Cloning Of Navel Orange Wax Gene CsLACS2 Promoter And Its Binding Characteristics To The Upstream Transcription Factor CsSHN1

The plant SHN1 transcription factor has the function of regulating the expression of downstream wax genes and controlling the synthesis of plant cuticular wax.The LACS family genes encode long-chain acyl-Co A synthetases that regulate the synthesis and metabolism of fatty acids in wax constituents.The Arabidopsis At SHN1 transcription factor binds directly to the At LACS2 gene promoter and regulates its expression.In order to study the molecular mechanism of navel orange Cs SHN1 transcription factor regulating the expression of downstream wax gene,the yeast one-hybrid technique was firstly used in this study to verify the transcriptional activation activity of Cs SHN1 transcription factor.Then,the promoter sequence of the downstream wax gene CsLACS2,which may bind to the Cs SHN1 transcription factor,was cloned from "Newhall" navel orange and subjected to cis-acting element analysis.The promoter fragment of CsLACS2 was designed and obtained based on the analysis of cis-elements distribution.Finally,the shortest core fragment of the CsLACS2 gene that binds to Cs SHN1 was screened using yeast one-hybrid technique and the core elements were postulated.At the same time,according to the full-length sequence of the Cs SHN1 gene promoter cloned in previous research of our group,the deletion fragment of Cs SHN1 promoter was designed and the deletion fragment activity detection expression vector of Cs SHN1 promoter was constructed,introduced into Agrobacterium tumefaciens,and genetically transformed into Arabidopsis.These results lay the foundation for obtaining the core fragment and core components of the Cs SHN1 promoter.The main research results are as follows:1.The verification of transcriptional activation activity of Cs SHN1 transcription factor : several yeast colonies appeared on SD-Leu/-Trp/-His plates,indicating that the DNA binding domain on the Cs SHN1-p GBKT7 vector can bind to the upstream activation sequence UAS of GAL4 in the yeast cellto activate the transcription of the downstream reporter gene,which demonstratied that Cs SHN1 has transcriptional activation activity.2.CsLACS2 promoter and its 5’ deletion fragment clone: We cloned the CsLACS2 promoter sequence of 2000 bp.Bioinformatics analysis indicated that the CsLACS2 promoter sequence contained multiple abiotic stresses and biotic stress response cis-acting elements,hormonal response-related cis-acting elements and photo responsive elements,indicating that CsLACS2 may be an abiotic stress and biotic stress response gene.According to the distribution of cis-acting elements on the CsLACS2 promoter,we cloned four CsLACS2 promoter 5’ deletion fragments,including CsLACS2-pro1(1368bp),CsLACS2-pro2(967bp),CsLACS2-pro3(649bp)and CsLACS2-Pro4(301bp).3.The identification of the combination of Cs SHN1 transcription factor with CsLACS2 promoter: on SD/-Leu/+150 ng/m L Ab A plate,the positive control plate colonies grew normally,the negative control did not show yeast colonies,and several yeast colonies appeared on CsLACS2 plate.Studies have shown that the Cs SHN1 transcription factor can bind to the CsLACS2 promoter.4.Identification of Cs SHN1 transcription factor binding to CsLACS2 deletion fragment: The results showed that the yeast colonies were detected on CsLACS2-pro1(1368bp),CsLACS2-pro2(967bp)and CsLACS2-pro3(649bp)plates,while no yeast colonies were detected on CsLACS2-pro4(301bp)plates,indicating that the binding site of Cs SHN1 transcription factor on CsLACS2 promoter is located between CsLACS2-pro3 and CsLACS2-pro4.Therefore,we hypothesized that the binding site of the Cs SHN1 transcription factor to the CsLACS2 promoter may be a cis-acting element such as TC-rich repeats or AAGAA-motif located between-649 bp～-301 bp.5.The screen of Cs SHN1 promoter core fragment: The Cs SHN1 promoter was divided into 6 fragment by 5’ deletion method: Cs SHN1-pro1(100bp),Cs SHN1-pro2(376bp),Cs SHN1-pro3(476bp),Cs SHN1-pro4(764bp),Cs SHN1-pro5(1062bp)and Cs SHN1-pro6(1310bp)were cloned and inserted into p BI121 expression vector,and the recombinant expression vector of Cs SHN1 promoter deletion fragment was successfully constructed,and introduced into Agrobacterium tumefaciens by freeze-thaw method and finally genetically transformed the model plant of Arabidopsis thaliana.