Cloning And Functional Identification Of Promoters From Cucumber Translationally Controlled Tumor Protein CsTCTP Genes

The translationally controlled tumor protein(TCTP)is a large family of ubiquitous organisms involved in the regulation of growth and development.It plays a key role in response to both biotic and abiotic stresses.The expression of TCTP is regulated by both the level of transcription and translation,and the promoter is the center of gene transcription regulation.At present,research on TCTP is mainly focused on humans and animals.There are few reports on the structure and function of TCTP in plants,and little is known about its promoters.In this experiment,the CsTCTPs promoter of cucumber was isolated and characterized using the cucumber strain B21-a-2-1-2 as material,combined with bioinformatics and transient expression analysis techniques.It is clear that this gene is regulated by a variety of cis-acting elements at the transcriptional level,which provides a basis for further exploration of the expression pattern of CsTCTPs gene transcriptional regulation and its regulatory mechanisms.At the same time,it is of great significance for further understanding the stress resistance of plants.The main results are as follows:(1)The promoter sequences of CsTCTP1 and CsTCTP2 were successfully isolated from cucumber genomic DNA using conventional PCR techniques,then the distribution and function of cis-acting elements in the promoter regions of CsTCTP1(-1,308 bp to +88 bp)and CsTCTP2(-1,305 bp to +69 bp)were predicted using bioinformatics methods.The results showed that the promoter region of CsTCTPs contained conserved core promoter elements such as TATA-box and CAAT-box,as well as upstream cis-acting elements such as hormone responsive elements(ABRE,ERE and TCA-element,etc.),defense-associated responsive elements(TC-rich repeats)and light responsive elements(G-boxe),etc.(2)Based on the position of the cis-acting elements on the CsTCTP1 and CsTCTP2 promoters,six different lengths of promoter deletion fragments were obtained: proT1-1.3kb(-1,308 bp to +88 bp),proT1-0.7kb(-714 bp to +88 bp),proT1-0.3kb(-306 bp to +88 bp),proT2-1.3kb(-1,305 bp to +69 bp),proT2-0.7 kb(-732 bp to +69 bp),proT2-0.3 kb(-324 bp to +69 bp).(3)Replacing the 35 S promoter in pCAMBIA 1301 with each promoter deletion,and GUS fusion vectors carrying each promoter deletion fragment were successfully constructed: proT1-1.3kb-GUS,proT1-0.7kb-GUS,proT1-0.3kb-GUS,proT2-1.3 kb-GUS,proT2-0.7 kb-GUS and proT2-0.3 kb-GUS.(4)The six GUS fusion vectors successfully constructed were respectively transferred into Agrobacterium strain GV3101.Tobacco was transiently transformed by Agrobacterium-injection permeation method,and histochemical staining and fluorescence GUS were performed.The results showed that all of the six promoter deletion fragments of CsTCTP1 and CsTCTP2 had the function of initiating gene expression,and the promoter activity was about 3 to 4-fold that of 35 S.Among them,the activity ofproT1-0.7kb was the strongest,and it was speculated that the sequence contains a cis-acting element similar to the enhancer;The function of other promoter sequences is not clear and needs further verification.(5)The transiently transformed tobacco leaves were treated with different exogenous hormones(ABA,SA and ETH),and promoter activity was measured 24 hours later.The results showed that the activity of promoters proT1-0.7kb of CsTCTP1 and proT2-1.3kb of CsTCTP2 were up-regulated in the ABA treatment group,it is speculated that the promoter sequence may contain ABA-related response elements;However,in the SA and ETH treatment groups,the activity of all the promoter fragments of CsTCTP1 and CsTCTP2 had declined to varying degrees,therefore,it can be inferred that SA and ETH may have a negative regulatory effect on both CsTCTP1 and CsTCTP2 promoters.