| Oxylipins are a class of oxidized fatty acids with diverse strucutres,which participate in the process of plant disease resistance through direct bactericidal action or producing defense signal substances.Oxylipins are catalyzed by the lipoxygenase(LOXs)in the fatty acid oxidation pathway.The previous transcriptome data has confirmed that some LOX-related genes were up-reg?Lated during the inoc?Lation of Botrytis cinerea,indicating that these genes may be involved in the defense response of Physcomitrella patens against Botrytis cinerea.However,the specific mechanism of this has not been reported yet.So,using Physcomitrella patens as the research material,this paper investigated the possible mechanism of LOX in the interaction between Physcomitrelle patens and Botrytis cinera.The conclusions are as follows:1.By bio-informatics analysis,it was found that Physcomitrella patens contains 8 lipoxygenase genes,the length of whose protein amino acid is between 640-840 aa;the sub-cellular location showed that except for Pp Lox7 located in the chloroplast,all others were located in the cytoplasm;most members of lipoxygenase have 7-8 introns,the secondary structure of whose protein is mainly composed of randomcoils and?-helices;and these members were scattered on 7 chromosomes,whose number and function continued to evolve in the course of evolution.2.By RT-PCR to analyze the transcription level of LOX-related genes after their infection of Botrytis cinerea,it was found that Pp Lox1and Pp Lox3 were significantly different at 24h and 6h respectively;PpLox2 was extremely different at 48h and 72h;Pp Lox8 was very different at 72h;other related genes had no significant differences.3.Nordihydroguaiaretic acid(NDGA)at a concentration of5×10-3m M and a soaking time of 3h has an inhibitory effect on the enzymatic activity of lipoxygenase by about 50%.After treating Physcomitrella patens plant with the above reagent and inoculate it with Botrytis cinerea for 3 days,then stain it with trypan blue,it was found that the number of invaded hyphae of the plant cells in NDGA treated plants was higher than that of the wild type.The relative quantification of hyphae also confirmed that the number of invading hyp Hae on the 3rd day of inoculation was about 3.5 times that of the wild type;after staining the phenolic substances by Safranin,it was found that the cell wall space and leaf vein in NDGA treated plants were stained deeper than that of the wild type,which indicated that the phenolic substances accumulated more in NDGA treated plants.Based on this,it is speculated that LOX may participate in the defense response of Physcomitrella patens against Botrytis cinerea by affecting the accumulation of phenolic substances.4.Detect the enzymatic activity of phenylalanine ammonia lyase(PAL),chalcone synthase(CHS)and cinnamyl alcohol in two plants(NDGA,WT)after their inoculation of Botrytis cinerea(0h,6h,12h,24h,48h,72h).The results showed that the PAL enzyme activity of WT plants increased first and then decreased after inoculation,the highest at 12h,186(OD290/min/g FW),the lowest at 72h,15(OD290/min/g FW).After NDGA treatment,the PAL enzyme activity was significantly lower than that of the control group during 0-24h.The CHS enzyme activity was significantly reduced after NDGA treatment compared with the control group within 6 to 72 hours,which was reduced by about 40%after 72hours of inoculation.The activity of CAD enzyme at 6-24h after NDGA treatment was different from that of the control group.The changes in the above enzyme activities indicate that LOX may participate in the defense reaction of Physcomitrella patens against Botrytis cinerea by affecting the synthesis of phenylpropanes,flavonoids and other substances.5.The changes of SA and ABA content of the two materials after inoculation with Botrytis cinerea were determined by high performance liquid chromatography.The results showed that the contents of SA and ABA in NDGA treated Physcomitrella patens decreased within 0-72h compared to the control group,while the wild type increased.In the process of treating NDGA plants with the above two hormones at the same time,the phenotype of the plants was restored,and the number of hyphae was reduced,which was similar to that of the wild type.All these showed that LOX may participate in the stress response of Physcomitrella patens to Botrytis cinerea by synthesizing two signal substances,SA and ABA,which increased plant resistance to pathogenic bacteria.6.The Pplox2 gene knockout vector was constructed,which was transformed into the protoplasts of Physcomitrella patens via polyethylene glycol-mediated protoplast transformation to obtain Pplox2-PTN182 mutants.In summary,the preliminary study on the disease resistance mechanism of the LOX gene family in Physcomitrella patens has laid the foundation for the follow-up in-depth study of the disease resistance mechanism of plants. |
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