Staswick P, Chrispeels M J
J Mol Appl Genet. 1984;2(6):525-35.
Phytohemagglutinin (PHA), the major lectin of the common bean Phaseolus vulgaris, is synthesized during the development of the seeds. In most cultivars PHA makes up 5-10% of the total seed protein, but certain cultivars do not contain PHA. In vivo labeling of a normal cultivar (Greensleeves) and a PHA-minus cultivar (Pinto 111) showed that PHA was not synthesized in the PHA-minus cultivar. To find out whether the lack of synthesis was due to the absence of mRNA for PHA, recombinant cDNA clones for PHA were obtained. Total poly(A)+ RNA was isolated from cotyledons of developing seeds of Greensleeves and used to direct cDNA synthesis. The double stranded cDNA was cloned in pUC8 and transformants of Escherichia coli screened with pPVL134, a recombinant plasmid which contains the complete coding sequence for a PHA-like protein. Two weakly hybridizing clones (pSC1 and pSC2) were selected. Hybrid selection experiments showed that these two clones selected mRNAs which could be translated into polypeptides identical in size to PHA and recognized by antibodies to PHA. The recombinant pPVL134 selected mRNA which translated into polypeptides which were slightly smaller than those of PHA, and poorly recognized by antibodies to PHA. The recombinant clones were used to demonstrate that the genes for PHA and for the PHA-like protein are under temporal control during seed development. The cultivar Pinto 111, which has no detectable PHA, also has greatly reduced levels of mRNA for PHA. However, the gene for the PHA-like protein encoded by pPVL134 is expressed to the same degree in the cultivars Greensleeves and Pinto 111.
植物血凝素（PHA）是菜豆（Phaseolus vulgaris）的主要凝集素，在种子发育过程中合成。在大多数品种中，PHA占种子总蛋白的5-10%，但某些品种不含PHA。对正常品种（Greensleeves）和PHA缺失品种（Pinto 111）进行体内标记，结果表明PHA缺失品种不合成PHA。为了确定PHA合成缺失是否是由于缺乏PHA的mRNA，获得了PHA的重组cDNA克隆。从Greensleeves发育种子的子叶中分离出总poly(A)+RNA，并用于指导cDNA合成。双链cDNA克隆到pUC8中，并用pPVL134筛选大肠杆菌转化体，pPVL134是一种重组质粒，含有PHA样蛋白的完整编码序列。选择了两个弱杂交克隆（pSC1和pSC2）。杂交选择实验表明，这两个克隆选择的mRNA可翻译成大小与PHA相同且能被PHA抗体识别的多肽。重组pPVL134选择的mRNA翻译成的多肽比PHA的略小，且很少被PHA抗体识别。利用重组克隆证明了PHA和PHA样蛋白的基因在种子发育过程中受时间控制。没有可检测到的PHA的品种Pinto 111，其PHA的mRNA水平也大大降低。然而，由pPVL134编码的PHA样蛋白的基因在Greensleeves和Pinto 111品种中的表达程度相同。
