| Caragana Korshinskii Kom is a leguminous plant,grows in a desert or a semi-desert region.Ithas high ecological adaptability to the environment,strong stress resistance,and In order to excavate the drought-resistant gene resources in Caragana Korshinskii,the cDNA library of drought-treated seedling was constructed and high-throughput sequencing was performed.Through bioinformatics analysis,49 drought up-regulated genes were found,including 6 WRKY transcription factors.On this basis,this study took the Col-0 ecotype of Arabidopsis thaliana as the receptor material,constructed expression vectors,transformed,obtained homozygous transgenic lines,and measured the drought-related phenotype to conduct preliminary functional analysis of the 6 WRKY transcription factors,and obtained the following results.(1)The sequences of the 6 WRKY transcription factors were compared with the Arabidopsis genome database.According to the homology alignment results of the sequences,the 6 WRKY transcription factors were named CkWRKY2,CkWRKY4,CkWRKY20,CkWRKY23,CkWRKY51 and CkWRKY75,respectively.The cDNA coding regions of these genes were 1614 bp,1386bp,1527 bp,1107bp,618 bp and951bp,encoding 537,461,508,368,205 and 316 amino acids,respectively.According to the number of structural domains and the structure types of zinc fingers,the 6 WRKY transcription factors are divided into three categories,among which CkWRKY2,CkWRKY4 and CkWRKY20 belong to class I,CkWRKY23 and CkWRKY51 belong to class II,and CkWRKY75 belong to class III.(2)The in-fusion method was used to connect the target gene with 35 S Red-Kan vector skeleton to construct the expression vector,which was obtained by agrobacterium-mediated dip-flowering transformation of Arabidopsis.Fifty-three transgenic lines were got based on these six vectors.After positive plants in T3 generation were screened,wild type and transgenic plants were used as research materials for screening and drought stress experiments.The transgenic plants were simulated with mannitol under drought stress.The results showed that under the100 mM mannitol stress,compared with the wild-type,the transgenic CkWRKY4 plants showed weaker performance,longer root length and slower growth rate than the wild-type,while the transgenic CkWRKY2 and CkWRKY75 plants grew well and had longer roots.Then,9 strains were further selected for subsequent experiments.(3)The transgenic lines and wild type were subjected to natural drought treatment.The water loss rate of isolated leaves was determined,and it was found that the water loss rate was lower than that of wild type.At the same time,the content of soluble sugar,proline,POD and other physiological indexes were determined.The content of soluble sugar,proline and POD was higher than that of wild type.The results showed that transgenic plants produced a lot of osmotic regulatory substances,antioxidant enzymes and other substances under drought stress to resist the drought environment.The survival rate of transgenic plants after drought rehydration was higher than that of wild type.(4)qRT-PCR experiment was conducted for natural drought transgenic lines.The results showed that WRKY gene expression in transgenic CkWRKY2,CkWRKY4 and CkWRKY75 lines increased significantly after drought induction.The results showed that CkWRKY2,CkWRKY4 and CkWRKY75 genes were involved in drought stress.The results got in this study showed that WRKY2,4 and 75 genes from the genome of Caragana Korshinskii had drought resistance,and the change of their expression patterns might be a way to change the drought resistance of plants.The results of this study provided the oretical basis for analyzing the molecular mechanism of drought resistance and breeding ofCaragana Korshinskii. |
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