Transformation Of Populus With CAPX Gene And Cloning And Analysis Of The CAPX Promoter Sequence

Chinese white poplar (Populus tomentosa Carr), which belongs to SalicaceaePopulus, is a deciduous tall arbor. It is native to China and widely distributed, growsfast and has straight trunk with beautiful shape, so is used as an excellent afforestationtree species.There will be a large number of reactive oxygen species generated byenvironmental stresses in plant cells. If not promptly removed, they would generate agreat damage to the plant, and damage the plants’ normal physiological function.Ascorbate peroxidase (APX) is a kind of important enzyme which participates in theascorbate-glutathione (AsA-GSH) cycle of the plant. The enzyme is ahaem-containing protein, which plays an important role in clearing hydrogen peroxide(H2O2) in plants and improving the resistance of plants. The expression of APX geneis a favorable condition for the plants to survive in the face of adversities, but thepromoter for regulating the expression of APX gene is few reported.The research on poplar gene promoter is gradually increased in recent years,which mainly related to poplar microtube tissue-specific promoter, floral organdevelopment gene promoter and adversity resistance gene promoter. These studieshave important theoretical and economic significance, which can not only help peopleto deepen understand how to regulate poplar growth and development, and themolecular mechanisms such as resistance to adversity stresses, but also contribute toshorten the period of poplar breeding and foster a new high degree of infertilityenvironmental variety to improve the poplar’s resilience.Using pBI121-cAPX recombinant plasmid, we integrated the target gene intowild type741Populus tomentosa by using leaf disc transformation method mediatedby agrobacterium, won transgenic plants of the cAPX gene expression, whichprovided materials for further study of the effect of cAPX gene on resistance ofPoplar. We took wild-type741Populus tomentosa as an experimental material, andselected four cytoplasmic sequences of APX isozyme gene(APX-Ⅱ, Ⅲ, Ⅶand IX)sequences. TAIL-PCR was done for the first four gene sequences to extend a promoter.We cloned three APX gene promoter sequence fragments, and analyed their features.The unknown specificity sequence of Ⅲ gene has not been obtained in the firstpromoter extension, in which we designed many primers to repeat and change a lot ofreaction conditions (temperature, time, number of cycles). On the basis of the firstpromoter amplification, three genes of APX-II, VII and Ⅸ were no further extended inthe second promoter extension. Following showed the main contents and results:1. Culture and identification of cAPX-overexpressed poplar: We used kanamycinto select obtained poplar differentiation plant. Then the genomic DNA from poplarwas extracted. NPTII primers were used for PCR detection. Results showed that, threecAPX-Ⅱ-overexpressed strains and two cAPX-Ⅲ-overexpressed strains could amplify specific DNA bands, and the wild type plants had no specific bands. So it explainedthat the before-mentioned cytosolic APX gene had been integrated into the poplargenome.2. The APX gene promoter cloning: By TAIL-PCR method, combined with theknown APX gene cDNA sequences of Populus tomentosa,we selected four genesequences of II, III, VII and IX.We used the software Primer Premier5.0to designspecific primers. We took the partial sequence fragment of Populus tomentosa totalDNA as a template, explored and optimized PCR conditions. Finally, we cloned threeAPX gene promoters of Populus tomentosa, they were II, VII and IX (length:331bp、322bp and299bp).3. The function analysis of the promoter fragment on three APX genes: Ⅱ genepromoter sequence analyses showed that the promoter fragment contained twopromoter elements CAAT-box,TATA-box and other cis-elements(GAG-motif、Sp1、CGTCA-motif and Skn-1-motif) analysised by use of PLACE and PlantCAREsoftware. In addition, promoter prediction site analysis showed it contained twopromoter sequences. Ⅸ gene promoter sequence comprised two promoter elementsCAAT box, TATA-box and other cis-elements(ABRE、ARE、Box I、CGTCA-motif、Skn-1-motif and TCT-motif). Ⅶ gene promoter sequence contained two promoterelements CAAT-box, TATA-box and other cis-elements(ABRE、ARE、Box I、CGTCA-motif、Skn-1-motif and TCT-motif). In addition, promoter prediction siteanalyses showed Ⅶ gene also contained a promoter sequence. The results laid thefoundation of the next step of building a different length of Populus tomentosa APXgene promoter expression vector, transforming the model plants, such as poplar,tobacco, studying the temporal and spatial expression of the promoter characteristics,and laid the foundation for the analysis of the molecular mechanisms that APX geneinvolved in plant abiotic stresses which provided theoretical support for geneticengineering to improve the resilience of trees and genetic material. The promotersequences of the three APX genes are short, so there are some limitations for theirfunctional analysis. The three gene promoter sequences remain to be further extended,and corresponding functional analysis needs more work to make it complement andperfect.