A Preliminary Study On The Regulation Of ABI3 Protein On The Expression Of AtBGLU19 Gene In Arabidopsis Thaliana

Plants need to undergo multiple challenges during their growth and reproduction.Seed germination and seedling formation provide the basis for the occurrence of these physiological processes,which greatly improve the possibility of plant survival and thus enable higher plants to evolve successfully.Seed is the basic unit responsible for survival?dispersal and reproduction,and its development process integrates the complex regulation of various genes.ABI3 is considered to be an important regulator in seed development.It regulates the expression of many genes related to seed development and plays a central role in the establishment of primary seed dormancy and seed maturation.Several studies have demonstrated that the conserved B3 domain in ABI3 is the key to this function,because the B3 domain can bind to the RY element,which exists in the promoter region of seed-specific genes,to control the expression of target genes.The RY element is present in the promoter in the form of multiple copies and it can control the transcription level of seed-specific genes.With the development of different omics techniques,some seed-specific genes have been gradually discovered.Recent studies have shown that some BGLU genes(such as At BGLU11,At BGLU18)in Arabidopsis thaliana can be specifically expressed in seeds,showing tissue-specific characteristics,suggesting that these genes play a potential role in seed establishment or germination.But,there are relatively few studies on At BGLU19 gene.The gene expression profile showed that Arabidopsis At BGLU19 gene was highly expressed at the stage of seed maturation,and its upstream promoter region contained several RY elements,which indicated that Arabidopsis At BGLU19 gene was seed-specific.This has been confirmed in our previous research.In order to further explore its internal regulation mechanism,we conducted this study.The main experimental results are as follows:(1)The acquisition of recombinant protein His-ABI3-B3.According to the information of At ABI3 gene in Arabidopsis proteomics provided by Tair website and the DNA sequence of coding B3 domain of ABI3 protein provided by NCBI website(http://www.ncbi.nlm.nih.gov/),Primer 5.0 was used to design ABI3-B3 primer.The plasmid of ABI3 puncture bacteria was used as a template for PCR amplification.The amplified product was ligated to the protein expression vector p ET-28 a and transformed it into E.coli BL21(DE3)strain.The sequencing verified that ABI3-B3 was successfully inserted into the p ET-28 a protein expression vector.IPTG was used to induce the prokaryotic expression of His-ABI3-B3 recombinant protein and then the recombinant protein was purified by Ni column affinity chromatography.(2)Preparation of RY elements and mutant RY elements.The promoter sequence of At BGLU19 was analyzed to determine the number and position of RY elements(sequence CATGCA).Primers were designed to amplify DNA fragments containing RY elements which were named as RY1,RY2 and RY3.DNA fragments containing mutant RY elements were amplified in the same way and named as mut RY1,mut RY2 and mut RY3.Then they were used as DNA probes for electrophoretic mobility shift assay.(3)Electrophoretic mobility shift assay investigated the binding of His-ABI3-B3 recombinant protein to RY element.After mixing the His-ABI3-B3 recombinant protein and DNA probes,the electrophoretic mobility shift assay was performed.The results showed that the His-ABI3-B3 recombinant protein can specifically bind to some RY elements of Arabidopsis At BGLU19 promoter,in which RY3 plays a major regulatory role,followed by RY2,and RY1 plays almost no role.(4)Detection of Arabidopsis At BGLU19 gene expression level in abi3 mutant.The genomic DNA of abi3 mutant was extracted,and PCR identification was carried out using the three-primer method to confirm this mutant was a homozygous mutant.RNA of the homozygous mutant was extracted and reversed transcription into c DNA.Using Tublin2 as the reference gene,q RT-PCR was performed to compare the expression differences of At BGLU19 gene in wild-type Arabidopsis plants and abi3 mutants.The results showed that the expression of At BGLU19 gene in abi3 mutant was significantly lower than that in wild-type plants indicating that At ABI3 gene mutation seriously affected the expression of Arabidopsis At BGLU19 gene.Based on the above research results,it can be seen that ABI3 protein can positively regulate the expression of At BGLU19 gene by combining with the RY2 and RY3 elements(RY3 plays a major regulatory role),which may make it play a role in seed development.