番木瓜诱导型基因CpPGIP2启动子的克隆与分析

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

【目的】获得诱导型基因Cp PGIP2启动子,并分析该启动子的功能。【方法】以番木瓜Cp PGIP2基因c DNA序列(HQ660394)搜索番木瓜基因组DNA序列,获得一段约2 000 bp的5’端上游DNA序列(ABIM01005077),参照该序列设计PCR特异引物,以番木瓜叶片DNA为模板克隆该启动子,并进行生物信息学分析。将3个启动子片段替换p CAMBIA1301载体中的Ca MV 35S启动子,构建缺失启动子表达载体,以GUS瞬时表达检测缺失启动子表达活性。【结果】获得了Cp PGIP2起始密码子上游长为1 981 bp的调控序列,经分析该序列含有真菌、脱落酸、赤霉素、光照等多种响应元件,为诱导型启动子。构建了1个启动子全长表达载体和2个5’端缺失启动子表达载体,分别命名为p D0-1981、p D1-1204和p D2-261。启动子在番木瓜果肉中的瞬时表达显示,长度为1 204 bp的启动子表达活性最强。并且,该启动子在根、茎、叶、果肉和愈伤组织中均有不同程度的表达,在近外果皮的果肉处和根部表达最明显。【结论】本研究获得了Cp PGIP2启动子序列,确定了表达活性最强的启动子长度,初步分析了启动子的表达模式和顺式作用元件,为进一步深入研究Cp PGIP2基因功能以及将该启动子应用于植物基因工程育种奠定了基础。

[ 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 overexpression of the target gene. The transgenes driven by tissue-specific or inducible promoters will only express in specific space and time where the transgene product is needed. Therefore, excavating new inducible and tissue-specific promoters has become a necessary component in plant genetic engineering research. Poly- galacturonase-inhibiting proteins （PGIPs） represent important plant defense proteins. PGIP genes are highly 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 CpPGIP2 gene （HQ660394） and papaya genome contigs （ABIMOlO05077）. Three promoter fragments, D0-1981, D1-1024, and D2-261 （DO, 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 （TaKaRa） us mon reverse ing forward primers （D0-F, D1-F, and D2-F） with the EcoR I restriction site and one com- 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℃ for 3 min followed by 35 cycles of 94 ℃ （30 s）, 55 ℃ （30 s）, and 72 ℃ （2 min）, and then a final extension at 72 ℃ for 7 min. The PERproducts were gel purified. The purified product was then cloned into a pMD 18-T vector （TaKaRa） and transformed into E. coli DH5a competent cells. The E. coli DH5a carrying recombinant plasmid, （pMD18- T＋PGIP2 promoter fragments）, were verified by PCR and DNA sequencing. Then, the sequence was searched for known transcription factor binding sites using the PlantCARE databases （http：//bioinformat- ics. psb.ugent.be/webtools/plantcare/html/）. The transcription start site （TSS） was predicted by using the Neural Network Promoter Prediction software （http：//www.fruitfly.org/seq tools/promoter.html）. The con- firmed recombinant plasmid were digested with EcoR I and Bgl II restriction enzymes and cloned into the plant expression vector pCAMBIA1301 by replacing the CaMV 35S promoter. Recombinant plasmid was extracted from the transformed E. coli DH5a and confirmed by PCR and restriction digestion. The pCAM- BIA1301 binary vector was used as a positive control. The binary vectors pCAMBIA1301, pD0-1981, pD1-1024, pD2-261 were transformed into the Agrobacterium strain EHA105 by using the freeze-thaw method. The confirmed Agrobacterium harboring pCAMBIA1301, the full-length promoter and its dele- tion fragments driving the GUS reporter gene were used for transient expression analysis in papaya. The papaya pulp slices （2 mm thick） were incubated in Agrobacterium engineering bacteria for 20 min, then were 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 CpPGIP2 promoter in different papaya tissues. [Results] A 1 981 bp upstream to the initiation codon of the CpPGIP2 was isolated and designated as the CpPGIP2 full-length promoter. The nucleotide sequence of the CpPGIP2 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 （204 bp 5＇ to ATG）. The PlantCARE analysis revealed a potential TATA box at 22 bp upstream to the potential TSS. We were able to find several CAAT boxes, light responsive elements, two fungal elicitor responsive elements （Box-W1）, a drought MYB binding site （MBS）, three anaerobic induction elements （ARE）, an abscisic 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 CpPGIP2 may response to fungi, drought, anaerobic, abscisic acid, salicylic acid, or gibberellin induction, and express in endosperms and leafs. It has been confirmed that PGIP expressed during the pathogen, salicylic acid, abscisic acid or trauma induction in some other plants. This suggested that the CpPGIP2 promoter responsed to a variety of signals and it is an inducible promoter. In order to characterize the CpPGIP2 promoter, promoter：：13- glucuronidase （GUS） reporter gene fusion constructs were prepared for its full-length and its 5＇ deletion fragments. Three expression vectors were constructed and named as pD0-1981, pD1-1024, and pD2- 261 respectively. Plasmid, which was isolated from the transformed E. coli （DH5c0 cultures, was con- firmed by PCR and restriction enzyme digestion analysis. The right length fragments 1 981, 1 024, and 261 can be amplified or cut out by EcoR I and Bgl II. The results showed that the three expression vectors were 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 of the reporter gene GUS in papaya. The pD 1-1204 vector expressed the strongest activity even higher than the positive control pCAMBIA1301. The pD0-1981 vector showed the lowest activity probably due to the fact that there are silencers between -1 981 to -1 204 bp 5＇ to ATG. The pD2-261 vector expressed the lowest activity on account of it being short of some important cis-acting elements. Moreover, the pD 1-1204 vector can also express GUS in the root, stem, leaf, and callus of the papaya. The expression in the root and the fruit were higher than the other tissues. [Conclusion] With the availability of a number of p!ant genome sequences, it has become relatively easier to isolate promoters. On the basis of the papaya draft genome, a 1 981 bp length of CpPGIP2 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 were fungi, drought, abscisic acid, gibberellin, salicylic acid and some other responsive cis-aeting elements in the sequence. The pD 1-1204 vector was confirmed expressing the strongest activity in the papaya fruit. The CpPGIP2 promoter deletion fragment D 1-1204 showed higher expression efficiency than the frequent- ly-used CaMV 35S promoter in the positive control pCAMBIA1301, so the promoter has good application potential in plant genetic engineering breeding.