria pollution probability.Using the optimized method,the over expression plants of OsMET04,OsMET07 and OsMET12 are obtained and RNA interference plants of OsMET07,OsMET12 are also obtained.However,RNA interference transgenic plant of OsMET04 has been transformated for three times,but it is still not successful to obtain the transgenic plant.it suggests that the interference of this gene may cause death or differentiation problems of transgenic rice.By PCR verification of transgenic plants,positive plants are screened.Compared with normal rice,OsMET07,OsMET12 transgenic plants didn’t show any obvious difference.But OsMET04 transgenic rice shows similar symptoms to which caused by inhibition of DNA methylation or infection of RGSV,such as multi-tillering,dwarf and low fruiting rate.By qPCR,GADPH an endogenousgene,which constant expressed in rice is selected as reference gene to quantitative analyze OsMET04 transgenic plants.And the expression level is increased by an average of 152 times.This study preliminarily proved that RGSV inhibits rice DNA methylation,result in multi-tillering,and some symptoms which may be relative to multi-tillering,such as dwarf and low fruiting rate,which may be a foundation of further studies. |
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