Establishment, Optimization And Validation Of Oil Palm Virus-induced Gene Silencing (VIGS) System

Oil palm（Elaeis guineensis）is one of the important woody oil crops in tropical regions.Because of its rich oil and fat widely present in the pulp and kernel of oil palm,it has the reputation of"the king of oil in the world".Brown is mainly distributed in southern Hainan Province,Xishuangbanna,Yunnan Province and other areas.Because oil palm is a perennial plant with a long growth cycle,the genetic breeding of oil palm is relatively difficult,and the development of in vitro rapid propagation technology of oil palm is conducive to breakthroughs in research.Virus-induced gene silencing（VIGS）technology is a simple and rapid tool to study gene function.Brown and related gene functions have not yet been reported.In this study,EgPDS gene was used as a reporter gene,and a plant expression vector was constructed after bioinformatics analysis,and the relevant parameters of the VIGS system of oil palm embryoid were optimized to explore the possibility of the application of viral vector TRV in oil palm embryoid.After the establishment of the system,specific gene verification was carried out.The main research results obtained are as follows:1.Combined with PDS genes in NCBI and other species,the EgPDS gene of oil palm was determined.Real-time quantitative PCR analysis showed that EgPDS was expressed in five stages of oil palm fruit and on oil palm embryoid bodies.The full-length EgPDS gene was cloned from the total RNA of the mesocarp of the fifth stage oil palm fruit.Through the bioinformatics analysis of EgPDS and the search for specific fragments,the specific target gene fragments of the expression vector were determined,and the p TRV2-EgPDS vector was constructed.2.On the basis of the oil palm embryo-type callus culture in our laboratory,through the preparation and selection of the infecting bacteria liquid,it was found that the embryo-type callus could be transformed when MSO was used as the infecting bacteria liquid;When the liquid concentration was OD₆₀₀=0.5 and the infection time was 5 min,the infection effect was the best;the co-cultivation group for 48 h had the highest transformation efficiency;tobacco system mediation was not suitable for the infection of oil palm embryoid bodies;infection bacteria The infection effect and growth state of oil palm embryo type callus were better than those of the other two strains when the species was EHA105;the transformation efficiency was the highest when the AS concentration was 20 mg/L;finally,the sampling time of the VIGS system was optimized,and it was found that the infected The infection effect was the best when the incubation time was 12 days;3.Select a pair of genes known to interact and may have positive regulatory effects:EgARF18 and EgFAD2 to verify the VIGS system,sample embryoid bodies and wild embryoid bodies transformed by the silenced vector p TRV2-EgARF18,and real-time fluorescence quantification PCR showed that the expression level of EgARF18 was significantly decreased,and the expression level of EgFAD2,the downstream gene of EgARF18,was also significantly decreased.This further verified the positive regulatory effect between EgARF18 and EgFAD2.Oil analysis was performed on the embryoid bodies of the experimental group and the control group,and it was found that compared with the embryoid bodies of the control group,the content of C18:1（oleic acid）in the experimental group was significantly increased,while the content of C18:2（linoleic acid）was significantly higher in the experimental group.significantly reduced.This is also consistent with previous reports that the FAD2 gene can catalyze the conversion of oleic acid to linoleic acid in plants.Through these experimental results,it is proved that the VIGS system established in this study is feasible in the verification of oil palm functional genes,which plays an important role in the study of oil palm gene function,and provides important information for subsequent oil palm genetics and breeding experiments.research Foundation.