Expression Analysis And Vector Construct Of A LRR Resistance Like Protein Gene From Cucumber

Cucumber,as a melon crop widely grown in the world, its yield and quality has attracted much attention. Cucumber blight,caused Fusarisum oxysporum f.sp. cucumerinum, is hard to prenvent and control,which can dramatically decrease yield and the quality. Thus,it is vital to research the resistance mechanism.Based on preliminary experimental study of root differential proteome of cucumber resistant and susceptible strains under blight stress,a resistance-associated protein has been identified in resistant varity and annotated as a member of LRR-type protein family in the NCBI, temporarily named as CsLRR-pf1 with the CDS length of 1317 bp (GenBank accession numbers:gi| 449450244). After cloning the cucumber CsLRR-pf1 coding sequence and promoter, gene expression patterns of CsLRR-pf1 are analysed.At the same time, cucumber CsLRR-pf1 is constructed into prokaryotic expression vector pET30a and into fusion expression vector pBI121-gfp to lay the basis for reseaching on the role and mechanism of LRR-type resistance-related proteins in cucumber defense responsesAfter cucumber CsLRR-pf1 CDS region and promoter being cloned, analysis by PlantCARE online software for CsLRR-pf1 promoter region sequence shows the prediction of regulatory elements in sense and antisense strand of this gene promoter upstream before the ATG:a 5UTR Py-rich stretch:high level of expression associated components; a enhanced xylem and phloem petals expression repressor portion expression element AC-Ⅱ; four AREs: cis-acting regulatory element essential for the anaerobic induction; a AT-rich element:binding site of AT-rich DNA binding protein (ATBP-1); a ATGCAAAT motif:cis-acting regulatory element associated to the TGAGTCA motif; a AuxRR-core:cis-acting regulatory element involved in auxin responsiveness; a Box-W1:fungal elicitor responsive element; two G-Box, two GA-motif, one GATA-motif, a GT1-motif,eleven MNF, two Spl:light-responsive elements; two HSE:cis-acting element involved in heat; stress responsiveness; two MRE: MYB binding site involved in light responsiveness; 37 CAAT-box:common cis-acting element in promoter and enhancer regions; 88 TATA-box:core promoter element around -30 of transcription start; four TC-rich repeats; defense and stress response element; a W box:wounding and pathogen-responsive element; a Skn-1-motif:elements required for endosperm expression; a circadian:cis-acting regulatory element involved in circadian control; a P-box:gibberellin-responsive element; CGTCA-motif and 2 TGACG-motif:cis-acting regulatory element involved in the MeJA-responsiveness. There is a substantially mean distribution of these regulatory elements in the promoter sequence.After Fusarisum oxysporum f.sp. cucumerinum infection, GA3, MeJA, SA, ETH and ABA treatments, CsLRR-pf1 gene expression patterns are being quantited to better understood by. Result shows CsLRR-pf1 gene is induced in F9(high resistance to bligh)and 995(high susceptibility to blight)after inoculated by Fusarisum oxysporum f.sp. cucumerinum while their expression peak is respectively in 11 days and 13 days. Substantial accumulation CsLRR-pf1 gene in F9 is earlier than in 995,which may be the main factor to their traits difference. At the same time it also suggests that CsLRR-pfl may participate in cucumber response to Fusarisum oxysporum f.sp. cucumerinum; CsLRR-pfl gene is induced and it presents the same change trend in F9, treated by GA3, MeJA and SA, perhaps it is due to the crosstalk of SA, GA3, MeJA signaling transduction pathways and coordination with each other in the induction process of plant defense response.While treated by ABA and ETH in F9 and 995, there is the similarity and dissimilarity in the expression pattern of CsLRR-pfl genes in response to different treatmens, suggesting that the biological mechanism of plant in response to different environments is extremely complicated.By colony PCR identification and Nde Ⅰ/HindⅢ double digestion, prokaryotic expression vector of cucumber CsLRR-pfl was successfully constructed. At the same time, by colony PCR identification and Xba Ⅰ/IBamH Ⅲ double digestion, this gene was successfully constructed with GFP fusion expression vector, laying the foundation for the subsequent research on the role of the LRR-type resistance-related proteins in stress responses.