Cloning Of Singlet Oxygen（¹O₂） Marker Genes Of Ageratina Adenophora And The Role Of ¹O₂ Signal During TeA-Caused Weed Death

Previously,our team obtained Alternaria alternata（Fr.）Keissler by single spore isolation technology,and the main pathogenic factor of this fungus was confirmed to be TeA.Further research found that TeA is a new type of PSⅡinhibitor,which significantly inhibits photosynthesis and causes disease by blocking the PSⅡelectron flow from Q_Ato Q_B.In addition,weeds that are difficult to manage such as Commelina communis and C.bengalensis are extremely sensitive to TeA,but plants such as Gossypium spp and Tagetes erecta L.show high resistance to it.The mechanisms of interaction between the toxin TeA and plants are different.In Arabidopsis,the toxin TeA activates the EXECUTER dependent¹O₂signaling pathway,but the role of the ¹O₂signal in the process of TeA killing the host plant Ageratina adenophora is still unclear.At the same time,it has been preliminarily determined that there is a cross-talk between the ¹O₂signal and the JA signal during the process of A.alternata pathogen inducing diseases in mature Arabidopsis plants.Thus,what is the relationship between the¹O₂signal and the JA signal during the killing of A.adenophora by TeA?With the experiment progress,it was found that the infection effect of TeA on A.adenophora in winter was far worse than that of in summer and warmer spring and autumn.The size of disease spots was reduced,the time of disease appearance was delayed,and the expression level of ¹O₂marker gene was decreased.It is unknown that if the ¹O₂signal is affected and how it can be affected during the TeA-caused weed death under low temperature stress pretreatment conditions.This thesis determined gene expression levels,SOSG to position ¹O₂,and JA contents to solve the problems raised through gene cloning,Imaging-PAM,q RT-PCR and HPLC-MS methods.The homologous cloning method was used to obtain conserved region sequences of the following genes in A.adenophora:Aa WRKY33（560 bp）,Aa Ferritin（362 bp）,Aa EX1（1743 bp）,Aa LOX3（1436 bp）,Aa AOS（986 bp）,Aa OPR2（501 bp）and Aa AOC3（332bp）.Due to the high similarity of sequence homology comparison results in the NCBI of database,the above conserved sequences can be used for q RT-PCR primers design.,and also provide the basis for the cloning of full-length c DNA by RACE method.Our results showed the full-length c DNA sequences of Aa AAA-ATPase and Aa WRKY33 were 1943 bp and 1802 bp in length,respectively.The sequence homology in the NCBI database showed a better comparison with the sequenced Compositae plants,establishing good experimental basis for subsequent functional research and applications of the Aa AAA-ATPase and Aa WRKY33 genes.In order to explore the role of the singlet oxygen signal in the interaction between TeA and A.adenophora and its relationship with the JA signal,we used Imaging-PAM to monitor the diseases of leaves at different time points under TeA treatment.It was shown that the disease began to appear at 1.5 h,and the disease further aggravated with the increasing infection time.At 12 h,the TeA-infected spots reached to 26 mm².Subsequently,the level of ¹O₂production was determined by SOSG dye.It was found that ¹O₂was produced in the leaves at 0.5 h,and with the infection time increased,the level of¹O₂production further increased.The expression levels of the ¹O₂marker gene were tested by q RT-PCR.It was suggested that TeA quickly activated the ¹O₂signal in A.adenophora,and the¹O₂signal reached to the peak at 3 h.The JA synthesis level under TeA treatment at different time points was measured by HPLC-MS.Results showed that the JA content increased with TeA infection and reached the highest level at 3 h,which was 238.24 ng/g FW,about 3 folds of normal conditions.At the same time,the expression levels of JA-related genes were detected,and it was found that JA signal can be activated by TeA quickly and reached to the peak at 3 h in A.adenophora,which was consistent with the ¹O₂signal.These results indicated that TeA activates the¹O₂signal in plant cells,leading to the disease aggrevation.There is a synergistic relationship between the ¹O₂signal and the JA signal,and the production of ¹O₂promotes the biosynthesis of JA,which in turn induces cell death.In addition,to explore the effect of low temperature stress on the pathogenicity of TeA and the influence of the ¹O₂signal,the expression level of the ¹O₂marker gene Aa AAA-ATPase after low temperature pretreatment was first detected.It was found that the¹O₂level increased after the treatment,but did not cause disease.Subsequently,the disease development of TeA-infected low-temperature pretreatment plant leaves were monitored by Imaging-PAM.It is indicated that the low-temperature pretreatment significantly reduced plant leaf disease,and the disease appearance time was postponed.The size of the leaf lesion was only 9.72 mm²at 12 h,which is only 0.37-fold of the unpretreated.Besides,the expression levels of¹O₂marker genes and JA-related genes in TeA-infected low-temperature pretreatment A.adenophora were determined.It was found that low-temperature pretreatment down-regulated the gene expression levels.It is preliminarily clear that low temperature pretreatment induced tolerance of A.adenophora to TeA by the induction of ¹O₂-mediated executer-dependent signal.The transcriptome analysis of A.adenophora leaves treated with TeA for 3 h indicated that TeA inhibited photosynthesis,which in turn induced the production of intracellular¹O₂,and a significant increase in JA levels,resulting in cell death under the synergistic effect of¹O₂signal and JA signal eventually.In summary,we successfully cloned the full-length c DNA sequences of 2¹O₂marker genes（Aa AAA-ATPase and Aa WRKY33）,the conserved region sequences of H₂O₂marker gene Aa Ferritin,D1 protein related gene Aa EX1 and 4 JA related genes（Aa LOX3,Aa AOS,Aa OPR2 and Aa AOC3）in A.adenophora.It is proven in the study that TeA can lead to cell death and diseasesusceptibility by activating the ¹O₂signaling pathway in the host plants,and the ¹O₂signal was synergistically with the JA signal.In addition,low temperature promoted a low level of¹O₂solely,but decreased the level of¹O₂induced by TeA in A.adenophora,thereby reducing the disease.