Baulcombe D, Verma D P
Nucleic Acids Res. 1978 Nov;5(11):4141-55. doi: 10.1093/nar/5.11.4141.
In soybean root nodules, leghaemoglobin (Lb) accounts for 25--30% of the total soluble protein but is not detected in other tissues. In order to determine whether the Lb genes are plant or bacterial in origin a cDNA probe for Lb was prepared from 9S poly (A) containing mRNA of root nodules. Although this 9S mRNA directed synthesis of predominantly three forms of Lb in vitro, the kinetics of hybridisation of cDNA and the 9S mRNA showed a transition at about 30% hybridisation which suggested that the 9S-cDNA was not pure Lb-cDNA. The abundant, Lb-cDNA was prepared by two cycles of hybridising 9S mRNA and cDNA to a Rot of 3 X 10(-3) and isolation of the hybridised cDNA on hydroxyapatite. The Lb-cDNA was homogeneous in hybridisation analysis with 9S mRNA and electrophoresis in 98% formamide gels. This cDNA hybridised with soybean DNA and not with Rhizobium DNA, thus directly demonstrating that Lb genes are of plant origin. Titration of Lb-cDNA with soybean DNA showed that Lb genes are reiterated about forty-fold per haploid genome.
在大豆根瘤中，豆血红蛋白（Lb）占总可溶性蛋白的25%至30%，但在其他组织中未检测到。为了确定Lb基因的起源是植物还是细菌，从含有根瘤mRNA的9S聚腺苷酸（poly(A)）制备了Lb的cDNA探针。尽管这种9S mRNA在体外主要指导三种形式的Lb的合成，但cDNA与9S mRNA的杂交动力学在约30%杂交时显示出转变，这表明9S-cDNA不是纯的Lb-cDNA。通过将9S mRNA和cDNA杂交至3×10⁻³的Rot并在羟基磷灰石上分离杂交的cDNA进行两个循环，制备了丰富的Lb-cDNA。Lb-cDNA在与9S mRNA的杂交分析和98%甲酰胺凝胶电泳中是均匀的。这种cDNA与大豆DNA杂交，而不与根瘤菌DNA杂交，从而直接证明Lb基因是植物起源的。用大豆DNA对Lb-cDNA进行滴定表明，Lb基因在每个单倍体基因组中重复约40倍。
