Application Of VIGS In TaADF7 Response To UV-B Radiation

Enhanced UV-B radiation can harm plants,affect the photosynthesis of plants,and lead to crop yield decrease.Wheat is an important food crop,so the response mechanism of wheat to UV-B radiation is of great significance to study.From the level of gene function to study the abnormality of wheat is still the molecular mechanism to reveal the wheat response to UV-B radiation,which is also the basic work for improving the resistance of wheat and other crops to UV-B.For a long time,due to the limitation of hexaplioid wheat materials and the lack of research methods,studying and exploring the mechanism of anti-UV-B molecules at the genetic level is very difficult.The technology of virus induced gene silencing?VIGS?has provided a new method for solving this mechanism.Wheat was used as the research material in this study.The VIGS vector in this paper was barley stripe Mosaic virus.Wheat Phytoene desaturase?PDS?and Actin depolymerization factor?ADF7?as a template to respectively constructed the VIGS expression vector.Tobacco was injected through agrobacterium-mediated BSMV,then the infection tobacco leaf provided a good source of the virus to inoculate wheat.Finally,VIGS system was established,which provided a new method for studying the function of wheat gene under the UV-B irradiation.The main research results are as follows:1.Construction of BSMV-?:PDS₄₀₀virus vector.2.p Ca BS-?,p Ca BS-?and p Ca BS-?:PDS₄₀₀were injected into four leaf-aged tobacco at1:1:1.By observing the phenotype of tobacco leaf after 20 days of injection,it found that the leaves of the upper layer was a weak bleaching phenomenon with light green,which indicated that the vector was basically successful.After injection 5-12 days,the infected tobacco leaves were ground to inoculated wheat,and found that new wheat leaf shown obvious bleaching phenomenon,which illustrated the VIGS system was basically successful in wheat.3.The instantaneous silence of PDS gene in wheat was identified from two aspects.On the one hand,it adopted the trypan blue staining method,and found that the fresh leaves of wild type and the bleaching wheat leaves were not color,but,dried leaves,since the cell loses its activity or the cell membrane were destroyed,which made the membrane permeability increases,and could be dyed blue.So the phenomenon of bleaching in wheat PDS silencing was not caused by cell death.Another,measured chlorophyll fluorescence parameters,and found Fo,Fm,Fv/Fm decreased successively in CK,BSMV:00?empty Vector?,BSMV:PDS(p Ca BS-?:PDS₄₀₀)and Fm,Fv/Fm in BSMV:PDS group was significantly lower than the two control groups?CK,BSMV:00??P<0.05?,which indicated the silencing of PDS gene caused the damage to the fluorescence yield of the PS?reaction center.At the same time,chlorophyll a and chlorophyll b were extracted,and found the chlorophyll content of wheat leaves in BSMV:PDS group was lower than that of two control groups?CK,BSMV:00?,and the difference was very significant?P<0.05?,which shown that the silencing of PDS in wheat leaves can significantly reduced the content of chlorophyll and that the reason for the wheat bleaching was the silence of PDS.4.Construction of BSMV-?:ADF7 virus vector.By the same method,p Ca BS-?,p Ca BS-?and p Ca BS-?:ADF7 were injected into four leaf-aged tobacco at 1:1:1.After 7-12days,used leaf fluid of tobacco to rub wheat,and used semi-quantitative PCR detected ADF7stripe was obvious dark in the silent plant,which indirectly indicated the construction BSMV:ADF7 vector was the basic success.5.Studied on the effect of ADF7 on phenotype,physiological and biochemical changes of wheat under UV-B radiation to explore the function of ADF7,and found the ADF7 silence didn't affect the growth of blade width,but influenced on plant height and the third leaf length of wheat,that is,it could inhibit the growth of wheat plant height and the prolong leaf length,and also found the silence ADF7 in plant height and leaf length of wheat was more sensitive to UV-B radiation.The increased speed of MDA content of silence ADF7 wheat under UV-B radiation was lower than the wild type and empty plasmid?BSMV:00?,by contrast,the activity of SOD in silence ADF7 wheat after UV-B radiation speed was higher than the wild type and empty plasmid,which meant that the ADF7 silence could relieve the accumulation of peroxide caused by the UV-B.Similarly,the wheat of silencing ADF7 affected the change of the soluble protein content and soluble sugar content causing by UV-B radiation.