Application of Salt-tolerant and Nitrogen-fixing Blue-green Algae to Agriculture in Sea Water

我们克隆了耐盐植物分子育种所必需的基因，如外生碱合成基因、水稻HKT基因和酵母ENA1基因。由于这些基因不足以达到最终目标，因此克隆了其他基因并分析了它们的功能。过量表达水稻钾离子转运体OsHAK5基因的烟草BY2细胞能在低钾离子培养基中生长，并表现出耐盐性。当过量表达参与脯氨酸积累的烟草HAL3基因时，BY2细胞的耐盐性增强。耐盐植物在海水中生长时，氮源的供应是一个重要的因素。从冲绳附近海水中分离到的单细胞蓝绿藻（Synechococcus sp.TU126）能固定大气氮，在无碳氮源的人工海水中生长。在25℃光照下人工海水中培养14天和45天的聚珠球菌细胞内总氮分别为19.22 mg/l、41.8mg/l，在培养基中排出的氮分别为3.2 mg/l、4.2 mg/l。耐盐绿藻Clamydomonas sp.W80在含NH_4 Cl的条件培养基中生长的程度与在条件培养基中含氮水平相同的新鲜培养基中生长的程度相同，表明聚球菌产生的氮化合物可以被植物利用。海藻酸钙中固定化的聚球菌细胞在培养30天和60天后分别分泌1.0 mg/l和5.6 mg/l的氮。综上所述，海水中固定化聚珠球菌可作为农业中耐盐植物的氮源。

We have cloned essential genes required for molecular breeding of salt-tolerant plants which can grow in sea water, such as ectoine synthetic genes, rice HKT gene and yeast ENA1 gene. Since these genes were not enough for the final goal, other genes were cloned and analyzed their function. Tobacco BY2 cell, which was over-expressed rice potassium ion transporter, OsHAK5 gene, can grow in low potassium ion medium and showed salt-tolerance. BY2 cell increased salt tolerance when it was over-expressed tobacco HAL3 gene which participate to praline accumulation.Supply of nitrogen source will be an important factor when salt-tolerant plants are grown in sea water. Single-cell blue-green algae, Synechococcus sp.TU126, which was isolated from sea water near from Okinawa can fix atmosphere nitrogen and grew in artificial sea water without carbon and nitrogen source. Total intracellular nitrogen in the Synechococcus grown in artificial sea water under light at 25℃ for 14 days and 45 days, was 19.22 mg/l、 41.8mg/l, respectively, and excreted 3.2 mg/l、4.2 mg/l nitrogen, respectively, in the medium.Salt-tolerant green algae, Clamydomonas sp.W80 grew in the conditioned medium of the Synechococcus in the same extent as that in the fresh medium contained NH_4 Cl in the same level of nitrogen in the conditioned medium, suggesting that nitrogen compounds produced by the Synechococcus can be utilized by plants. Immobilized Synechococcus cells in calcium alginate secreted nitrogen of 1.0 mg/l and 5.6 mg/l in culture medium after 30 days and 60 days, respectively. In conclusion, salt-tolerant plants can be supplied nitrogen source from immobilized Synechococcus sp.TU126 in agriculture in sea water.