Screen,Cloning And Characterization Of Suppressors Of Var6-1 In Arabidopsis Thaliana

The correct expression of plant nucleus genes ensures the normal development of chloroplasts and the effective photosynthesis of chloroplasts.In this study,series of var6-1suppressors were screened and their mutation were cloned respectively.We found that these gene products are important functional proteins in spliceosome of eukaryotes.Most of the splicosomal proteins are highly conserved in eukaryotes,which mediate the pre-mRNA splicing along with assembly-activation-disassembly cycles.Under these backgrounds,studies on several suppressors have been made and the mainresults achieved are as follows:(1)S16-04,S17-09 and S20-10 mutants was screened for the suppressors of var6-1,and we found that the mutation of G to A occurred at 1394 bp of PRL1 of S16-04,resulting in the termination codon formed by the tryptophane mutation at 178 th of its protein.The mutation of G to A at 2589 bp of PRL1 of S17-09,results in the change of the 3’ splicing site of the 12 th intron,which may lead to abnormal splicing of transcription products.The mutation of G to A occurred at 1853 bp of PRL1 gene of S20-10,which changed the3’splicing site of the eighth intron of PRL1.(2)Genetic analysis and sequence of the S29-02 showed that the deletion of G at 1442 bp in the AAR2 gene(AT1G66510)and resulted in frame shift mutation of AAR2.(3)The mapping and whole-genome resequencing of S01-20 A revealed that the 203 rd G was changed to A in SmD1b(AT4G02840),resulting in the replacement of the 27 th amino acid of its protein from glycine to arginine.(4)The plant expression vectors p CDC5-CDC5-GFP,p AAR2-AAR2-GFP and p SmD1b-SmD1b-GFP were constructed and genetic complementation experiments proved that the mutation of AT1G66510(AAR2)leads to the phenotype in S29-02,the mutation of AT4G02840(SmD1b)leads to the phenotype in S01-20 A,and the mutation of AT1G09770(CDC5)leads to the phenotype in cdc5-1 var6-1.(5)The result of subcellular localization showed that SmD1b-GFP are located in in the nucleus of the root cells.(6)Based on the statistics of alternative splicing of Arabidopsis ribosomal proteins,it was found that nearly 68 of the 252 ribosomal proteins in Arabidopsis had the possibility of alternative splicing.(7)The total RNA were extracted from S01-20 A,S29-02,cdc5-1,prl1 and Col-0respectively.After that,we examined the alternative splicing and the accumulation of the first intron of ribosomal gene RPS21 B,RPS5B,RPL7 B and RPL13 B by RT-PCR and semi-quantitative PCR,whether splicing in these mutants will be affected.Then we found that compared with wild type,there are no significant difference in these mutants.