# Investigation of nodulin genes expressed during soybean root nodule development.

 Title: Investigation of nodulin genes expressed during soybean root nodule development. Author: Gottlob-McHugh, Sylvia Gabriele. Abstract: The infection of soybean roots by the soil bacterium Bradyrhizobium japonicum results in the formation of specialized organs on the roots called nodules. Within the nodules, a differentiated form of the bacterium (the bacteroid) reduces atmospheric nitrogen to ammonia. Nodule development involves a complex interaction requiring the coordinated expression of nodule-specific bacterial (bacteroidin) and nodule-specific plant (nodulin) genes. A number of nodulin gene products from soybean have been characterized, but the functional roles of only a few have been determined. This thesis describes the isolation and characterization of four nodule-specific cDNA clones (and a leghemoglobin clone) from a cDNA library that was derived from soybean nodule poly (A)$\sp+$ enriched RNA. mRNA corresponding to these cDNA clones, could be detected in RNA isolated from the soybean nodule; but could not be detected in RNA isolated from the root. The pattern of expression of the genes corresponding to the cDNA clones was investigated at various times following infection of the soybean Glycine max (L.) Merrill by Bradyrhizobium japonicum. It appears that these nodulin mRNAs are expressed just prior to the onset of nitrogen fixation and accumulate to their maximum levels shortly thereafter. Sequence characterization of the four clones indicated that they represented members of a previously described nodulin multi-gene family: the "Nodulin A" family (Verma and Delauney, 1988). The "Nodulin-A" family represents a group of abundantly transcribed nodulins, whose function is unknown. Three of these clones, 9-11-B, 36-1-A, and 6-9-F, support the previously published sequences of nodulin-23 (Mauro et al., 1985; Sengupta-Gopalan et al., 1986), nodulin-22 (Sandal et al., 1987), and nodulin-44 (Sengupta-Gopalan et al., 1986) respectively. A fourth clone, 15-9-A, represents a new member of this multi-gene family. Northern blot hybridization with oligo-nucleotide probes specific to this cDNA clone, indicates that 15-9-A represents an abundantly transcribed 1 kb mRNA. Our analyses suggest that 15-9-A is a member of a subfamily of three closely related genes within the larger "Nodulin-A" family. 15-9-A is related to the nodulin-20 sequence of Sandal et al. (1987), which our analyses show is also a member of this subfamily. The 15-9-A cDNA is 100% identical to nodulin-20 in the region that is 5$\sp\prime$ to the position of the nodulin-20 intron, and 89% similar in the region that is 3$\sp\prime$ to the intron. Although the region of greatest divergence between the two sequences is in the 3$\sp\prime$ coding region, each sequence codes for a proline-rich carboxy terminus. In addition, we have determined that a 0.8 kb mRNA that cross-hybridizes to 15-9-A represents a third gene within this subfamily. We have also detected the presence of "Nodulin-A"-like sequences in the DNA of alfalfa, several actinorhizal plants, spinach, and corn. This raises the possibility that "Nodulin-A" like sequences have a functional role in plants other than soybean. We have isolated four alder genomic clones which cross hybridize to the cDNA clone 9-11-B, as an approach to characterizing a "Nodulin-A" sequence from another plant. Further characterization of these clones should determine their relatedness to the soybean sequences and may provide clues as to their possible functional significance. Date: 1990 URI: http://hdl.handle.net/10393/5902

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