Cloning And Functional Study Of Var6-1 Suppressor In Arabidopsis And Preparation Polyclonal Antibody

Chloroplasts are one of important organelles in plant cells and are the main places for photosynthesis.PAC is a chloroplast protein encoded by the nucleus and is a necessary factor in chloroplast development.In the early experiment,the mutant var6-1 seeds of the PAC lower expression were mutagenized by EMS,and the suppressor mutant with green first true leaf were screened.One of the suppressor mutants S04-05 was map-based cloned and functionally studied,and polyclonal antibodies to PRL1 were also prepared.The main results achieved are as follows:（1）The S04-05single was isolated from the suppressor S04-05DM and there are pleiotropic phenotypes in S04-05single such as shorter petiole,wrinkled leaves,delayed flowering,shorter siliques and reduced seeds in individual siliques.Statistical analysis revealed that S04-05single began to bolting when developed to 17±2 true leaves;and that the number of seeds per silique S04-05single was 35±5.（2）Map-based cloning of S04-05single and sequencing of candidate genes revealed that there is G to A mutant in CAND1 of S04-05single.Constructed CAND1 transgenic complement plants,S04-05single turned to wild-type phenotype,S04-05DM turned to var6-1phenotype,and further confirmed that the mutant gene was CAND1.（3）Histochemical staining revealed that the CAND1 was expressed in the 7-day-old whole plant,especially in the veins,the root tip and the junction the root and hypocotyl showed higher expression.Similarly,there are also expressed at styles,filaments and the base of siliques.The subcellular localization results showed that GFP-CAND1 was located in the cytoplasm and nucleus.（4）The content of PAC in wild type,var6-1,S04-05DM and S04-05single was detected.It was found that the content of PAC in S04-05DM increased.It was inferred that CAND1regulate PAC accumulation,and thus regulate the chloroplasts development.（5）To construct the transgenic plant with accumulation of AXR2,the T₂ generation were not suppressed the phenotype of var6-1.But to construct the transgenic plant with site-directed mutagenesis of abnormal accumulation of AXR2,the T₁ generation has a distinct developmental phenotype,and the plants shrink and curl,unable to grow normally.（6）The p ET28a-PRL1 prokaryotic expression vector was constructed and expressed in E.coli expression strains.Recombinant PRL1 was purified via Nickel affinity chromatography,and polyclonal antibodies were prepared by immunizing rabbits.Construct PRL1 transgenic complement plants with N-strep tag to test the validity of the antibody.It was found that PRL1 was accumulated higher in tissues where cell division is active.