CLoning And FunctronaL AnaLysis Of RcPDAT1-2 Gene Promoter In Castor

Plant transgenic engineering is one of the important methods and strategies for crop genetic improvement.The flexible application of promoters can provide effective tools for the efficient and specific expression of foreign genes.People use different types of promoters to drive the expression of downstream target genes to achieve efficient and stable expression.In this study,the promoter of RcPDAT1-2 gene,a key gene for ricinoleic acid synthesis,was used as the research object to explore the expression regulation mechanism of RcPDAT1-2,and the castor variety Tong Ri No.5 was used as the material to analyze the RcPDAT1-2 gene and clone RcPDAT1-2 to start The full-length and deleted sequences were verified by Arabidopsis transformation and the activity of RcPDAT1-2 promoter was analyzed.The experimental results are as follows:The bioinformatics analysis of the RcPDAT1-2 gene showed that the RcPDAT1-2 gene encoded a total of 660 amino acids.The encoded protein had no signal peptide,was a non-secreted protein,had a transmembrane domain,and contained 2 LCAT domains.Predictive analysis of cis-acting elements of the RcPDAT1-2 gene promoter showed that the promoter sequence contains not only the core elements TATA-box and CAAT-box,but also multiple light response elements,hormone response elements,stress-induced response elements and MYB transcription factors The binding site of the RcPDAT1-2 promoter was verified by the activity of the full-length activity of the experiment For transient expression analysis of onion epidermal cells,the RcPDAT1-2 promoter can initiate the expression of GFP gene in the cell membrane and nucleus,and the promoter strength is slightly higher than the CaMV35S promoter.The RcPDAT1-2 promoter has promoter activity.The cloned RcPDAT1-2 promoter deletion sequence and Arabidopsis genetic transformation experiments showed that:7 promoter deletion sequences were successfully cloned,including 5 5 'deletions,1 3' deletion sequence and 1 5 ',3'co-deletion Sequence;successfully constructed 7 RcPDAT1-2 promoter deletion expression vectors;using Arabidopsis thaliana as host material,the deletion promoter activity was detected by Arabidopsis genetic transformation to obtain T3 generation Arabidopsis transgenic plants.GUS staining analysis of T3 generation transgenic Arabidopsis seedlings,flowers and pods,in addition to RcPDAT1-2(5)and RcPDAT1-2(8)promoters,other RcPDAT1-2 promoters of different lengths have promoter downstream The activity of gene expression' may 'contain.negative regulatory elements in the promoters of RcPDAT1-2(5)and RcPDAT1-2(8),which needs further analysis and verification.The experimental results of the drought and low temperature response element verification in the RcPDAT1-2 promoter showed that the GUS enzyme activities of transgenic plants with different length promoters were different,and the GUS enzyme activities of the transgenic plants were also different at different times;In the GUS staining analysis,RcPDAT1-2(8)did not produce blue precipitate.The GUS enzyme activity under drought-induced conditions was higher than that of other promoters.The RcPDAT1-2(8)promoter was affected by drought stress;RcPDAT1-2(1),RcPDATl-2(2),RcPDAT1-2(3)and RcPDAT1-2(8)promoters showed the same trend at different treatment times,first increased and then decreased and then increased Trend,and the GUS enzyme activity reached the highest at 48 h,and the promoter was affected by the MBS drought response element;for RcPDAT1-2(1),RcPDAT1-2(2),RcPDAT1-2(3)containing LTR low temperature response element,RcPDAT1-2(4)and RcPDAT1-2(5)promoters were subjected to low temperature treatment at 4 ? In uninduced GUS staining analysis,RcPDAT1-2(5)did not produce a blue precipitate.After induction,RcPDATl-2(5)GUS enzyme activity reaches the highest,the RcPDA T1-2(5)promoter was affected by low temperature stress;the GUS enzyme activity of transgenic plants showed different trends at different treatment times in the five promoters,but the GUS enzyme activity appeared after 4 hours of low temperature treatment at 4 ? Increased,the promoter is affected by the LTR low temperature response element,and its GUS enzyme activity will also vary with treatment time.