Cloning, Expression And Mechanism Of Action Of Plant The Elicitors The Protein Harpinn <sub> Csds001 </ Sub>

Harpins are a class of proteins which is ecoded by hrpN (hrpZ,hrpW) andproduced in nature by certain plant Gram-negative bacteria and some animalpathogens. They cause hypersensitive response (HR) of plants and elicit a complexnatural defense mechanism. There are homolog and wide diversity among the hrpgenes. This work began with the isolation and characterization of wild type bacteriaErwinia carotovora subsp.carotovora strains CSDS001, then the genomic libraries ofthe CSDS001 was constructed and the hrpN_CSDS001 gene was cloned. HarpinN_CSDS001protein was overproduced in E.coli JM109. The biological activities andmultifunctionality of HarpinN_CSDS001 and HarpinN_CSDS001-Zn were tested and analyzedby series controlled experiments. Potential mechanism of inducing systemic acquiredresistance (SAR) and promoting plant growth by HarpinN_CSDS001 andHarpinN_CSDS001-Zn was explored by analyzing Arabidopsis treated by the two proteinsby Genechip. The main results are:1,CSDS001 was isolated from soft-rotting Chinese cabbage. It caused soft rot inChinese cabbage and celery, and produced exoenzymes such as Pel,Peh,Cel,Prt. 2×10⁸ cfu/ml CSDS001 inoculated into tobacco leaves causing HR.2,CSDS001 genomic library was constructed in vector pLARF5, harbored in E.colistrain HB101 and contained 1000 colonies. Each colony possessed 20—30kb ofchromosomal DNA. Southern blot revealed 1.5-kb EcoRI segments containinghrpN_CSDS001.3,The hrpN_CSDS001 nucleotide sequence is 1071bp. Registered number is AY939927.HarpinN_CSDS001 contains 556 amino acids and its molecular weight is 35.55kD andpI is 5.5.4,pT7-hrpN_CSDS001, in protein over expression vector, was constructed by cloninghrpN_CSDS001 into pET—28a (+). Then the pT7-hrpN_CSDS001 was transferred intoE.coli strain JM109. IPTG induced hrpN_CSDS001 to over express.5,The over expressed protein was identified as HarpinN_CSDS001 by Western blot.Northern blot analysis showed the expression of hrpN_CSDS001 were influenced by pH,carbon and nitrogen source.6,Over expressed HarpinN_CSDS001 formed as inclusion body and can elicit HR intobacco leaves. It means this protein in inclusion body form also had biologicalactivity.7,In some conditions, HarpinN_CSDS001 chelated with Zn²⁺ and formedHarpinN_CSDS001-Zn, which had the same biological activity-eliciting HR intobacco leaves.8,30mg/L HarpinN_CSDS001-Zn and 30mg/L HarpinN_CSDS001 were applied oncapsicum,aubergine,cucumber,potato,and tomato respectively. The resultsshowed that the yields increased by HarpinN_CSDS001—Zn were 13.69%,16.52%,18.86%,11.55%,11.64% respectively and by HarpinN_CSDS001 were 11.73%,13.27%,16.3%,8.26%,10.25%. Statistical analysis of the plant disease,growthand development showed that both HarpinN_CSDS001—Zn and HarpinN_CSDS001 mayinduce plant defense, boost growth and promote development, moreoverHarpinN_CSDS001-Zn had more powerful biological functions than HarpinN_CSDS001.9,Based on the cluster analysis of mRNA level tested by Genechip of Arabidopsistreated by HarpinN_CSDS001, 52 genes (clustered into five groups) were regulated in3h,12h,24h,36h and 48h, and the fourth and fifth group genes related to thefunctions of plant defense,growth and development.10,Analysis of genes which expression changed significantly (log ratio≤1 or≥1) showed that after treatment with HarpinN_CSDS001 at 3h,12h,24h,36h and 48h,there were 912,1787,2393,1833 and 1755 genes correlatively changedsignificantly. The results revealed that the level of mRNA regulation byHarpinN_CSDS001 increased gradually, most of the reaction happened at 24h andcontinual physiological responses also were drawn out.11,Analysis of transcription factors (TF) families showed that 13 TF familiesresponded to HarpinN_CSDS001. They were ZIM,BES1,TCP,C2C2,AP2/EREBP,WRKY,bHLH,bZIP,GARP,MYB,NAC,HB,C2H2. The families mainlyregulate reactions about plant defense, photosynthesis, growth, development andflowering. 12,Used KEGG2, provided by http://www.genome.ad.jp/ to analyze theflavonoid biosynthesis pathway revealing that chalcone synthase (CHS) gene andflavanone 3-hydroxylase (F3H) gene expression increased.13,24h after treatment by HarpinN_CSDS001-Zn to Arabidopsis, there were 2741genes regulated significantly, 35 TF families responding to HarpinN_CSDS001-Zn andthe chalcone synthase gene and flavanone 3-hydroxylase gene expression alsoincreased. The results indicated that HarpinN_CSDS001-Zn had more powerfulbiological function than HarpinN_CSDS001. It was consistent with the results ofcontrolled experiments in the fields.