Plant Molecular Biology Laboratory, McGill University, 1205 Doctor Penfield Avenue, H3A 1 B1, Montreal, Canada.
Plant Mol Biol. 1984 Jan;3(1):21-8. doi: 10.1007/BF00023412.
Cloned cDNAs corresponding to mRNAs which accumulate in nitrogen-fixing root nodules of soybean (nodulin mRNAs) were used as probes to investigate the sizes, sequence relationships, tissue specificities and developmental accumulations of individual nodulin mRNA sequences. Northern blot analysis indicated that the NodB, NodC and NodD mRNA sequences are 1 150, 770, and 3 150 nucleotides long, respectively, which is consistent with the previously determined sizes of the hybrid-selected translation products (27 000, 24 000 and 100 000 MW, respectively). The NodA clones pNodA15 and pNodA25 hybridized to two mRNAs of lengths 1 600 and 1 100 nucleotides, indicating that they contain significant sequence homologies. However, increasing the hybridization stringency showed that the pNodA15 clone encodes the 1 600 nucleotide mRNA corresponding to the major NodA hybrid-selected translation product (44 000 MW) while pNodA25 encodes an mRNA of 1 100 nucleotides. The latter probably corresponds to one of two smaller (23 500 and 24 500 MW) in vitro translation products. RNA dot-blot hybridizations indicated that nodulin and leghemoglobin mRNAs began to appear and accumulate in Rhizobium infected root tissue very early (day 3 to 5) and reached fully induced levels by day 11. This accumulation was specific for nodule tissue (except for the NodD sequence) and preceded the accumulation of nitrogen fixation activity. Nodules produced by different effective Rhizobium strains accumulated similar levels of leghemoglobin and nodulin mRNAs while ineffective strains had a pleiotropic affect. While one ineffective strain (61A24) gave reduced levels of all these mRNAs, the other (SM5) gave levels which were nearly normal by the time nitrogen fixation activity should have reached its maximal level (day 17). Thus, leghemoglobin and nodulin genes are switched on soon after infection, prior to nodule morphogenesis, and the switch occurs prior to and is independent of nitrogen fixation activity.
克隆的与在大豆固氮根瘤中积累的 mRNA 对应的 cDNA 被用作探针，以研究单个豆球蛋白 mRNA 序列的大小、序列关系、组织特异性和发育积累。Northern 印迹分析表明，NodB、NodC 和 NodD mRNA 序列分别长 1150、770 和 3150 个核苷酸，与先前确定的杂交选择翻译产物的大小（分别为 27000、24000 和 100000MW）一致。NodA 克隆 pNodA15 和 pNodA25 与长度为 1600 和 1100 个核苷酸的两种 mRNA 杂交，表明它们含有显著的序列同源性。然而，增加杂交的严格性表明，pNodA15 克隆编码与主要 NodA 杂交选择翻译产物（44000MW）相对应的 1600 个核苷酸的 mRNA，而 pNodA25 编码 1100 个核苷酸的 mRNA。后者可能对应于两个较小的（23500 和 24500MW）体外翻译产物之一。RNA 点印迹杂交表明，豆球蛋白和豆血红蛋白 mRNA 早在 Rhizobium 感染根组织中开始出现并积累（第 3 至 5 天），并在第 11 天达到完全诱导水平。这种积累是特定于根瘤组织的（除了 NodD 序列），并且早于氮固定活性的积累。不同有效 Rhizobium 菌株产生的根瘤积累相似水平的豆血红蛋白和豆球蛋白 mRNA，而无效菌株则具有多效性影响。虽然一种无效菌株（61A24）降低了所有这些 mRNA 的水平，但另一种（SM5）在氮固定活性应该达到其最大水平（第 17 天）时，其水平几乎正常。因此，豆血红蛋白和豆球蛋白基因在感染后很快被激活，在根瘤形态发生之前，并且这种激活发生在氮固定活性之前并且独立于氮固定活性。
