Preliminary Study On The Function And Expression Localization Of AsA8and Nip43Involved In Symbiotic Nitrogen Fixation In Astragalus Sinicus

With two genes AsA8and nip43of Astragalus sinicus which are supposed to be involved in symbiotic nitrogen fixation, considering the previous basis and research goals, this study has carried out the continuous laboratory work in two aspects, which can be summarized as follows.Firstly, in our previous work, a valid fragment sequence of AsA8was isolated from a cDNA library of A. sinicus nodules. Homology comparison showed its amino acid had a high similarity to12-oxophytodienoic acid reductase, which plays a critical role in jasmonic acid biosynthesis pathway. It is hypothesized that this gene would be involved in nodule formation and development, but further study is necessary to analyse the biological function of AsA8in symbiotic nitrogen fixation. In the present continuous work, some progresses and results have been obtained.1. By using RACE method, a full length sequence of AsA8was obtained. Homology comparsion and phylogenetic analysis confirmed that AsA8encoded12-oxophytodienoic acid reductase.2. By using Western Blotting, it was found that AsA8mainly expressed in the infected roots and nodules of A. sinicus. A further immunolocalization experiment was conducted to determine the cellular localization of AsA8in nodules. The results showed that fluorescence signals were detected in both infected cells and plastids of the uninfected cells, which sugguested that these sites possess capacity of JA biosynthesis.3. To investigate whether AsA8affects root nodule development, A. sinicus roots were transformed with AsA8-RNAi and overexpression constructs respectively. Through the observations of symbiotic phenotypes for these transformed plants, it was found that when AsA8expression decreased, the root nodules number in the transgenic roots significantly decreased. Meanwhile, the transgenic plants with overexpressed-AsA8grew better, and the morphology and size of corresponding nodules changed remarkably. The results indicated that AsA8would be involved in nodulation and nodule development. Secondly, a target protein Nip43interacting with NopP, an â…¢-type-secreted effector protein in Mesorhizobium huakuii7653R, has been isolated via yeast two-hybrid system. More evidences are needed to verify the interactions of NIP43-NopP in plant living cells.4. By using bimolecular fluorescence complementation (BiFC) method in this study, it was confirmed that NIP43could interact with NopP in tobacco plant living cells. Two proteins interacted at the cell membrane. The critical region of NIP43required for the interactions with NopP was determined to the N terminal by constructing a partial deletion vector for NIP43.5.In addition, nip43-targeting RNAi and overexpression vectors were constructed, which provided a good basis for future study on the function of nip43in nodulation and symbiotic nitrogen fixation.