United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705.
Plant Physiol. 1992 Sep;100(1):374-81. doi: 10.1104/pp.100.1.374.
A full-length cDNA clone encoding aspartate aminotransferase (AAT) has been identified from a carrot root cDNA library. Degenerate oligo primers were synthesized from the known amino acid sequence of AAT form I from carrot (Daucus carota L. cv Danvers). These primers were utilized in a polymerase chain reaction to amplify a portion of a carrot AAT gene from first strand cDNA synthesized from poly(A)(+) RNA isolated from 5-d-old cell suspension cultures. The resulting 750-bp fragment was cloned, mapped, and sequenced. The cloned fragment, mpAAT1, was used as a probe to identify a full-length cDNA clone in a library constructed from poly(A)(+) RNA isolated from carrot roots. A 1.52-kb full-length clone, AAT7, was isolated and sequenced. AAT7 has 54% nucleotide identity with both the mouse cytoplasmic and mitochondrial AAT genes. The deduced amino acid sequence has 52 and 53% identity with the deduced amino acid sequences of mouse cytoplasmic and mitochondrial AAT genes, respectively. Further analysis of the sequence data suggests that AAT7 encodes a cytoplasmic form of carrot AAT; the evidence includes the (a) absence of a transit or signal sequence, (b) lack of "m-residues," or invariant mitochondrial residues, in the carrot AAT sequence, and (c) high degree of sequence similarity with the amino acid sequence previously obtained for form I of carrot, a cytoplasmic isoenzyme. High- and low-stringency hybridizations to Southern blots of carrot nuclear DNA with AAT7 show that AAT is part of a small multigene family. Northern blot analysis of AAT7 suggests that AAT is expressed throughout cell culture up to 7 d and is highly expressed in roots but not in leaves.
已从胡萝卜根 cDNA 文库中鉴定出编码天冬氨酸氨基转移酶（AAT）的全长 cDNA 克隆。从胡萝卜（Daucus carota L. cv Danvers）的 AAT 形式 I 的已知氨基酸序列合成了简并寡聚引物。这些引物用于聚合酶链反应，从 5 天大的细胞悬浮培养物中分离的 poly(A)+RNA 合成的第一链 cDNA 中扩增胡萝卜 AAT 基因的一部分。产生的 750bp 片段被克隆、定位和测序。克隆的片段 mpAAT1 被用作探针，以鉴定从胡萝卜根分离的 poly(A)+RNA 构建的文库中的全长 cDNA 克隆。分离并测序了全长 1.52kb 的克隆物 AAT7。AAT7 与鼠细胞质和线粒体 AAT 基因具有 54%的核苷酸同一性。推导的氨基酸序列与鼠细胞质和线粒体 AAT 基因的推导氨基酸序列分别具有 52%和 53%的同一性。进一步的序列数据分析表明，AAT7 编码胡萝卜的细胞质形式的 AAT；证据包括（a）不存在转运或信号序列，（b）胡萝卜 AAT 序列中缺乏“m-残基”或不变的线粒体残基，以及（c）与以前获得的胡萝卜形式 I 的氨基酸序列高度相似，细胞质同工酶。用 AAT7 对胡萝卜核 DNA 的 Southern 印迹进行高和低严格杂交表明，AAT 是一个小基因家族的一部分。AAT7 的 Northern 印迹分析表明，AAT 在细胞培养物中表达，直至 7d，在根中高度表达，但在叶片中不表达。
