Molecular Cloning And Expression Regulatory Of AnGolS2 Gene From Ammopiptan-thus Mongolicus In Xinjiang

In recent years,the research on plant stress resistance has been more and more in-depth,and the response of plants to adversity and the mechanism of adaptation have also received more and more attention.Plants respond accordingly in the face of adversity,thus better adapting to harsh environments?including low temperature,high salt,drought,etc.?.The promoter plays an important role in the regulation of gene expression.Therefore,the research on the relationship between gene promoters and stress response mechanisms has become a hot topic.In this study,Ammopiptanthus nanus was selected as the experimental material.Using gene cloning technology,An GolS2 and the promoter of this gene were successfully cloned.The cis-acting element of this promoter was predicted by software,and the deletion vector was designed according to the cis-acting element.Finally,a transient expression analysis was performed.The test results obtained are as follows:1.The gene-specific primers were designed based on the EST sequence of the GolS gene found in the drought stress transcriptome.The RACE amplification technique was used to amplify the 3'and 5'ends of the gene using the cDNA as a template,and the sequence was then sequenced.After splicing,the full-length gene sequence was 1168 bp,and the coding region was 987 bp.2.According to the coding region of An GolS2,specific primers were designed and the genomic DNA was used as a template.The 821 bp promoter sequence was cloned using chromosome walking technique.Then the cis-acting element was obtained through the PLACE and plant CARE website.The predicted results indicate that the promoter sequ ence contains some cis-acting elements associated with abiotic stress and hormonal responses in addition to the TATA-Box and CAAT-Box core elements.3.Real-time fluorescent quantification of three parts of root,stem,and leaf of Ammopiptanthus sphaerocarpa after treatment at different times of low temperature,high salinity and drought stress.It was found that the expression level of An GolS2 gene in rhizome leaves under different stress conditions had different changes.It is speculated that An GolS2may involve the transportation and storage of leaf photosynthate.Using ABA,IAA,NAA,GA₃,MeJA,and ETH to induce quantitative analysis of Ammopiptanthus mongolicus plants,it was found that the expression level of AnGolS2 gene changes under different hormone induction.Therefore,we hypothesized that hormones may involve An GolS2 gene expression.Regulation.4.Construction of a deletion vector for ethylene-related elements containing a deletion of the ethylene element?385 bp?and a deletion?378 bp?containing no ethylene element.The two deletions and the full length sequence of the promoter were linked together to the expression vector pCAMBIA 3301.Three recombinant expression vectors were obtained.Agrobacterium-mediated transformation was used to perform transient expression analysis using the GUS gene as a reporter gene,The results showed that the leaves that were injected with the ethylene-containing promoter deletion fragment expression vector exhibited a color reaction after ethylene induction.The injection of a promoter containing no ethylene element in the expression vector of the deletion fragment showed no color reaction with ethylene and no ethylene reaction.The expression vector of the promoter deletion fragment of the induced leaves was not colored,indicating that the ethylene transcription factor binding site on the AnGolS2 promoter may regulate the expression of the An GolS2 gene,but the activation of this element was significantly lower than that of the whole plant.The long promoter implied that there may also be ethylene-induced cis-acting elements in other parts of the full-length promoter.