The Exploration And Establishment Of Virus Induced Gene Silencing System In Artemisia Sphaerocephala

Artemisia sphaerocephala,a sandy shrub belonging to the Artemisia genus of the Compositae family,which is important for the maintaintion and restoration of the desert ecosystem,and this shrub contains linoleic acid,vitamin E,has the health functions.The cognition of gene functions was needed in A.sphaerocephala,with the sequencing of transcriptome completed.Virus-induced gene silencing?VIGS?technology does not require to establishing the system of genetic transformation,which was quickly identified gene function and widely used in non-model plants.The PDS?Phytoene desaturase,PDS?gene is selected,and it act as a key gene for the synthesis of carotenoid.When the gene is silenced,the chlorophyll synthesis pathway is breaked,plant phenotype is easily to distinguish and has the characteristic of"photobleaching".In this study,we used A.sphaerocephala as material,tobacco rattle virus?TRV?as vector and PDS gene as target gene to construct the positive and negative recombinant vector in agrobacterium.Three inoculation methods of injection method?I?,vacuum infiltration?V?and vacuum infiltration+ultrasonic?V+U?,four bacterial concentrations of OD₆₀₀?0.5,0.8,1.0 and 1.5?,two recombinant vectors were combined,in total 24 treatment groups,to treat the A.sphaerocephala in cotyledon stage and the true leaf to exploring the feasibility of the VIGS system.The results obtained are as follows:?1?The AsPDS gene of 441 bp was cloned in A.sphaerocephala,the forward and reverse recombinant vectors of pTRV₂-AsPDS-F,pTRV₂-AsPDS-R were successfully constructed based on TRV virus.?2?In the cotyledon stage,the establishment of the VIGS system was significantly affected by the inoculation methods,bacterial concentrations and directions of gene insertion.The optimal treatment was V+U-1.5-R,the degree of whitening was 0.72,and the photobleaching characteristics occured at the first and second pairs of true leaves.?3?In the true leaf stage,the establishment of the VIGS system was significantly affected by the inoculation methods and bacterial concentrations,the directions of gene insertion had no effects.The optimal treatment was I-1.5-R,the degree of whitening was 0.34,and the photobleaching characteristics occured at the third and fourth pairs of true leaves.?4?In the cotyledon stage,14 groups treatment produced positive pla nts,and the total average of intensity of photobleaching was 0.15;In the true stage,5 groups treatment produced positive plants,and the total average of intensity of photobleaching was 0.05.The cotyledon stage positive plants and total average of intensity of photobleaching were better than the true leaf stage in A.sphaerocephala.In this paper,the rapidly and efficient functional gene silencing system were established in A.sphaerocephala,which provided technical support for the subsequent study of gene function in this species.