Cloning And Functional Analysis Of Soybean GmCBL7 Gene Promoter

In a complex environment,plants need to deal with a large number of stimuli throughout their life cycle.In this process,Ca²⁺is a ubiquitous second messenger.It plays a role in many kinds of cell metabolism processes in response to plant biotic and abiotic stress responses.Calcineurin-like B（CBL）protein,as a calcium sensor,can sense stress Ca²⁺signals and bind to Ca²⁺,thereby activating its downstream protein kinase CIPK.By interacting with CBL-interacting protein kinases（CIPKs）,it can interact with each other in plants.It plays an important role in response to abiotic stress and growth and development.In the process of transcriptional regulation,gene promoters and their cis-acting elements can activate or inhibit gene expression.Therefore,analyzing the function of promoters that regulate gene expression at the transcriptional level is essential to reveal the regulatory mechanism of genes in plant growth and development and adversity defense,and can provide a basis for the improvement of stress-resistant varieties of plants.In this study,the full-length sequence of GmCBL7 gene promoter（Cbl P7-1）and its 4 different length 5’-end deletion fragments were cloned from soybean.After constructing plant expression vectors,the stable transformation of tobacco and the transient transformation of soybean hairy roots provided a basis for the functional verification of the GmCBL7 gene promoter.The main findings of this study are as follows:1.Primers were designed according to the upstream sequence of soybean GmCBL7 gene in the plant genome database Phytozome,and the GmCBL7 gene promoter sequence was cloned from soybean.The sequence length was 1523 bp and named Cbl P7-1.Predicted by online promoter analysis software PLACE and Plant CARE,it was found that there are many promoter core elements in this sequence,such as TATA-box related to transcription initiation and CAAT-box related to transcription frequency.The sequence also has some regulatory elements related to stress and plant hormone induction:HSE（high temperature response element）,MBS（drought response element,combined with MYB transcription factor）,MYB（involved in drought,salt and ABA response）,MYC（drought）,And ABA response elements),ABRE（ABA response related elements）,ERE（ethylene response related elements）,etc.,as well as some cis-acting elements related to light regulation,endosperm formation,meristem expression and injury response.The prediction results show that the GmCBL7gene promoter can be induced to express under stress.At the same time,a plant binary expression vector p CAMBIA1301-Cbl P7-1 driven by the promoter was constructed.2.The promoter plant expression vector p CAMBIA1301-Cbl P7-1 was stably transformed into tobacco by the leaf disc method mediated by Agrobacterium,and two positive transformed plants with Cbl P7-1 fusion gus reporter gene were obtained.Analysis of GUS activity in the roots,stems and leaves of transgenic plants showed that the GmCBL7 gene promoter showed different tissue expression patterns in transgenic plants,especially the strongest expression activity in stems.The results of GUS enzyme activity assay showed that the GmCBL7 gene promoter is involved in regulating the response of soybean to high salt and drought stress.3.In order to find the core functional region of the promoter,the 5’end deletion analysis of the promoter was further carried out,and 4 5’end deletion fragments of different lengths were cloned.The sequence lengths were 1202bp（Cbl P7-2）and 902bp（Cbl P7-3）,454bp（Cbl P7-4）,251bp（Cbl P7-5）.The plant binary expression vectors driven by the 5’-end deletion fragment were constructed,respectively,p CAMBIA1301-Cbl P7-2,p CAMBIA1301-Cbl P7-3,p CAMBIA1301-Cbl P7-4 and p CAMBIA1301-Cbl P7-5.4.Using Agrobacterium-mediated method to transiently transform tobacco and soybean hairy roots with 5 kinds of promoter plant expression vectors.After GUS histochemical staining,it was found that the full-length fragment Cbl P7-1（1523bp）and two 5’deletion fragments Cbl P7-2（1202bp）,Cbl P7-3（902bp）has the priming activity,and Cbl P7-3 has the strongest priming activity.However,Cbl P7-4（454bp）has weak priming activity,Cbl P7-5（251bp）has no priming activity.It is speculated that the core regulatory region of this promoter may be located in the region of-1202～-454bp.