Received 10 May 2003 Accepted 26 Aug. 2003 Supported by the Hi-Tech Research and Development 863 Program of China (2001AA212051) and the National Special Program for Research and Industrialization of Transgenic Plants (JY03-A-13). * Author for correspondence. E-mail: <gywang@cau.edu.cn>. http://www.chineseplantscience.com Acta Botanica Sinica 2004, 46 (6): 751- 756 Cloning of Plastid Acetyl-CoA Carboxylase cDNA from Setaria italica and Sequence Analysis of Graminicide Target Site ZHAO Hu-Ji 1 , WANG Jian-Hua 1 , GAO Peng 1 , GU Ri-Liang 1 , ZHANG Jing-Qiang 1 , WANG Tian-Yu 2 , WANG Guo-Ying 1* (1. State Key Laboratory of Agrobiotechnology and National Center for Maize Improvement , China Agricultural University, Beijing 100094, China; 2. Institute of Crop Germplasm Resources, The Chinese Academy of Agricultural Sciences, Beijing 100081, China) Abstract: Acetyl-CoA carboxylase (ACCase) is a biotinylated enzyme that catalyzes the first committed step in fatty acid biosynthesis. Graminaceous ACCase in plastid is the target site of two classes of graminicide herbicides. Two full-length cDNAs of plastid ACCase from sethoxydim-resistant and sensitive Setaria italica Beauv., named foxACC-R and foxACC-S, have been cloned. cDNA sequencing showed that they encode a protein of 2 321 amino acids long with a pI of 5.89 and a calculated molecular mass of 256 kD. The sequences of foxACC-R and foxACC-S have been compared with their homologs from other plants and analyzed for conserved amino acid regions and their functional domains. It is found that the amino acid at position 1 780 is Leu in foxACC-R other than Ile in foxACC-S and other cereal plastid ACCase. It is suspected that the change of Ile to Leu residue is critical for interaction of plastid ACCase with cereal herbicides APPs and CHDs. According to Southern hybridization, foxACC-R and foxACC-S are both estimated to be single copy in the genome of S. italica. Key words: Setaria italica; Acetyl-CoA Carboxylase; cloning; herbicide; target site Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a biotinylated enzyme that catalyzes the first committed step in fatty acid biosynthesis and provides malonyl-CoA for the synthesis of a variety of important secondary metabo- lites and for malonylation (Brownsey et al., 1997; Herbert et al., 1997). In plants, these primary and secondary meta- bolic pathways are located in different compartments. It has been shown that plants have two forms of ACCase. One of them locates in plastids, the primary site of plant fatty acid synthesis, which is a “prokaryotic-type” multisubunit enzyme, and contains four subunits: a biotin carboxyl carrier protein (BCCP), a biotin carboxylase (BC), and two subunits of a carboxy transferase (CT). Another plant ACCase locates in cytoplasmsol, which is a “eukary- otic-type” multifunctional enzyme containing all four of the prokaryotic subunits in a single chain, in the order of BC, BCCP, CTβ and CTα. But there is an exception, Graminae ACCases in plastids and in cytoplasmsol all belong to eu- karyotic type (Alban et al., 1994; Konishi and Sasaki, 1994; Gornicki et al., 1997; Zhao and Wang, 2003). Recently, plant ACCases have attracted particular at- tention because graminae ACCase in plastid is the action site of two chemically dissimilar classes of graminicide: aryloxyphenoxy propionates (APPs) and cyclohexanediones (CHDs). The basis of selectivity for these graminicide lay in the structure of the plastid ACCase. The herbicides reduce ACCase activity in meristematic, as a result, inhibit de novo fatty acid biosynthesis and cause plant death in sensitive plants. It has been reported that graminae ACCase in plastid is sensitive to these graminicides, but dicots and nongraminaceous monocots ACCase exhibits less sensitive to them (Herbert et al., 1997; Christoffers et al., 2002). A recent report indicated that the sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equiva- lent position changes the wheat plastid ACCase from the sensitive to resistant (Zagnitko et al., 2001). A foxtail millet (Setaria italica ) line Chum BC6-1, which is highly resistant to sethoxydim, has been developed by distant hybridization (Wang and Darmency, 1997). In this paper, we report the cloning of the full-length cDNA of foxtail millet plastid ACCases from Chum BC6-1 and wild lines and the comparison of their amino acid sequences.