The Detection Of Lily Symptomless Virus In The Chloroplast Of Lily

It is generally believed that the plant virus that infect the host plants and mainly destroy the cell chloroplast structure affects the accumulation of chlorophyll and photosynthesis. As early experiments suggested in our lab, it was not obvious that the external symptoms of lily plant by Lily Symptomless Virus (LSV) infection alone, at the same time, the degree of influence compared with lily mottle virus (LMoV) infection lily plant was lighter in terms of the physiological index, photosynthesis and the ultrastructure of chloroplast, this study is proving whether the LSV exist in the chloroplast or not, it was necessary to find out the acting site of LSV and provide the basis theoretical foundation for further explore the pathogenic mechanism of LSV, by those ways of extraction the chloroplasts of LSV infected plants, purification of total protein in the chloroplast and molecular detectior of coat protein (CP) accumulated in the chloroplast.In this study, we took a species of Lilium oriental,’Sorbonne’ as experimental material, leaves respectively infected on LSV and healthy control were used. PEG precipitation method was first introduced in the separation and purification of LSV particle, which followed by the method of sucrose density gradient centrifugation for further purification. By the observation of electron microscope and the analysis of Microplate reader, we found the concentration of LSV particle was very low and the amount of impurity was very high by PEG precipitation extraction. After further purification by sucrose, density gradient centrifugation, the concentration of LSV particle was very high and the amount of impurity was very low. The rate of purified LSV ultraviolet absorption value at260nm and280nm was1.331. Therefore, the results met the pure standards. The concentration of purified LSV was0.328mg/ml. Virus production was14.53(μg each gram in fresh lily leaves.The extraction of complete chloroplast respectively was using buffer extraction method and enzymatic hydrolysis method, and optimized. The optimization results of buffer extraction method was that with5%sorbitol at the centrifugal speed of2500r/min the highest concentration of complete chloroplast can be got. With some calculation, complete chlorophyll production was130.4μg each gram in fresh lily leaves. The optimization results of enzymatic hydrolysis method was that with the cellulose enzyme concentration of1%and4℃cold treatment for the lily leaves, the highest concentration of complete chloroplast can be got. With some calculation, complete chloroplyll production was182.7μg each gram in fresh lily leaves. Synthesis method was used combine the optimal scheme of these two extraction methods. With some calculation, complete chloroplyll production was191.7μg each gram in fresh lily leaves. Therefore, synthesis method was the best plan to extract complete chloroplast.In this experiment, trypsin used to remove the CP might stick on the surface of chloroplast, which would avoid the contamination of the CP in the cytoplasm. From the study, both the test of SDS-PAGE and Western blot were confirmed that LSV-CP did exist in the chloroplast protein of host plants, and might affect the photosynthesis of host plants. The determination of chlorophyll content and the analysis of fluorescence dynamics proved the infection of LSV inhibits lily PS Ⅱ photochemical activity.