Gornicki P, Podkowinski J, Scappino L A, DiMaio J, Ward E, Haselkorn R
Department of Molecular Genetics and Cell Biology, University of Chicago, IL 60637.
Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6860-4. doi: 10.1073/pnas.91.15.6860.
cDNA fragments encoding part of wheat (Triticum aestivum) acetyl-CoA carboxylase (ACC; EC 6.4.1.2) were cloned by PCR using primers based on the alignment of several biotin-dependent carboxylases. A set of overlapping clones encoding the entire wheat ACC was then isolated by using these fragments as probes. The cDNA sequence contains a 2257-amino acid reading frame encoding a 251-kDa polypeptide. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is 52-55% identical to ACC of yeast, rat, and diatom. Identity with the available C-terminal amino acid sequence of maize ACC is 66%. The biotin attachment site has the typical eukaryotic EVMKM sequence. The cDNA does not encode an obvious chloroplast targeting sequence. Various cDNA fragments hybridize in Northern blots to a 7.9-kb mRNA. Southern analysis with cDNA probes revealed multiple hybridizing fragments in hexaploid wheat DNA. Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants, indicating significant conservation among plant ACCs.
利用基于几种生物素依赖性羧化酶比对设计的引物，通过聚合酶链式反应（PCR）克隆了编码小麦（普通小麦）乙酰辅酶A羧化酶（ACC；EC 6.4.1.2）部分序列的互补DNA（cDNA）片段。然后，以这些片段为探针，分离出了一组编码完整小麦ACC的重叠克隆。该cDNA序列包含一个2257个氨基酸的可读框，编码一个251 kDa的多肽。与原核生物生物素羧化酶相对应的最高度保守结构域的氨基酸序列，与酵母、大鼠和硅藻的ACC有52% - 55%的同一性。与玉米ACC的可用C端氨基酸序列的同一性为66%。生物素附着位点具有典型的真核生物EVMKM序列。该cDNA不编码明显的叶绿体靶向序列。各种cDNA片段在Northern杂交中与一个7.9 kb的信使核糖核酸（mRNA）杂交。用cDNA探针进行的Southern分析揭示了六倍体小麦DNA中有多个杂交片段。一些小麦cDNA探针也与来自其他植物的ACC特异性DNA杂交，表明植物ACC之间存在显著的保守性。
