The Influence To The Photosynthetic Genes' Expression In Tobacco Leaves Infected By Tobacco Mosaic Virus Under Different Light Intensity

Tobacco mosaic virus is one kind of viruses with cosmopolitan distribution, which can infect about 300 types of plants and cause serious damage. Virus infection usually leads to the change of important physiological functions of plants, especially the key faction to influence the photosynthesis. Currently, there are more and wider research targets on the TMV's influence on photosynthesis in plants but fewer on the virus' influence on related gene from molecular level. This paper takes natural & healthy and infected tobacco leaves as materials, successfully builds the method to exact the total RNA of tobacco leaves and analyzes the influence of TMV infection toward some photosynthesis related genes' expression, which can be concluded briefly as follows:First, an improved protocol was developed to isolate total RNA in good yield and integrity from TMV infected Tobacco leaves containing high levels of secondary metabolites. Our experiments mainly improved the traditional RNA isolation method in our Lab. Polyvinylpolypyrrolidone at 2% and 2% P-mercaptoethanol were added to the standard extraction buffer, and we make the reaction time longer in chloroform and phenol extraction. Through the comparison in the RNA quality, extraction efficiency, extraction time and the reagent fees etc, it demonstrates that the improved extraction method is the best extraction way for isolating total RNA from TMV infected tobacco leaves and the extracted RNA is complete with high purity (the ratio of OD₂₆₀ and OD₂₈₀ is between 1.8 and 2.0 and the radio of OD₂₆₀ and OD₂₃₀ is more than 2.0), especially the yield is as high as 80μg·g^-1·FW^-1. According to the scan of UV absorption spectrum (200～300 nm), it appeared one single hump at 260nm which indicated the high purity. Meanwhile, the cDNA of the cellular nuclear gene Lhcb1 and chloroplast gene rbcL had been amplified through RT-PCR, suggesting the integrity of isolated RNA. The results of the research demonstrated that the total RNA isolated by this protocol is of sufficient quality for gene expression and some other molecular biology research such as gene clone etc.Second, inorder to study the influnce to the photosynthetic genes' expression in Tobacco leaves infected by tobacco mosaic virus under different light intensity, we detected the expression of some key Photosynthetic gene in transcriptional level. We get kown the specificity and extend of photosynthetic genes' expression through isolating total RNA from tobacco leaves, reverse transcription, designing primers to amply genes, and the semiquantitative analysis of gene expression under different light intensity. PSII is believed to play an important role in the responses of leaf photosynthesis to environmental stresses. We studied Lhcb1 and psbA genes, coding for the Lhcb1 and D1 protein components of photosystem II. And rbcL & rbcS genes coding for Rubisco protein.