Molecular Cloning Of Increased Mobile Gene Silencing Mutant Imos1 In Arabidopsis Thaliana

The stable expression of exogenous genes in transgenic plants is one of the important means to improve agronomic traits such as nutritional fortification,biotic and abiotic stress resistance,etc.However,in the case of successful introduction and structural integrity,there is still a situation in which the target gene cannot be well-expressed.Normally,this phenomenon is associated with as gene silencing.The phenomenon of gene silencing regulated at RNA level is often occurring,and recent studies show that this phenomenon is non-cell autonomous,possibly mediated by plant vascular system,e.g.the phloem.Phloem transports more than assimilates from a source to sink tissues,but also biological macromolecules such as m RNA,small RNA PTGS mediated by the 21-nt small RNA can be transmitted between cells through plasmodesmata,and also to the shoots via long distance transfer.At present,there is no clear mechanism on the long-distance transportation of gene silencing.To understand this process,our experiment is based on the fact that the long-distance transportation of gene silencing can be stably inherited in Arabidopsis thaliana mutants,including imos1 and GS6.We then perform sequence cloning,and search for the genes related to rapid transportation silencing.The main results of this study are as follows:1)In this experiment,a F3 population was constructed by genetic cross of the stable genetic mutants imos1 and GS6 with different silencing rates obtained by EMS induced transgene Rt SS.Nine candidate genes,AT1G32230,AT1G47560,AT1G62580,AT3G09405,AT4G28050,AT4G30170,AT5G45960,AT5G56470 and AT5G59740 were obtained by comparing and analyzing the differential SNPs by whole genome sequencing with WT and Rt SS.2)On the basis of previous studies,the CRISPR/ Cas system p Cas Kana was constructed.The plant of this system was resistant to Kana and the fluorescent protein could be directly observed by microscope to screen the CRISPR/ Cas-free mutant.The CRISPR/ Cas system,p KEE401E-m Cherry were constructed,which used EC1.2-1.1as the fusion promoter and inserted into the screening protein m Cherry,which was a binary vector easy to observe and high in T1 generation homozygous mutants.3)9 candidate genes were successfully constructed on plasmid p HDE-35S-Cas9-m Cherry-UBQ to complete infection and obtain T₀ generation seeds,among which five genes AT1G32230,AT1G47560,AT5G45960,AT5G56470 and AT5G59740 were screened to obtain 3,24,6,31 and 6 T₀ generation positive plants,respectively,in which 8 AT1G47560 positive plants were selected for PCR and sequencing,and no mutation was found.AT1G32230 and its homologous gene AT2G35510 are constructed into plasmid p KEE401,T0 generation is obtained through transgene,13 resistant seedlings are obtained through screening,three plants are found to have obvious variation during phenotypic observation,DNA is extracted,specific primer amplification is designed according to the vicinity of a target point,a sample is sent for sequencing,a comparison result is obtained,among them,plant rcd1:sro1-1 appeared at anticipated sites.4)The RCI3 gene is related to the transport rate of a silencing signal,a primer is designed according to the CDS sequence of the gene to construct a prokaryotic expression vector p DEST15-RCI3,and the inserted fragment is determined to be correct through by using IPTG induction,no protein fragment was obtained.After multiple experiments and protein structure prediction and analysis of RCI3 gene,the segmented expression and transmembrane structure removal were performed.However,the effect was still not ideal.Preliminary judgment is that this gene is not suitable for prokaryotic expression.5)Under the condition of successful construction of the entry vector,a bait vector p DEST32-RCI3 was constructed by means of Gateway technology,which lays a foundation for screening the protein interacting with the bait vector p DEST32-RCI3 through a yeast two-hybrid system,thus laying a foundation for researching the action mechanism and function of the gene.