Gene cloning of the novel mannose-binding rice lectin and its possible role in expression of the disease resistance

经Sephadex G-50亲和层析纯化的新型甘露糖结合水稻凝集素（MRL）在双向聚丙烯酰胺凝胶电泳中分离完全，主要斑点位于pI 4.85和4.74，次要斑点位于pI 4.66，4.56和4.44，而所有斑点均分布在分子量约45，000处。Western blot分析表明，所有斑点均与MRL特异性单克隆抗体反应，pI分别为4.85、4.77、4.66和4.56的蛋白具有凝集素活性。在pI为4.85和4.77的蛋白质中，N端的氨基酸序列TLVKIGPWGGNGGSAQDISV与水稻盐胁迫和干旱胁迫诱导的salT基因产物基本一致。在MRL亚基的cDNA和基因组克隆中，在pI 4.85处的高度同源性也是保守的。这些结果表明，MRL可能是由一些与胁迫相关的蛋白质组成的，它们可能是一个胁迫诱导蛋白超家族，MRL在所有供试品种幼苗的所有生长部位都有活性，但主要分布在叶肉和表皮细胞的细胞壁和周质上。无论是亲和小种还是不亲和小种，接种后提取物MRL的含量变化都很快。用单克隆抗体、α-D-甘露糖或0.15M氯化钠预处理，亲和菌菌丝伸长较不亲和菌明显。相比之下，ConA预处理抑制了附着胞的形成和感染菌丝的伸长。用单克隆抗体和诱导剂如糖精、丙苯咪唑或菌丝提取物共同处理诱导剂，抑制了它们的诱导活性。这些结果有力地支持了我们的假设，即MRI可能在寄主-寄生虫相互作用中起植物抗体或受体的作用。

The novel mannose-binding rice lectin (MRL) finally purified by Sephadex G-50 affinity chromatography was not homogeneous, but separated completely in two dimensional polyacrylamide gel electrophoresis ; the major spots were located at pI 4.85 and 4.74, the minor at pI 4.66,4.56, and 4.44, whereas all spots distributed at about MW 45,000. In western blot analysis, all the spots reacted with the monoclonal antibodies specific to MRL.The proteins at pI 4.85,4.77,4.66, and 4.56 recovered from the gel had lectin activity, respectively. The major proteins at pI 4.85 and 4.77 had also common amino acid sequence at N-terminus, TLVKIGPWGGNGGSAQDISV, which is almost identical to salt and drought stress-inducible salT gene products in rice plant. The high homology was also conserved in both of the cDNA and genomic clones of the MRL subunit at pI 4.85. These results suggested that MRL are composed of some isolectins, if not, related proteins to remain a common epitope, and may belong to a superfamily of stress-inducible proteins.MRL activity was found in all growth parts of seedlings in all cultivars tested, but localized at cell wall and periplasm around mesophyll and epidermal cells. The content of extractive MRL rapidly changed when inoculated with both compatible and incompatible races of M.grisea. Pre-treatment with the monoclonal antibody, a-D-mannose or 0.15M NaCi enhanced the elongation of infection hypha in the compatible rather than in the incompatible. In contrast, pre-treatment with ConA suppressed the appressorium formation and the elongation of infection hypha. Co-treatment with the monoclonal antibody and an inducer to elicit resistant reaction such as saccharin, Probenazole, or mycelial extract suppressed their inducer activity, These results strongly supported our hypotheses that MRI.may function as plant antibodies or receptors in host-parasite interaction..