Isolation and analysis of the promoter for an inducible gene CpPGIP2 from Carica Papaya

Abstract: 【Objective】Promoters play a key role in the regulation of gene expression. Targeted gene expres-sion,using constitutive promoters,usually causes physiological disorders because of the overexpressionof the target gene. The transgenes driven by tissue-specific or inducible promoters will only express inspecific space and time where the transgene product is needed. Therefore,excavating new inducible andtissue-specific promoters has become a necessary component in plant genetic engineering research. Poly-galacturonase- inhibiting proteins(PGIPs) represent important plant defense proteins. PGIP genes arehighly induced by pathogens to inhibit the activity of fungal PGs and retard the invasion of plant tissues.The objective of this study is to isolate and characterize the PGIP2 promoter from C. papaya【Methods】Genomic DNA was extracted from leaves using the Plant Genomic DNA Miniprep Kit Protocol(Solarbio).Specific primers were designed according to the sequences of the Cp PGIP2 gene(HQ660394) and papayagenome contigs(ABIM01005077). Three promoter fragments,D0-1981,D1-1024,and D2-261(D0,D1,and D2 indicate deletions,the numbers after the hyphen indicate the number of nucleotides up-stream to ATG which is present in the deletion fragment) were amplified with Ex Taq DNA Polymerase(Ta Ka Ra) using forward primers(D0-F,D1-F,and D2-F) with the Eco R I restriction site and one com-mon reverse primer(D-R) with the Bgl II site at their 5'end. Genomic DNA was used as the template,and amplification was carried out as follows: initial denaturation at 94°C for 3 min followed by 35 cyclesof 94 ℃(30 s),55 ℃(30 s),and 72 ℃(2 min),and then a final extension at 72 ℃ for 7 min. The PCR products were gel purified. The purified product was then cloned into a p MD18-T vector(Ta Ka Ra) andtransformed into E. coli DH5 a competent cells. The E. coli DH5 a carrying recombinant plasmid,(p MD18-T + PGIP2 promoter fragments),were verified by PCR and DNA sequencing. Then,the sequence wassearched for known transcription factor binding sites using the Plant CARE databases(http:// bioinformat-ics. psb.ugent.be/webtools/plantcare/html/). The transcription start site(TSS) was predicted by using theNeural Network Promoter Prediction software(http://www.fruitfly.org/seq_tools/ promoter.html). The con-firmed recombinant plasmid were digested with Eco R I and Bgl II restriction enzymes and cloned into theplant expression vector p CAMBIA1301 by replacing the Ca MV 35 S promoter. Recombinant plasmid wasextracted from the transformed E. coli DH5α and confirmed by PCR and restriction digestion. The p CAM-BIA1301 binary vector was used as a positive control. The binary vectors p CAMBIA1301,p D0-1981,p D1-1024,p D2-261 were transformed into the Agrobacterium strain EHA105 by using the freeze-thawmethod. The confirmed Agrobacterium harboring p CAMBIA1301,the full-length promoter and its dele-tion fragments driving the GUS reporter gene were used for transient expression analysis in papaya. Thepapaya pulp slices(2 mm thick) were incubated in Agrobacterium engineering bacteria for 20 min,thenwere cultured on the MS solid medium for 48 h in dark. After GUS staining,the blue coloration was ob-served to measure the efficiency of the full-length promoter and its deletion fragments. The leaf,stem,root,and callus of the papaya were infected with Agrobacterium engineered bacteria to measure the ex-pression efficiency of the Cp PGIP2 promoter in different papaya tissues.【Results】A 1 981 bp upstreamto the initiation codon of the Cp PGIP2 was isolated and designated as the Cp PGIP2 full-length promoter.The nucleotide sequence of the Cp PGIP2 promoter cloned in this study was deposited at NCBI(Genbank:HQ660396). The promoter sequence was analyzed for transcription start sites and potential cis-acting tran-scription factor binding sites. The Neural Network Promoter Prediction identified a TSS at 1777 bp(204bp 5'to ATG). The Plant CARE analysis revealed a potential TATA box at 22 bp upstream to the potentialTSS. We were able to find several CAAT boxes,light responsive elements,two fungal elicitor responsiveelements(Box-W1),a drought MYB binding site(MBS),three anaerobic induction elements(ARE),anabscisic acid responsive element(ABRE),two gibberellin responsive elements,and a salicylic acid re-sponsive element,as well as an element involved in the control of leaf morphology development and sever-al elements required for endosperm expression. This suggested that Cp PGIP2 may response to fungi,drought,anaerobic,abscisic acid,salicylic acid,or gibberellin induction,and express in endospermsand leafs. It has been confirmed that PGIP expressed during the pathogen,salicylic acid,abscisic acid ortrauma induction in some other plants. This suggested that the Cp PGIP2 promoter responsed to a varietyof signals and it is an inducible promoter. In order to characterize the Cp PGIP2 promoter,promoter::β-glucuronidase(GUS) reporter gene fusion constructs were prepared for its full-length and its 5'deletionfragments. Three expression vectors were constructed and named as p D0-1981,p D1-1024,and p D2-261 respectively. Plasmid,which was isolated from the transformed E. coli(DH5α) cultures,was con-firmed by PCR and restriction enzyme digestion analysis. The right length fragments 1 981,1 024,and261 can be amplified or cut out by Eco R I and Bgl II. The results showed that the three expression vectorswere successfully constructed. Then,the recombinant plasmid were transformed into Agrobacterium. Us-ing a transient transformation,promoter expression efficiency was examined. A qualitative GUS assay re-vealed that the full-length promoter as well as its deletion fragments were able to drive the expression ofthe reporter gene GUS in papaya. The p D1-1204 vector expressed the strongest activity even higher thanthe positive control p CAMBIA1301. The p D0-1981 vector showed the lowest activity probably due to thefact that there are silencers between-1 981 to-1 204 bp 5'to ATG. The p D2-261 vector expressed thelowest activity on account of it being short of some important cis-acting elements. Moreover,the p D1-1204 vector can also express GUS in the root,stem,leaf,and callus of the papaya. The expression in theroot and the fruit were higher than the other tissues.【Conclusion】With the availability of a number ofplant genome sequences,it has become relatively easier to isolate promoters. On the basis of the papayadraft genome,a 1 981 bp length of Cp PGIP2 promoter was obtained by using a general PCR(Genbank:HQ660396). Bioinformatics analysis showed that a TSS was at the site of 204 bp 5'to ATG and there werefungi,drought,abscisic acid,gibberellin,salicylic acid and some other responsive cis-acting elementsin the sequence. The p D1-1204 vector was confirmed expressing the strongest activity in the papaya fruit.The Cp PGIP2 promoter deletion fragment D1-1204 showed higher expression efficiency than the frequent-ly-used Ca MV 35 S promoter in the positive control p CAMBIA1301,so the promoter has good applicationpotential in plant genetic engineering breeding.