Analysis Of Key DNA Functional Segments Of Arabidopsis ?-glucosidase 19 Gene Promoter

In order to meet the needs of growth and development,plants in nature can correspondingly drive specific genes to be expressed in a tissue site according to changes in the external environment,thereby adapting to the complex and constantly changing growth environment.A promoter is a sequence in a DNA sequence that can initiate transcription,recognize RNA polymerase,and bind to DNA,that is,a sequence located at the 5'end upstream region of a structural gene to initiate gene transcription.There are many important cis-acting elements in plant gene promoters.These elements are involved in regulating the expression of corresponding downstream genes at the transcription level,so that plants can resist the stress of the external environment and grow better.According to research,when the transcriptional regulation of promoters in plants is affected,it may cause gene expression or even adverse reactions to their growth and development.Therefore,the study of plant promoters is very important for us to better understand the expression patterns and regulatory mechanisms of gene transcription regulation.Previous research in our laboratory found that the ?-glucosidase19(Beta-glucosidase,BGLU19)gene promoter of Arabidopsis thaliana is a promoter with seed-specific promoter,which can control the highly efficient expression of foreign genes in plant seeds rather than other parts of the plant,as well as avoid the energy load and material waste caused by non-specific expression in transgenic plants,and reduce the adverse effects on plants.In this study,we will obtain homozygotes of transgenic Arabidopsis thaliana through the deletion mutation of the AtBGLU19 promoters at the molecular level,to determine the seed-specific expression elements and key DNA regions,and to study the?-glucosidase 19 genes promoter and their expression regulation mechanisms.In this study,after analyzing the key cis-acting elements in the At BGLU19 promoter,the cloning vectors and plant expression vectors of the deleted sequences of the At BGLU19 promoter were constructed,which were transformed into Arabidopsis thaliana by inflorescence dip method,and finally the transgenic homozygous lines were selected.GUS histochemical staining analysis showed that all the deleted fragments could drive the GUS reporter gene expression in the AtBGLU19 promoter and the seeds of each deleted promoter.The main research results of this paper are as follows:(1)Analysis of tissue-specific elements of the AtBGLU19 promoter sequence.After analyzing the DNA sequence of the AtBGLU19 gene promoter through the database of PLACE and PlantCARE cis-acting regulatory elements,this study obtained some elements related to tissue-specific expression,mainly: 2 ABRE elements,3 RY elements(RY1 element,RY2 element,RY3 element),SEF4 element,SEF3 element and ESP element,etc.(2)Analysis of deletion mutations of AtBGLU19 promoter and each deletion promoter.Compared with the AtBGLU19 promoter,the tissue-specific elements in different deleted promoters are reduced.The deleted promoter ?AtBGLU19-1 lacks the sequence of the RY1 element and SEF4 element;?AtBGLU19-2 in turn lacks the sequence of the SEF3 element;The shortest ?AtBGLU19-3 still lacks the sequence of the RY2 element.(3)The cloning vector of each deletion sequence of AtBGLU19 promoter was successfully constructed.Based on the sequence analysis results of the key cis-acting elements of the At BGLU19 gene promoter,amplified deletion promoter primers for the 5' deletion mutation series were designed.After PCR amplification,three deletion fragments with sequence sizes of 562 bp,424bp and 264 bp were obtained,and the deletion fragments were sequentially designated as ?BGLU19-1,?BGLU19-2,and?BGLU19-3.After purification,the amplified product was cloned and transformed,and its plasmid sequencing results showed that the deleted fragment was correctly cloned into the vector.The recombinant cloning vectors were named BGLU19P-1,BGLU19P-2,and BGLU19P-3 in order of the length of the deleted sequence from large to small.(4)The expression vector of each deletion sequence of AtBGLU19 promoter was successfully constructed.The corresponding expression vectors were ligated with the digested fragments of each of the deleted sequence cloning vectors,and the positive clones were screened,colony PCR,and double-digested electrophoresis were detected.After obtaining positive clones and comparing the sequencing results,it was found that three plant expression vectors with missing fragments were successfully constructed.According to their length from small to large,they are named p?BGLU19-1-GUS,p?BGLU19-2-GUS,and p?BGLU19-3-GUS.It was transformed into Arabidopsis thaliana by inflorescence infection method,and finally the transgenic lines were screened.(5)GUS histochemical staining analysis of AtBGLU19 promoter and different tissues of each deleted promoter.According to the results of observing the staining ofdifferent lengths of the deleted promoter and the AtBGLU19 promoter in different tissues and the depth of staining,it was found that both the AtBGLU19 promoter and the deleted promoter series can drive the expression of downstream GUS reporter genes in seeds,indicating that both of them can affect gene expression.In addition to the seed part of the full-length promoter AtBGLU19,other parts are not stained,and all tissue parts of the deleted promoter are stained,of which 264bp's shortest deleted promoter ?AtBGLU19-3 in the seeds,seedlings,flowers and siliques staining the deepest.Based on the above experimental results,it is speculated that the cis-acting elements that regulate the specific expression of the GUS gene in seeds are mainly present in in ?At BGLU19-3,and the ?AtBGLU19-3 promoter segment is a key DNA segment required for At BGLU19 promoter function.However,the regulation of promoters between specific elements needs to be further explored and identified through the activity of mutant promoters and so on.