Functional Study On AsIA257 And AsIB259 Genes Related To Symbiotic Nitrogen Fixation Of Astragalus Sinicus

The AsIA257 is a nodule-specific expressed diphosphonucleotide phosphatase, which was firstly isolated from A. sinicus by suppressive subtractive hybridization technique. Amino acids composing the putative active center of PPD1 from yellow lupin (Lupinus luteus L.) are similar to those present in known purple acid phosphatases (PAPs), which suggests that AsIA257 genes might encode a novel group of specific metallophosphatases.In this stuy, by using real-time quantitative PCR, the temporal-spatial exprseeion characteristics of target genes in A. sinicus symbiotic conditions and some stress conditions were testigated.And DsRed-AsIA257 fusion was constructed to perform the subcellular localization of AsIA257. Importantly, we have got overexpressing AsIA257 plants by hairy root transformation in A. sinicus and investigate the total number of nodules.Here, AsIA257 cDNA was cloned.Upon M. huakuii infection, AsIA257 expression was induced starting from 9 day after inoculation and reached the highest level at 27 dpi. AsIA257 was only expressed in the mature nodules and roots rather than in other organs or tissues. The transcript levels of AsIA257 in wild-type nodules under the stress such as NaCl,(NH4)SO4 Pi and dark were investigated.The expression levels of AsIA257 was significantly suppressed under NaCl,(NH4)2SO4 and dark treatment. However, the transcript levels of AsIA257 were significantly increased under Pi stress.It might related to phosphorus metabolism. In addition, DsRed-AsIA257 fusion was constructed to perform the subcellular localization of AsIA257, and it indicated the AsIA257 was putative membrane protein.The efficiency of overexpressing was detected by real-time RT-PCR,the nodules transformed with the empty vector were used as the control.The results indicated that AsIA257 was overexpressed effectively in nodules harvested at 15,27and 32 days after inoculation. The overexpressing plants were small and weak, the leave turn yellow, showing threefold less nodules with respect to control trangenic plants. The AsIB259 is specifically expressed nsLTP1 genes in A. sinicus root nodules and related to symbiotic nitrogen fixation.In this suudy, transgenic roots overexpressing AsIB259 developed more nodules with respect to control roots.It still need futher experimental data to convince. In this work, some substantial and convincing data were obtained to confirm that AsIA257 was specifically expressed PPD gene in A. sinicus root nodules,and related to symbiotic nitrogen fixation and likely participate in phosphorus metabolism.It provides good experimental basis for their function and mechanism in symbiosis and nitrogen fixation between A. sinicus and M. huakuii.