Cloning Of LpWRKY20 Gene From Lilium Pumilum And Genetic Transformation Of Tobaccos

Lilium pumilum is an important ornamental flower with anti-roll petals and bright color.It is a significant parent of lily resistant breeding.In this study,we selected the WRKY transcription factor family from the transcriptome of Lilium transcriptome.The genes that regulate the dormancy release were selected and analyzed for their response to abiotic stress.The gene was cloned and analyzed by bioinformatics.The plant expression vector was constructed and transformed into tobacco.The transgenic plants were subjected to drought stress.Phenotypic observation and physiological indicators under drought stress determine whether the gene has drought resistance.The main findings of this paper are as follows:1.40 WRKY transcription factors were separated by analyzing the WRKY transcription factor family of Lilium transcriptome.These genes are divided into 3 groups,which Group1 has 6 genes,Group2 has 28 genes,and Group3 has 6 genes.The relative expression of LpWRKY20 gene gradually increased with storage time,which proved that it was involved in the regulation of dormancy release.2.Abiotic stress is accompained in the peocess of dormancy.qRT-PCR analysis shows that the expression of LpWRKY20 is not the same in different abiotic stresses.In addition,the expresssion patterns are different in leaves,roots and bubls.Significant up-regulation of bulbs in drought stress suggests that the it is more sensitive to drought response.3.The open reading frame of this gene is 1899 bp,and the predicted encoding encodes 632 amino acids.Conserved domain analysis revealed that LpWRKY20 has two highly conserved WRKY domains belonging to the Group1.Bioinformatics analysis showed that the relative molecular weight of the protein was 68.36kDa,the theoretical isoelectric is 5.45,which was a hydrophilic protein,no signal peptide and no transmembrane structure;subcellular localization was predicted in the nucleus;protein secondary structure was mainly "Mixed "tyPe protein exists.4.The pBI121-LpWRKY20-GFP plant expression vector was constructed by double enzyme digestion,and transferred into tobacco by Agrobacterium-mediated leaf disc method to obtain resistant plants.The fusion expression vector and pBI121-GFP expression vector were bombarded into onion epidermal cells by gene gun method for subcellular localization analysis.The results showed that the fusion expression vector was only expressed in the nucleus.5.The activities of SOD and CAT in transgenic plants were higher than control under drought stress,and the ability to scavenge free radicals in the body increased significantly with the prolongation of time.The content of MDA was lower than the control,indicating that the it had a low degree of damage to the cell membrane and had a strong self-repairing ability.The changes of physiological indicators under drought conditions reflect that the resistance of transgenic plants is stronger than that of controls.It is preliminarily concluded that LpWRKY20 has the function of drought resistance.