| The effect of heavy metal stress on symbiotic nitrogen fixation is mostly study in phenotype observation and physiological data analysis,and its molecular mechanism is rarely reported.In this study,Medicago lupulina was used as a research object.The differentially expressed genes of Medicago lupulina under copper stress were obtained by RNA-seq in the early stage of the experiment.And the genes expression were analyzed under copper stress and in symbiotic process as well as the relationship between copper stress and symbiosis.At the same time,RNAi technique was used to identify the chitinase gene Cu33611,which was related to copper stress and was specifically expressed in the inoculated root.The contents of the study and the results are summarized as follows:1.The cysteine cluster protein(CCP)is thought to be associated with defense and symbiosis.The expression of 9 CCP genes was divided into three groups: ML68936_c0 and ML94029_c0 were expressed and reached the highest level at 7dpi(day past inoculation),and then down-regulated,reach the highest at 30 pdi,mainly involved in the early and late stage of nodule formation;The expression level was high in the middle stage of nodule formation,reached the highest at 30 days,which may play a role in the middle and late stage of nodulation(ML67778_c0?ML85596_c0?ML98221_c0?ML101635_c0 and ML68801_c0).The expression level was higher in the late stage of nodule formation,and the expression level was not expressed or at very low level in the other detection time points,which may play a role mainly in the late stage of nodule formation(ML54082_c0 ? ML64613_c0 and ML90197_c0).2.Expression analysis of genes related to ethylene synthesis and signal transducti on showed that the genes related to ethylene synthesis ML98991_c3(1-aminocycloprop ane-1-carboxylate oxidase,ACO)and ML93355_c1(1-aminocyclopropane-1-carboxylat e synthase3,ACS)were different induced in nodule formation,ML98991_c3 was mainl y inhibited in the late stage of nodule formation,while ML93355_c1 was inhibited at10 and 30 dpi under copper stress.One ethylene receptor gene ML46877_c0 and oneEIN3-targeted F-box gene(ML46356_c0)were induced in different stage of nodule for mation well the other ethylene receptor gene ML46877_c0 was inhibited at the late sta ge of nodule formation.In addition,the expression of ML42013_c0?ML99097_c0?ML99511_c0?ML85546_c0?ML98388_c0 were expressed in roots and nodules at 30 dpi.I n addition to ML99097_c0,the expression of the other four genes was inhibited in th e roots and nodules.3.The genes involved in Cu stress and nodule formation may play an important role in the process of Cu transport in plants.The gene expression analysis of the selected three related genes shows that under the Cu stress condition,ML98670_c0 ? ML52550_c0expression may be related to the uptake and transport of Cu under the condition of Cu deficiency.The expression of ML98423_c0 is up-regulated and may be related to the detoxification of Cu under stress.The three genes were up-regulated after inoculation,which may be related to the increase in the demand for Cu in nodules.The expression of ML85004_c0 and ML89530_c0,which are related to the two macrophages,are relatively high in the nodules.4.The result of RNAi showed that the plant was shorter,the number of nodules was reduced and the nodules was smaller after Cu33611 silencing.The infection event of 10 dpi after Cu33611 silencing showed that the root hair curling and the number of infected lines were significantly decreased compared with the control.The number of nodules was much lower than that of the control nodules,the infected cells and the nitrogen fixation areas were reduced and nodulaton were significantly affected.These results suggest that Cu33611 is involved in the regulation of the nodule formation well its specific function needs further study... |
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