The Study On The Mechanism Of Lilium Regale Wilson Chitinase Gene LrCHI2 Responding To Fusarium Oxysporum

Lilium regale Wilson is a unique wild lily species in China.It shows strong resistance to Fusarium oxysporum,which is the main pathogen causing lily root rot.It is an important germplasm resource for breeding disease-resistant lily cultivar and studying the molecular mechanism of lily disease resistance.In the previous analysis of the transcriptome of L.regale Wilson after infection by F.oxysporum,a pathogenesis-related protein（PR）gene LrCHI2 induced by F.oxysporum was identifie.The purpose of this study is to further understand the function of LrCHI2.The expression characteristics and subcellular localization was analyzed.The prokaryotic recombinant protein LrCHI2 was expressed and purified,and its inhibitory activity against several pathogenic fungi was analyzed.LrCHI2 was overexpressed in the model plant tobacco（Nicotiana tabacum）to verify its role in the infection of F.oxysporum.WRKY transcription factors often act as upstream regulators of PR genes in plant defense system,therefore,the promoter of LrCHI2was further cloned and the transcriptional regulation of LrWRKY2 on LrCHI2 was studied.The research work of this paper and the main results obtained are as follows:1.Based on the transcriptome sequencing results of L.regale infected by F.oxysporum,a L.regale chitinase gene was obtained and named LrCHI2.The full length of LrCHI2 is 1313 bp,which contains a 933 bp open reading frame（ORF）and encodes a protein with 310 amino acids.QRT-PCR analysis showed that LrCHI2 was expressed at a high level in roots and at a low level in other L.regale organs;the expression level of LrCHI2 responded to the treatment of ethephon（ETH）,salicylic acid（SA）,methyl jasmonic（MeJA）and hydrogen peroxide（H₂O₂）;the expression level of LrCHI2 also responded to the infection of F.oxysporum.2.The subcellular localization vector pBIN m-gfp5-ER-LrCHI2 was constructed and transformed onion（Allium cepa）epidermal cells.After co-culture,the LrCHI2-GFP fusion protein was observed to be located on the cell wall under laser confocal microscope.The prokaryotic expression vector p ET-32a-LrCHI2 was constructed and transformed into E.coli BL21（DE3）to express LrCHI2 protein.The results showed that the recombinant protein LrCHI2 inhibited the growth of F.oxysporum,Colletotrichum gloeosporioides,F.solani and Alternaria panax.The plant overexpression vector p CAMBIA2300s-LrCHI2 was constructed and overexpressed in tobacco to obtain the T2 generation LrCHI2 transgenic tobacco.QRT-PCR results showed that LrCHI2 was stably expressed in T2 generation transgenic tobacco lines.Leaf inoculation experiment showed that transgenic tobacco had strong resistance to F.oxysporum.3.The promoter of LrCHI2 was cloned by genome-walking.The analysis of cis acting elements showed that the promoter contained a cis acting element w-box.Electrophoretic mobility shift assay（EMSA）results showed that the recombinant protein LrWRKY2 bound to the probe containing the W-box fragment of LrCHI2promoter in vitro.The bait vector pAbAi-pLrCHI2 and prey vector pGADT7AD-LrWRKY2 were constructed for yeast one hybrid（Y1H）experiment.The results showed that LrWRKY2 had a transactivation effect on the LrCHI2 promoter.The LrCHI2 promoter was fused toβ-glucuronidase（GUS）reporter gene to constructed pLrCHI2-GUS plant expression vector,and then the pLrCHI2-GUS vector was transformed into LrWRKY2 overexpressed transgenic tobacco and wild type（WT）tobacco,respectively.GUS quantitative fluorometric assays showed that the LrCHI2promoter responded to several abiotic（Sodium chloride（Nacl）,Cadmium Chloride（Cdcl₂）and wound treatments）and biotic stresses（F.oxysporum,F.solani and Nigrospora oryzae infection）,as well as MeJA treatment.In addition,compared with the single pLrCHI2 transgenic tobacco lines,the GUS activity of pLrCHI2 and LrWRKY2 co-expressedg transgenic tobacco lines was significantly improved.These results showed that LrWRKY2 can positively regulate the activity of LrCHI2promoter.In summary,LrCHI2 responds to plant signal molecule treatments and F.oxysporum infection.The prokaryotic recombinant protein LrCHI2 has obvious inhibitory activity against several pathogenic fungi including F.oxysporum.Overexpression of LrCHI2 gene in tobacco confers resistance to F.oxysporum.In addition,MeJA treatment,biotic and abiotic stresses can enhance the LrCHI2promoter activity.LrWRKY2 positively regulates the transcriptional activity of LrCHI2.The above results indicate that LrCHI2 is regulated by WRKY transcription factors and plays an important role in the defense of L.regale against F.oxysporum.