| Salix psammophila is one of the main tree species for sand fixation and afforestation in Northwest my country,and it maintains the stability of the sandy ecosystem.Salix psammophila has good stress resistance,and the study of its resistance-related genetic mechanism is beneficial to explore the potential value of Salix psammophila.At the same time,genetic engineering technology can be used to open up a new way for the cultivation of highly resistant garden plants.Therefore,this thesis takes Salix psammophila as the main research object to study the AP2 family DREB subfamily genes that can regulate stress resistance.In this study,the SpsDREB4 and SpsDREB8 genes were cloned from Salix psammophila.Use software to analyze biological information,analyze the specific expression of different tissues,analyze the expression of abiotic stress,and construct and transform gene overexpression vectors.The main findings are as follows:1.Design degenerate primers based on the conserved regions of the DNA binding domain of the DREB gene in plants,extract the whole plant RNA of Salix psammophila,reverse transcription to obtain c DNA,and use RT-PCR to amplify two DREB gene CDS sequences,the sequence sizes are 1368 bp and 864 bp,encoding 455 and 287 amino acids,respectively,named SpsDREB4 and SpsDREB8 genes.The phylogenetic tree further showed that the two cloned genes were DREB A-2 group genes.2.Bioinformatics analysis shows that: SpsDREB4 and SpsDREB8 protein molecular weight are 50021.41 Da and 31615.84 Da,theoretical isoelectric point are 5.57 and 5.21;SpsDREB4 and SpsDREB8 protein secondary structure predictions include α helix and extension Chains and random coils,both of which are hydrophilic proteins,have no signal peptide and no transmembrane structure;the tertiary structure shows that they all contain 1 α helix and 3 β-sheets,which are located in the second β-sheet The 14 th valine and 19 th glutamic acid are very conservative.3.Using quantitative PCR technology to analyze the tissue-specific expression of SpsDREB4 and SpsDREB8 genes in Salix psammophila.The results showed that the expression level of SpsDREB4 gene in leaves was the highest,while the expression levels of tender stems,roots,and flowers decreased sequentially,and the expression level of stem tips was the lowest;the expression of SpsDREB8 gene was the highest in tender stems,and that of leaves,flowers and stem tips The amount decreases successively,and the expression amount of root is the lowest.4.Through abiotic stress simulation,the expression levels of SpsDREB4 and SpsDREB8 genes under different stress conditions were analyzed by quantitative PCR.The results showed that the expression level of SpsDREB4 gene in abiotic stress was weak.While the expression of SpsDREB8 gene was induced by abiotic stresses such as drought,high salt,low temperature and high temperature.5.Using genomic DNA as a template,the genomic sequences of SpsDREB4 and SpsDREB8 genes were amplified.Sequence analysis of the amplified products showed that the SpsDREB4 and SpsDREB8 genes had no introns.6.Through the Gateway homology replacement technology,the plant expression vectors PMDC32::SpsDREB4 and PMDC32::SpsDREB8 were successfully constructed,which were transformed into Populus hopeiensis Hu et Chow by the leaf disc method.The above research results indicate that the SpsDREB4 and SpsDREB8 genes cloned in this study belong to the DREB A-2 group.Genome gene amplification shows that there are no introns.Tissue specificity and stress expression analysis showed that the two genes were expressed differently in different tissues of Salix psammophila,and their responses to stress were also different.This research lays a foundation for further exploration of DREB gene bioinformatics and functional research in woody plants,and provides guidance for the use of molecular breeding techniques to cultivate resistant garden plants. |
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