The Effect Of RY Element Of Arabidopsis Mannose Lectin Gene Promoter On Gene Expression

Arabidopsis is an important model plant for plant genetic engineering and biotechnology research.With the in-depth study of Arabidopsis,it provides a theoretical basis for the further research of transgenic technology of various crops.Each process of the growth and development of higher plants is a complex process of gene regulation,which starts from the promoter.Promoter is the switch of gene regulation.The abnormal promoter has great influence on the growth and development of plants.With the continuous development of biochemistry,molecular biology and gene engineering,the function of promoters and the functional elements of promoters have become the focus of biological research.Our laboratory analyzed the gene annotation information published on TAIR website?https://www.arabidopsis.org/?,and found that At3g21380 gene is a mannose binding lectin superfamily protein and name it AtMBL1.Further experimental analysis confirmed that AtMBL1 promoter is seed specific expression promoter.In this study,we analyzed the tissue-specific elements of the AtMBL1 promoter sequence,and found that there were four RY elements.The previously cloned AtMBL1 promoter sequence was mutated and digested by enzyme,it was connected to the plant expression vector,and it was infected to Arabidopsis thaliana var Columbia by Agrobacterium mediated infection.The mature seeds were collected and sprinkled onto MS medium containing the antibiotic Hygromycin B to screen for homozygous lines.Then,GUS histochemical staining was used to observe each period of the selected wild-type homozygous strains and four mutant promoter homozygous strains.The results of the four mutant promoters were compared with those of AtMBL1 natural promoter,the GUS enzyme activity was determined at the seed stage of wild type and four mutant promoter homozygous lines.The main experimental results of this study are as follows:?1?Sequence analysis of AtMBL1 promoter and RY elements.First,the RY element site of the natural AtMBL1 promoter was analyzed using biological software,and four RY elements RY1,RY2,RY3,and RY4 were identified.?2?Construction of plant expression vector for each mutant sequence of AtMBL1 promoter.The site-directed mutation primers were designed by primer5 software,and the natural promoter was used as a template for two-step PCR amplification experiments to obtain each mutant promoter sequence.After the product is analyzed by gel electrophoresis,the correctly band is connected to the expression vector.The recombinant plasmid product was digested by double digestion and verified by sequencing,and finally an expression vector ligated with the mutant RY sequence was obtained.?3?GUS staining analysis of each mutation sequence.The results of GUS histochemical staining showed that the natural promoter of AtMBL1 was a seed specific promoter,that is,only the seed stage was stained,but the four leaf seedling stage,flowering stage and silique stage were not stained.Therefore,GUS staining was performed on the natural promoter of AtMBL1 in this study,and GUS staining was performed on each mutation sequence as a control.The GUS staining of PMUTRY1-GUS,PMUTRY2-GUS,PMUTRY3-GUS,PMUTRY4-GUS mutant lines did not appear staining phenomenon in seed stage.The results showed that the gene expression of AtMBL1 promoter was down regulated after the mutation of RY element,that is,the regulation of RY element played a positive role,which indicated that RY element played an important role in the gene transcription activity of AtMBL1 promoter.In the histochemical staining experiment,the transgenic lines with four mutated promoters were not dyed blue,so the specific role of four RY elements,that is,the contribution of four RY elements to promoter activity,could not be determined according to the staining results.Although there is no staining in the seeds of the mutant promoter transgenic lines,slight staining occurs in the four leaf seedling stage,flowering stage and silique stage,indicating that the promoter mutant RY element drives GUS reporter gene to express in other parts,so we speculate that RY element may inhibit the expression of promoter driven gene in other tissues,or the mutation of RY element may affect other cis-acting elements and cause the emergence of non-seed-specific expression.This result needs to be verified by further experiments.?4?GUS activity analysis of natural promoter and each mutant promoter.The amount of fluorescent substance produced by the reaction is used to detect the GUS content quantitatively.The results showed that the GUS activity of each mutant promoter was significantly lower than that of the natural promoter,which indicated that the four cis elements RY1,RY2,RY3 and RY4could drive the expression of downstream GUS gene and play a positive regulatory role in the AtMBL1 promoter.At the same time,the GUS activity of the four mutant promoters was compared.The results showed that P_MUTRY3-GUS>P_MUTRY2-GUS>P_MUTRY2-GUS>P_MUTRY4-GUS,we conclude that RY3 element plays a positive regulatory role in the promoter of AtMBL1,followed by RY2,RY1 and RY4.