The Function Of A Cysteine Proteinase Gene, Asnodf32, In Astragalus Sinicus Nodule Senescence

Asnodf32,a late nodulin gene in Astragalua sinicus,was firstly isolated by Natio et al..It encodes a nodule-specific cysteine proteinase.Its transcription level increased in mature nodules.The result of in situ hybridization showed that it was located in the inter zone and the senescence zone of mature nodule.So Natio suggested that AsNODF32 was linked with nodule senescence.But it is a hypothesis and has not been proved. RNA silencing was applied in A.sinicus to testify this hypothesis and investigate the relationship between Asnodf32 and nodule senescence.At the same time,tissue culture and transformation systems ofA.sinicus were established.The efficiency of silencing was detected by real-time RT-PCR.,the nodules transformed with the empty vector were used as the control.The result indicated that Asnodf32 was silenced effectively in nodules harvested at 30,45 and 60 days after inoculation. RNA silenced nodules should some differences with the control nodules in symbiotic effect and internal structure.For the symbiotic effect,the difference in average nodule length between the two samples was significant at the harvest time of 60 days after inoculation.Nitrogenase activities in nodules at various harvest times were estimated by acetylene reduction activity.After incolated for 60 days,nitrogen fixation capacity of control nodules clearly declined compared with it in nodules harvested at 45 days after inoculation.While the nitrogenase activity of control nodules just decreased slightly.For the internal structure,Microscopic analysis of nodule paraffin-embedded slides showed that at the time of 60 days after inoculation,up to 80%control nodules senesced seriously.Moreover,the senescence zone enlarged to the middle of these nodules.In contrast,the silenced nodules were normal with small senescence zones.DNA fragmentation in both plant cell nuclei and bacteroid nucleoids were analysed by TUNEL method.It was found that DNA fragmentation of the bacteroid nucleoids was observed in half of the control nodules at the time of 60 days.While in the silenced nodules,no signals indicated bacteroid fragmentation.Further identifications were carried out by TEM.At the time of 60 days after inoculation,the nodules of control senesced seriously.. But in the silenced nodules,most nodule cells were normal. The results above indicated that silencing of Asnodf32 delayed nodule senescence, suggesting the importance of this gene and its product in the regulation of nodule senescence.Many cysteine proteinases were up-regulated under stress conditions,so they were regared to involve in the responses of plants against stresses.Here the transcript levels of Asnodf32 in wild-type nodules under the stresses such as NaC1,(NH₄)₂SO₄ and dark were investigated.After the treatment with 80 mmol/L NaCl for 3 days,the change of Asnodf32 mRNA was small.While 3-day treatment of 1 mmol/L(NH₄)₂SO₄ and dark made 2.5 and 5-fold accumulation in Asnodf32 mRNA,respectively.These results implied that Asnodf32 or its product was likely involved in plant responses under stresses.