| The plant SABATH methyltransferases are able catalyze both O-methylation and N-methylation of phytohormones and other small molecules with the formation of corresponding methylated ester.SABATH methyltransferases indirectly involved in signal transduction by catalyzing important plant hormones,such as salicylic acid(SA)and jasmonic acid(JA).SABATH methyltransferases play an important role in regulation of plant secondary metabolism,growth and development,and defense response.Great progresses have been made for jasmonic acid(JA)and methyl jasmonate(MeJA)in the regulation of flower spike development,signal transduction,fungal and pest defense responses.Studies have shown that the JA signal transduction and metabolism pathway can induce the rice defense system against brown planthoppers(BPH),but the regulation machanisms are still largely unknown.In this work,four rice SABATH methyltransferase genes induced by methyl jasmonate were selected and their roles in rice responses to biotic(BPH)and abiotic stresses were investigated by using molecular biological techniques.It provided basic results elucidate the biological function of SABATH methyltransferase genes in rice,and find the potential defense genes.Through gene chip analysis,four MeJA highly induced rice SABATH methyltransferase genes were found,and they were temporarily named as OsSAMT1,OsSAMT2,OsSAMT3,and OsSAMT4.The sequences of the four genes were obtained using molecular cloning technology,initial study the biological functions of the four genes in BPH induced defense response in rice,the preliminary work was carried out as follows:1、qRT-PCR analysis revealed that OsSAMTs were all induced by MeJA and expressed in different tissues of rice.OsSAMT1,OsSAMT2 and OsSAMT3 were mainly expressed in rice stems,OsSAMT4 was mainly expressed in floret.In addition,the expression levels of OsSAMTs gradually increased along with brown planthopper feeding in a certain period of time,and the induced expression level of OsSAMTs in a JA signal deficient rice mutant were significantly decreased.2、The sGFP subcellular localization vector and prokaryotic protein expression vector were constructed.OsSAMTs were found be located in the cytoplasm when transient expressed in rice protoplast cells.After 4 hours of IPTG induction,the expression levels of GST and His-tag fusion proteins reached the maximum amount,and formed as insoluble inclusion bodies.3、The promoter sequences about 2.5 kb upstream of the OsSAMTs gene were conneted with the GUS reporter gene,and the OsSAMTs:GUS transgenic lines were observed.GUS histochemical staining revealed that OsSAMT2/3/4 had different expression patterns in rice roots,stems,leaves,flowers and embryos.MeJA could significantly induce the expression of OsSAMT2 and OsSAMT3 in rice leaves.In addition,it was found that damage and feeding of brown planthoppers can also induce OsSAMT2 and OsSAMT3 expression in stems.It suggested that OsSAMT2 and OsSAMT3 may participate in the JA signal transduction pathway and the brown planthopper induced defense response.4、The over-expression and CRISPR/Cas9 vector of the OsSAMTs genes were constructed and transgenic plants were obtained.The over-expression of OsSAMTs were verified by PCR and obtain homozygous lines.The target region of the CRISPR/Cas9 transgenic plants was amplified for sequence analysis and homozygous mutation lines were obtained.Brown planthopper feeding test revealed that OsSAMT1 plays a positive regulatory role in the defense against brown planthopper in rice,while OsSAMT2 and OsSAMT3 play a negative regulatory role.5、Using the dual luciferase reporter system,it was found that OsMYC2,the key regulator of JA signaling pathway,can activate the expression level of OsSAMT2,but not the expression of OsSAMT3 and OsSAMT4 genes.In order to know whether the OsMYC2 regulates the expression of OsSAMTs through the JA signaling pathway,OsMYC2-Cas9 transgenic lines were constructed and obtained. |
