Genes Regulating Nitrogen Fixation Ability of Nodulated Plants and Their Applications
A plant and root nodule technology, applied in the field of plant molecular biology, can solve the problems of little understanding of rhizobia infection
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[0058] As an embodiment of the present invention, the gene encoding the RINRK protein is cloned into an appropriate vector by conventional methods, and the recombinant vector with the foreign gene is introduced into the plant cells that can express the RINRK protein , making the plant cell express RINRK protein. Plants overexpressing the RINRK protein can be obtained by regenerating the plant cells into plants. Preferably, the RINRK protein coding gene or antisense gene is transferred into plants by using the Agrobacterium transformation method.
[0059] As used herein, the forward connection refers to: the connection between the coding gene of RINRK and the expression vector is a positive connection, that is, the coding gene is connected to the vector in the direction of 5'→3'. Usually, the coding gene of RINRK is located downstream of the promoter in the expression vector, that is, the 3' end of the promoter is connected downstream to the 5' end of the coding gene. The cod...
Embodiment 1
[0066] Example 1, RINRK encodes an LRR receptor kinase
[0067] In the present invention, the model leguminous plant lotus japonicus is mutated with ethyl methanesulfonate, and a mutant with defective infection line is screened. The phenotype of the mutant was observed and counted in detail.
[0068] 1. Observation of symbiotic phenotype of rinrk-1
[0069] The mutant seedlings (seedlings 7 days after germination) were inoculated with Rhizobium bacterium Mesorhizobium lotiR7A from japonicus japonicus. After three weeks, there was no significant difference between the wild type and mutant plant growth ( figure 1 , A). Observation of its root nodules found that the wild type can form pink root nodules that can fix nitrogen ( figure 2 , A), while the mutants at the same period only had the formation of white root nodule organs ( figure 2 , A). After analysis of these root nodule sections, it was found that rhizobia could colonize normally in the wild type, but no rhizobia...
Embodiment 2
[0083] Embodiment 2, the research of RINRK expression pattern
[0084] Nodulation factor (NF) is lipochitosan isolated and purified from rhizobia M.loti R7A. Here, the inventors sprayed NF evenly on the plant roots, and after 12-24 hours of treatment, the plant roots were taken and RNA was extracted.
[0085] The present inventors analyzed whether RINRK is induced by rhizobia or nodulation factors by using fluorescent real-time quantitative PCR. Studies have shown that the expression of RINRK begins to increase after 12 hours of treatment with nodulation factor (NF), and the expression level further increases after 24 hours, which indicates that nodulation factor can induce the expression of RINRK ( Figure 8 A). After inoculation with rhizobia M.loti R7A, the expression of RINRK was also significantly up-regulated ( Figure 8 B), which shows that both nod factor and rhizobia can induce the expression of RINRK.
[0086] In order to further explore the spatiotemporal expres...
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