Preliminary Analysis Of The Promoter Function Of The Arabidopsis Mannose Binding Lectin Gene At3g21380

The growth and development of higher plants are the result of selective gene expression,and gene expression is regulated by the promoter.The promoter acts like a "switch" and determines the activity of the gene.Once the promoter activity is abnormal,it usually leads to the regulation of gene expression disorder,which may lead to abnormal plant growth and development.Although many plant promoters with tissue-specific or inducible activity have been obtained in the present study,the number of promoters that are really suitable for a specific purpose of genetic improvement is small or inactive.Therefore,the potential promoters in plant genome need to be analyzed and identified.Gene annotation information provided on the Tair website(http://www.arabidopsis.org/)shows that the Arabidopsis thaliana At3g21380 gene is a coding gene of mannose-binding lectin superfamily protein(MBL).In this study,At3g21380 gene was tentatively named AtMBL1 gene.The gene expression data of AtMBL1 gene was analyzed by online website.It was found that AtMBL1 gene was a seed-specific expression gene,so it is speculated that AtMBL1 promoter is a seed-specific expression promoter.The AtMBL1 promoter was first cloned,then digested and ligated into a plant expression vector.The plant expression vector was transformed into wild Arabidopsis thaliana by Agrobacterium-mediated flower dipping.After seed maturation,the seeds were harvested,and then the harvested seeds are sprinkled on the screening medium containing hygromycin for generation-by-generation screening.Finally,the T3 generation homozygous lines without character separation were obtained.The tissue expression pattern of AtMBL1 promoter was analyzed by GUS histochemical staining.The results showed that AtMBL1 promoter could drive the specific expression of exogenous genes in seed sites.Then,based on the analysis of the full-length sequence of AtMBL1 promoter,a series of primers with 5'deletion promoter were designed and three deletion fragments were cloned.Then three plant expression vectors with deletion fragments were successfully constructed and transformed into wild Arabidopsis thaliana,and then screened into transgenic homozygous lines.GUS histochemical staining analysis showed that all the deleted fragments could drive the expression of GUS reporter gene in transgenic Arabidopsis thaliana seeds.The main experimental results of this paper are as follows:(1)Construction of AtMBL1 promoter expression vector.According to the upstream base sequence of the Arabidopsis thaliana AtMBL1 gene provided by Tair website,the primers of the AtMBL1 promoter were designed by using Primer 5.0 software,then PCR amplification.The amplified products were digested into plant expression vector pGFPGUSplus and transformed into Escherichia coli DH5? strain.The positive clones were preliminarily verified by colony PCR and restriction enzyme digestion.Finally,by comparing the results of sequencing,the plant expression vector of AtMBL1 promoter was successfully constructed and named pMBL1-GUS.pMBL1-GUS was transformed into wild Arabidopsis thaliana by Agrobacterium-mediated flower dipping.Homozygous lines were obtained through multi-generation screening.(2)Sequence analysis of promoter of AtMBL1 promoter.The sequence of AtMBL1 promoter was analyzed by using online analysis software such as PLACE and PlantCARE,and many regulatory elements related to seed-specific expression were identified.It includes RY element(CATGCA),ACGT element,ACGTCA element,as-1 element(TGACG),GCN4 element(TGAGTCA),skn-1 base sequence(GTCAT),AACA element(AACAAAA).(3)Construction of cloning vectors with deletion sequences of AtMBL1 promoter.Based on the sequence analysis of AtMBL1 promoter,a 5'terminal deletion primer was designed.Three deletion fragments of 621 bp,514bp and 351 bp were obtained by PCR amplification.The three deletion fragments were named ?MBL1,?MBL2 and ?MBL3 in turn.The purified amplified product was cloned into pEASY-Blunt Cloning Kit vector and transformed into Escherichia coli DH5? strain.The positive clones were preliminarily screened by using the results of colony PCR and double digestion electrophoresis and finally sequenced.Sequencing results showed that the cloning vectors of the three deleted fragments were successfully constructed.According to the length of the fragments from large to small,they were named MBL1P-1,MBL1P-2 and MBL1P-3 in turn.(4)Construction of expression vectors with deletion sequences of AtMBL1 promoter.After the cloning vector of the deleted sequence is digested,the target fragment is recovered by electrophoresis,and the target fragment is ligated to the pGFPGUSplus vector.The positive clones were preliminarily screened by colony PCR and double digestion electrophoresis.Finally,the results showed that the plant expression vectors with three deleted fragments had been constructed successfully by comparing the results of sequencing.According to the length of the missing fragments from large to small,they are named p?MBL1-GUS,p?MBL2-GUS and p?MBL3-GUS in turn.Plant expression vector with deleted promoter was transformed into wild Arabidopsis thaliana by Agrobacterium-mediated flower dipping.The homozygous lines of the deleted sequences were finally obtained by screening.(5)AtMBL1 promoter is seed-specific expression promoter.GUS histochemical staining was performed on selected transgenic homozygous lines with AtMBL1 promoter by X-Gluc solution.The detection period included seed stage,seedling stage,flowering stage and silique stage.The staining results showed that AtMBL1 promoter could drive gene expression,and could effectively drive the specific expression of GUS reporter gene in Arabidopsis thaliana seeds.(6)GUS histochemical staining of each deletion sequence of AtMBL1 promoter.The results showed that both AtMBL1 promoter and deletion series could drive downstream GUS reporter genes to express in Arabidopsis thaliana seeds,indicating that all of them had the activity of initiating gene transcription.? MBL3 can still drive the downstream GUS reporter gene expression,which is the key region of AtMBL1 promoter to play a regulatory role.It is presumed that seed-specific expression elements mainly exist in ?MBL3.The missing parts of the sequence can not inactivate the promoter,but whether the elements in these sequences can promote or inhibit gene expression,the interaction between the elements and the molecular mechanism of the elements regulating promoter activity need to be verified by experiments such as site-directed mutagenesis of promoter,yeast single hybridization and gel retardation analysis.