Studies On The Phenotype Recovery Mechanism Of Intragenic Suppressors Of Jhs1 And The Function Of GC1 Gene

The protein JHS1(Jing He Sheng 1)in Arabidopsis is homologous to the DNA2 in human and yeast.Previous studies have shown that JHS1 plays an important role in DNA damage repair,cell cycle regulation,and meristem maintenance.In jhs1 mutant,the mutation at the 3’ splice site of the intron resulted in aberrant splicing of m RNA,leading to reading frame shift and premature termination of the protein.The jhs1 mutant has severe growth defects,such as belt-like stems,abnormal arrangement of siliques,short roots and so on.To further investigate the function of the JHS1 and its regulatory network,suppressors of jhs1 by EMS(ethyl methane sulfonate)mutagenesis were screened and three candidate suppressors were obtained: k28-5 j,k48-1-1 j,and k109-6 j.These candidate suppressors not only restored the growth phenotype to wild-type-like levels,but also largely restored their response to DNAdamaging agents.DNA sequencing and genetic transformation experiment results indicated that these suppressors were caused by secondary mutations in JHS1.In order to learn the recovery mechanisms,the m RNA level of JHS1 in suppressors was analyzed and found to be restored partly.Then RT-PCR and SDS-PAGE silver staining assays were carried out to detect the m RNA splicing of JHS1 in suppressors,and the results showed that there were c DNA bands in suppressors different from that in jhs1.c DNA sequencing results suggested that alternative splicing make JHS1 to be translated normally in the correct reading frame,with only a small amino acid sequence insertion.In addition,we also evaluated 3’ splice sites of introns in the Arabidopsis genome,and found that the secondary mutations in suppressors affected the selection of 3’ splice site of 11 th intron,leading to an increased probability of correct splicing types directly or indirectly.In order to further reveal the recovery mechanism at protein level,protein structures of jhs1 suppressors were analyzed by homology remodeling.The results indicated that their structures were similar to that of the wild type and the small insertions were not located in the core regions,but in an outer loop region.Therefore,the inserted sequences may not affect the function of JHS1.TUNEL assay was carried out to investigate the DNA damage in suppressors.The results showed that the degrees of DNA damage in suppressors were similar to that of the wild type,indicating that the JHS1 protein in suppressors was functional.This study not only revealed the recovery mechanism of jhs1 suppressors by the secondary mutation in JHS1 gene at m RNA and protein levels,but also provided materials and a new strategy for studying 3’ splice site selection mechanism of introns.The chloroplast is the main place of photosynthesis in plants.Chloroplasts maintain their number in cell by binary fission which involves a series of division proteins.Previous studies have shown that AT2G21280 is an important chloroplast division gene,but the results of different studies are contradictory.In order to reveal the true function of AT2G21280 protein,this work analyzed the chloroplast division phenotype of the knockout mutants and over-expressing lines,and found that AT2G21280 has little effect on chloroplast division,thus correcting the mistake of naming AT2G21280 as GC1(Giant Chloroplast 1).Phylogenetic analysis showed that AT2G21280 is not a homologous protein of Sul A,and they belong to different protein families,thus correcting the mistake of naming AT2G21280 as At Sul A.Immunofluorescence staining is an experimental method widely used in protein subcellular localization study.Previously,this method was mainly applied to the study of animal cells.For plant materials,due to the existence of cell walls,it is necessary to use paraffin to embed leaves and do tissue sectionings,which is tedious and time-consuming.In order to improve the immunofluorescence staining method in plant cells,this study used protoplast system.Through the optimization of multiple experimental procedures,an immunofluorescence staining method for chloroplast division proteins was developed,which makes the operations much faster and more convenient.