Cloning And Functional Analysis Of Soybean GmWRI1a Promoter

Promoters of higher plants are important components of gene expression vectors,affecting the expression of exogenous gene in plants.The promoter sequence contains a number of cis-acting elements that regulate the expression of the corresponding downstream genes at the transcriptional level and enhance the ability of plants to adapt to complex external growth environments.The research on plant promoters can help to understand the expression pattern of gene transcription regulation and its regulation mechanism,and can also be directly applied to plant genetic engineering to improve or improve the expression of exogenous genes.The AP2/EREBP transcription factor plays an important role in stress resistance such as cold resistance,drought resistance,high temperature tolerance and salt tolerance,and can also regulate the response of plants to hormones such as ethylene?ETH?and gibberellic acid?GA₃?.It plays an important role in stress resistance.The WRINKLED1?WRI1?transcription factor,which contains two AP2 domains,belongs to the AP2/EREBP family.WRI1 plays a key role in regulating fatty acid metabolism,controlling the carbon source from sugar to oil synthesis in seeds.In the early stage,our group cloned the GmWRI1 a gene from soybean,which increased the oil content of soybean seeds by regulating the related genes of soybean fatty acid anabolic pathways BCCP2,FAE1,KAS1,ACP1 and related genes of glycolytic pathways Pl-PK?1,PDHE1?.In addition,the gene is also involved in soybean drought and salt and alkali stress response.The function of the gene can be further identified by studying the promoter of the gene.Therefore,to explore the function of GmWRI1 a,this study firstly isolated the promoter of GmWRI1 a gene extracted soybean Dongnong 47 genomic DNA from genomic DNA of soybean cultivar Dongnong 47,firstly isolated the promoter of soybean GmWRI1 a gene,And,the promoter of GmWRI1 a gene was used todirectly replace the CAMV35 S constitutive promoter of expression vetor p BI121 to drive the expression of GUS reporter gene.To analyze the structure and functions of pGmWRI1 a promoter,four pGmWRI1 a promoter deletion-GUS fusion expression vectors were transformed into Arabidopsis thaliana.The regulation of GmWRI1 a promoter in plant growth was explored,which laid a good foundation for the in-depth study of GmWRI1 a gene.The research results are as follows:1.The promoter pGmWRI1 a of soybean GmWRI1 a gene was cloned by PCR.The sequence of the promoter was 1669 bp length.It was predicted that the promoter not only contained TATA-box,CAAT-box,but also had multiple cis-acting motifs using the PLACE website,such as ethylene-related motif?ERE?,gibberellin-related motif?P-box ? GARE-motif?,jasmonic acid-related motif?CGTCA-motif?,ABA response element CE3 and photoresponsive element: G-Box,GA-motif,ACE,AE-Box,ATC-motif,CATT-motif,and GT1-motif.2.In transgenic Arabidopsis,full-length GmWRI1 a promoter at normal growth conditions could drive the GUS gene express in roots,stems,leaves,flowers,petals,sepals,stamens and pistil stigma.GUS gene was strongly expressed in the hypocotyls and shoot tips of transgenic Arabidopsis seedlings.The results showed that the pGmWRI1 a promoter can drive GUS gene expression during the whole growth period of transgenic Arabidopsis.3.According to the positions of the main cis-elements of pGmWRI1 a promoter,the 1138 bp,1087 bp,690 bp and 437 bp length pGmWRI1 a mutants were amplified by PCR.And the four pGmWRI1 a mutants were transformed into Arabidopsis using inflorescence method.The transcription initiation site of pGmWRI1 a promoter was predicted to be between-690 and-437 bp.The four Arabidopsis thaliana mutant plants were respectively treated with 2 m M gibberellin,0.1 m M jasmonic acid and 0.2 m M ethylene.The results of GUS enzyme activity assay showed that the ethylene's cis-elements located at-1669 and-1138 bp,the jasmonic acid's cis-elements located at-1087 and-690 bp,the gibberelli's cis-elements located at-690 and-437 bp and drought's cis-elements located at-1087 and-690 bp.4.To analyze the response of GmWRI1 a to gibberellin,jasmonic acid and ethylene treatment,the soybean cultivar DN47 was respectively treated with 2 m M gibberellin,0.1 m M jasmonic acid and 0.2 m M ethylene.The results of q RT-PCR showed that GmWRI1 a gene was up-regulated by gibberellin and ethylene,and there was no response to the treatment of jasmonic acid..