| In higher plants,cell wall invertase(CWI)and vacuolar invertase(VI)are essential players in sugar signaling and sugar metabolism.They can affect plant development,source-sink interactions and plant response to environmental cues.The activities of CWI and VI are regulated at transcriptional and posttranscriptional levels.C/VIF1(cell wall/vacuolar inhibitor of fructosidase 1)and C/VIF2 are two invertase inhibitor in Arabidopsis,they can control CWI and VI activity,thereby regulating the activities of CWI and VI at posttranscriptional levels.Abscisic acid(ABA)is involved in the regulation of several aspects of plant growth and development,including the occurrence of germ,seed germination and dormancy,seedling growth,reproductive organ development and fruit ripening,etc.Meanwhile,ABA regulates abiotic stress tolerance in plants via activating/repressing gene expression.Through transcriptome sequencing,we found that the expression of C/VIF1 and C/VIF2 are regulated by ABA.In this thesis,the effects of C/VIF1 and C/VIF2 on plant response to ABA and salt stress in Arabidopsis thaliana were systematically studied.RT-PCR and qRT-PCR were used to detect the expression of C/VIF1 and C/VIF2.The results showed that both C/VIF1 and C/VIF2 were up-regulated by ABA at different levels.To study the functions of C/VIF1 and C/VIF2,we generated35S:C/VIF1 and 35S:C/VIF2 overexpression plants and single and double mutants for C/VIF1 and C/VIF2,respectively.In order to obtain mutants of C/VIF1 and C/VIF2 as soon as possible,we optimized the CRISPR/Cas9 gene editing system.The GmFT2 a expression cassette was inserted into the pHEE CRISPR/Cas9 vector to generate the pHEE-FT vector,for which early flower phenotype was used as a screening marker.After plant transformation,early flowering plants in the T1 generation were selected and sequenced to check the edited status.Normal flowering plants in T2 generation from edited T1 plants were sequenced to identify homozygous transgene-free mutans.In this system,transgene-free mutants can be obtained by phenotype observation.Meanwhile,due to the addition of GmFT2 a gene,the life cycle of plants is greatly shortened,thus reduced the total time length for mutant acquisition.Single and a double mutant of C/VIF1 and C/VIF2 were obtained by using the optimized pHEE-FT CRISPR/Cas9 gene editing system.NBT staining results showed that the activities of invertases in muant plants increased,possible due to the decrease/loss of invertase inhibitor activities.Phenotypic analysis showed that theroot length of the single and double mutants was longer than that of the wild type.Both single and double mutants produced more siliques than the wild type.The results of ABA sensitivity experiments showed that the sensitivity of the C/VIF1 and C/VIF2 overexpression plants were enhanced to ABA.After ABA treatment,seed germination rate of these plants was lower than that of wild type,indicating that both C/VIF1 and C/VIF2 participate in the regulation of response to ABA.Meanwhile,in the experimental results of salt stress,we found that the germination of c/vif1-c2,35S:C/VIF2 and c/vif1c/vif2 transgenic plants were less tolerance to salt than that of Col wild type,but that of 35S:C/VIF1 and c/vif2 were largely not affected.In summary,we found that C/VIF1 and C/VIF2 are ABA responsive genes,and C/VIF1 and C/VIF2 are involved in the regulation of plant response to ABA and salt stressed in Arabidopsis thaliana. |
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