Isolation Of Copper Sterss-related Genes Involved In Symbiotic Nitrogen Fixation In Medicago Lupulina

ABSTRACTDue to the ability of nitrogen fixation and stress-resist ability of legumes---rhizobia symbiosis, it plays an important role in aiding phytoremediation of mining wasteland. The nitrogen shortage is a pivotal factor to the phytoremediation of some metal contaminated soils. So it’s very important to reveal the mechanism of legumes--rhizobia symbiotic system and improve the nitrogen content of metal contaminated. To carry out a preliminary discussion of mechanism of legumes--rhizobia symbiosis under copper stress, the infection process and internal structure of the nodule was observed by fluorescence microscopy and paraffin section. In this study, 137 genes related to copper stress was screened by RNA-seq combined with q RT-PCR and the symbiotic genes needs future screening.1. To observe the infection process in medicago lupulina, fluorescent protein gene was used to mark the rhizobia. The result shows that the starting time of infection has no significant difference at low concentration of copper(50mg/kg), but there is obvious delay in the beginning of infection at moderate or high copper level(100、150mg/kg). The statistics of root hair、infection threads and nodule primordia in inoculated root of medicago lupulina at 11 d shows that they have no significant difference at low copper level(50mg/kg), but the number of root hair、infection thread、nodule primordia decreased significantly at moderate or high copper concentration. These researches reveal that heavy metal-copper directly affect the legumes--rhizobia symbiosis. The microscopic observation of inter structure of nodule under different copper concentration shows that although there is no difference at low copper level compared to control group, the number of infected cell or nitrogen fixing zone declines significantly at moderate or high copper stress. At the same time, senescent zone tends to expand at moderate or high copper stress.2. According to result of RNA-seq and BLASTX description, the 137 genes were classified into 10 functional categories: putative protein(24%), Metabolism(23%), nodulin(13%), Transcription factor(12%), Binding protein(8%), Defense/stress response(5%), cell structure and maintenance(4%), energy(4%), Transport protein(4%), Hormone regulated(4%).3. 61 genes related to symbiosis under copper stress were screened by q RT-PCR in the future screening. According to result of RNA-seq and BLASTX description, the 61 genes were classified into 9 functional categories: putative protein(25%), Metabolism(23%), nodulin(23%), Binding protein(10%), energy(7%), Hormone regulated(3%), Defense/stress response(3%), cell structure and maintenance(3%), Leghmoglobins(3%).