Cloning And Functional Analysis Of Chitinase Gene EuCHIT30.7 From Eucommia Ulmoide

The study of molecular mechanisms of plant-microbe interactions,mainly including pathogenicity and plant resistance,is essential for the development of new or safer strategies for effective plant disease control.Based on the previous studies on rubber granule protein identification in our laboratory,the gene Eu CHIT30.7containing peptide-VYLSAAPQCVFPDAHLDR-was screened,and the full sequence of this gene was identified from the whole genome annotation database of Eucommia ulmoides constructed in our laboratory for cloning and identification studies to analyze the function of this gene,and the following results were obtained:1.Eu CHIT30.7 gene cloning and sequence analysisIn this study,the full-length c DNA sequence of the Eu CHIT30.7 gene was cloned by the RACE technique,with a length of 1069 bp,open reading frame ORF867 bp,5’UTR 44 bp,and 3’UTR 158 bp,encoding 288 amino acid residues.The results of the raw signal analysis showed that the protein encoded by this gene belongs to the GH18 family,does not contain introns,is an acidic stable hydrophobic protein,contains a signal peptide,and does not contain a transmembrane structural domain.2.Eu CHIT30.7 Spatio-temporal expression analysisThe results of spatial expression analysis showed that the expression of this gene was significantly tissue-specific,with the highest relative expression in male flowers.The results of temporal expression analysis showed that the expression of the gene was different in different parts of the plant in different months,indicating that the expression of the protein encoded by the gene was influenced by the environment during the growing period,and the expression of the gene was significantly higher in the pericarp from July to October than in other parts of the plant.It is assumed that this gene is related to the flowering and fruiting process of Eucommia.3.Eu CHIT30.7 subcellular localization analysisRCR amplified the coding sequence of Eu CHIT30.7,constructed the fusion protein vector p CAMBIA1300-Eu CHIT30.7-EGFP,and demonstrated that the protein encoded by this gene is located in the cell membrane and cytoplasm by transient expression in Nicotiana tabacum leaf epidermal cells.4.Eu CHIT30.7 upstream regulatory sequence analysisPredictive analysis of the 2000bp promoter upstream of the Eu CHIT30.7gene using a laboratory-constructed genome-wide annotation database revealed the presence of light-responsive,abscisic acid-responsive,methyl jasmonate-responsive,anaerobic-inducible,ethylene-responsive,drought-inducible,trauma-and pathogen-responsive,and circadian-rhythm-regulating response elements in the upstream sequence.When 100μmol·L^-1Me JA was applied to three-month-old juniper seedlings at the 5-leaf stage,Eu CHIT30.7 gene expression was highest at 24 h,2.97times higher than that of untreated,demonstrating that Eu CHIT30.7 gene expression could be induced by Me JA;whereas when Eucommia ulmoides seedlings were sprayed with 200μmol·L^-1ABA,Eu CHIT30.7 gene expression decreased,and at 12 h The expression of Eu CHIT30.7 gene was reduced by spraying with 200umol/L ABA,and the expression at 12h was 0.52 times that of untreated Eu CHIT30.7 gene,indicating that the gene expression was inhibited by ABA.The expression of the gene was significantly increased at 6 h,16.4 times higher than that of untreated seedlings,and then gradually decreased,indicating that the gene could respond to low-temperature induction,but the expression was inhibited again with time.5.Analysis of Eu CHIT30.7 for resistance to tobacco powdery mildewThe plant overexpression vector p CAMBIA1301-35S-Eu CHIT30.7 was constructed and 32 trans-Eu CHIT30.7 plants were obtained from Samsung tobacco by using Agrobacterium-mediated genetic transformation of leaf disc method.The results showed that after 12d of powdery mildew inoculation,the relative spot area of trans-Eu CHIT30.7 plants was 11%and 12%of that of wild-type and trans-empty plants,respectively,and the number of powdery mildew spores was 28%and 29%of that of wild-type and trans-empty plants;the SOD,POD and CAT activities of trans-Eu CHIT30.7 tobacco increased and then decreased after powdery mildew infection The expression of PR1a,PR2,PR5,MLP423 and PDF1.2,a key gene of the jasmonic acid pathway,was significantly higher in Eu CHIT30.7 tobacco than in wild-type and empty vector tobacco,while the expression of MLO2,a gene related to powdery mildew resistance in tobacco,was significantly higher in Eu CHIT30.7tobacco than in wild-type and empty vector plants.The expression of MLO2 was also significantly higher after inoculation,and was 10.08 and 10.11 times higher than that of wild-type and empty-vector tobacco,respectively,at 72h.This indicates that overexpression of Eu CHIT30.7 gene enhances the suppressive effect of tobacco plants against powdery mildew,not by suppressing the expression of MLO genes related to powdery mildew resistance,but by increasing the ability of tobacco to scavenge reactive oxygen species,improving protective enzyme activity and increasing the expression of key genes in salicylic and jasmonic acid-dependent signaling pathways in the plants.6.Eu CHIT30.7 and isoprene-like anabolismThe expression of Eu HMGR,Eu GGPPS,and other genes related to gum synthesis in Eucommia ulmoides was significantly increased in TP plants compared with wild type by using Agrobacterium-mediated hypocotyl transformation of Eucommia ulmoides and introducing Eu CHIT30.7 gene overexpression,indicating that the expression of Eu CHIT30.7 gene is closely related to isoprene-like anabolism in Eucommia ulmoides.