Hondred D, Hanson A D
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing 48824.
Proc Natl Acad Sci U S A. 1990 Sep;87(18):7300-4. doi: 10.1073/pnas.87.18.7300.
In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase [LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27]. Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a lambda gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified approximately 3000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1305 and 1166 base pairs) revealed open reading frames encoding 10 peptide fragments of LDH. The 1305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3' noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.
在大麦和其他谷物的根部，缺氧会诱导产生一组五种L-乳酸脱氢酶同工酶[LDH；（S）-乳酸：NADH氧化还原酶，EC 1.1.1.27]。生化和遗传数据表明，这五种LDH同工酶是四聚体，由两个Ldh基因座的产物随机结合产生。为了研究这个系统，从缺氧处理的大麦根来源的λgt11 cDNA文库中分离出LDH的cDNA克隆。用针对通过改进的三步法纯化约3000倍的大麦LDH产生的抗血清筛选该文库。用由聚合酶链反应合成的cDNA探针重新筛选免疫阳性克隆，该探针使用从两种胰蛋白酶LDH肽的氨基酸序列模拟的引物。发现了两种类型的LDH克隆。对每种类型的一个代表性插入片段（分别为1305和1166个碱基对）进行核苷酸序列分析，揭示了编码LDH的10个肽片段的开放阅读框。1305个碱基对的插入片段包含一个356个残基的LDH单体的完整编码区。两个LDH cDNA的核苷酸序列在编码区有92%相同，但在3'非编码区高度不同，因此可能对应于两个假定的Ldh基因座。两种大麦LDH的推导氨基酸序列彼此96%相同，并且与脊椎动物和细菌LDH的序列非常相似。RNA印迹杂交显示有一条1.5千碱基的单一mRNA条带，其水平在缺氧诱导期间根部上升约8倍，可翻译的LDH信息水平也是如此。
