Hu Z, Poulton J E
Department of Biological Sciences, The University of Iowa, Iowa City, Iowa 52242, USA.
Plant Physiol. 1999 Apr;119(4):1535-46. doi: 10.1104/pp.119.4.1535.
The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10), which plays a key role in cyanogenesis in rosaceous stone fruits, occurs in black cherry (Prunus serotina Ehrh.) homogenates as several closely related isoforms. Biochemical and molecular biological methods were used to investigate MDL microheterogeneity and function in this species. Three novel MDL cDNAs of high sequence identity (designated MDL2, MDL4, and MDL5) were isolated. Like MDL1 and MDL3 cDNAs (Z. Hu, J.E. Poulton [1997] Plant Physiol 115: 1359-1369), they had open reading frames that predicted a flavin adenine dinucleotide-binding site, multiple N-glycosylation sites, and an N-terminal signal sequence. The N terminus of an MDL isoform purified from seedlings matched the derived amino acid sequence of the MDL4 cDNA. Genomic sequences corresponding to the MDL1, MDL2, and MDL4 cDNAs were obtained by polymerase chain reaction amplification of genomic DNA. Like the previously reported mdl3 gene, these genes are interrupted at identical positions by three short, conserved introns. Given their overall similarity, we conclude that the genes mdl1, mdl2, mdl3, mdl4, and mdl5 are derived from a common ancestral gene and constitute members of a gene family. Genomic Southern-blot analysis showed that this family has approximately eight members. Northern-blot analysis using gene-specific probes revealed differential expression of the genes mdl1, mdl2, mdl3, mdl4, and mdl5.
黄素蛋白（R）-（+）-扁桃腈裂解酶（MDL；EC 4.1.2.10）在蔷薇科核果类植物的氰化物生成过程中起关键作用，在黑樱桃（Prunus serotina Ehrh.）匀浆中以几种密切相关的同工型存在。采用生化和分子生物学方法研究了该物种中MDL的微观异质性和功能。分离出了三个具有高度序列同一性的新型MDL cDNA（命名为MDL2、MDL4和MDL5）。与MDL1和MDL3 cDNA（Z. Hu，J.E. Poulton [1997] Plant Physiol 115: 1359 - 1369）一样，它们具有开放阅读框，预测有一个黄素腺嘌呤二核苷酸结合位点、多个N-糖基化位点和一个N端信号序列。从幼苗中纯化的一种MDL同工型的N端与MDL4 cDNA推导的氨基酸序列匹配。通过基因组DNA的聚合酶链反应扩增获得了与MDL1、MDL2和MDL4 cDNA对应的基因组序列。与先前报道的mdl3基因一样，这些基因在相同位置被三个短的保守内含子打断。鉴于它们的总体相似性，我们得出结论，mdl1、mdl2、mdl3、mdl4和mdl5基因来源于一个共同的祖先基因，构成一个基因家族成员。基因组Southern杂交分析表明，这个家族大约有八个成员。使用基因特异性探针的Northern杂交分析揭示了mdl1、mdl2、mdl3、mdl4和mdl5基因的差异表达。
