* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Cloning of Plastid Acetyl-CoA Carboxylase cDNA from Setaria italica
Deoxyribozyme wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Molecular Inversion Probe wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Community fingerprinting wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Butyric acid wikipedia , lookup
Proteolysis wikipedia , lookup
Metalloprotein wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Peptide synthesis wikipedia , lookup
Protein structure prediction wikipedia , lookup
Citric acid cycle wikipedia , lookup
Point mutation wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Genetic code wikipedia , lookup
Fatty acid metabolism wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
Acta Botanica Sinica 植 物 学 报 2004, 46 (6): 751-756 http://www.chineseplantscience.com Cloning of Plastid Acetyl-CoA Carboxylase cDNA from Setaria italica and Sequence Analysis of Graminicide Target Site ZHAO Hu-Ji1, WANG Jian-Hua1, GAO Peng1, GU Ri-Liang1, ZHANG Jing-Qiang1, WANG Tian-Yu2, WANG Guo-Ying1* (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 metabolites and for malonylation (Brownsey et al., 1997; Herbert et al., 1997). In plants, these primary and secondary metabolic 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 “eukaryotic-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 eukaryotic type (Alban et al., 1994; Konishi and Sasaki, 1994; Gornicki et al., 1997; Zhao and Wang, 2003). Recently, plant ACCases have attracted particular attention 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 equivalent 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. 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: <[email protected]>. 752 Acta Botanica Sinica 植物学报 Vol.46 No.6 2004 1 Materials and Methods 1.1 Plant materials Seeds of sethoxydim-resistant foxtail millet ChumBC 6-1 and wild line Sda 11were from the Institute of Crop Germplasm Resources, The Chinese Academy of Agricultural Sciences. 1.2 Methods 1.2.1 RNA isolation Total RNA was isolated from 2week-old foxtail millet using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. 1.2.2 RACE Total RNA of foxtail millet seedling was reverse-transcribed, and the cDNA ends of foxACC were amplified using “System for Rapid Amplification of cDNA Ends”(Invitrogen). The 5' RACE/3' RACE gene-specific primers (Table 1) were designed according to known conservative regions of graminae plastid ACCase cDNAs. The PCR products were cloned into pGEM-T Easy Vector (Promega) and sequenced. 1.2.3 Cloning the full-length cDNA of foxACC THERMOSCRIPTTMRT-PCR System (Invitrogen) was used to reverse transcribe the first strand of foxACC cDNA. One µL of the above cDNA was used for amplification by PCR with Advantage cDNA PCR Kit (Clontech). After denatured at 94 ℃ 2 min, the PCR reaction was carried out for 30 cycles as follows: 94 ℃ 15 s, 68 ℃ 8 min, followed by a 7 min final extension at 68 ℃. The full-length primer (Table 1) was designed based on the sequence obtained by the 5' RACE/3' RACE. The PCR products were cloned into TOPO XL PCR Cloning Kit (Invitrogen). 1.2.4 Sequence analysis and comparison of foxACC with its homologs Some internet and biology softwares were used to analyze foxACC sequences, which include OMIGA, DNAMAN, BLAST, http://www.cbs.dtu.dk/services/ TargetP/, psort.nibb.ac.jp, http://www.ncbi.nlm.nih.gov/ Structure/ and so on. 1.2.5 Southern blotting analysis Genomic DNA of foxtail millet was extracted from leaves by using the method of SDS (Fu et al., 1994). About 20 µg of genomic DNA was digested with 40 U of restriction endonucleases (Promega) at 37 ℃ overnight. The digested DNA was separated by electrophoresis on 0.8% of agarose gel and blotted on Hybond-N+ nylon membrane (Amersham Pharmacia) in 0.4 mol/L NaOH. Probe was random-primed labeled with [α-32P] dCTP. Southern hybridization was carried out overnight at 42 ℃. The hybridized filters were washed in 2 × SSC, 0.5% SDS at 42 ℃ for 30 min, twice in 1×SSC, 0.1% SDS at 65 ℃ for 30 min, twice in 2×SSC at 65 ℃ for 10 min, and exposed to X-ray film (Fuji) at -70 ℃. 2 Results 2.1 Cloning of foxACC full-length cDNAs The first 5' RACE generated three bands (about 1 000 bp, 750 bp and 420 bp) in sethoxydim-resistant foxtail millet, but only one band (380 bp) in wild foxtail millet (Fig.1a). Sequencing of the longest three bands revealed that all these products showed high homology to known graminae plastid ACCase cDNA, especially to that of maize, but the start codon and 5' UTR sequence of ACCase gene could not be found. The second 5' RACE and 3' RACE generated a single band both in sethoxydim-resistant and susceptible foxtail millet as we suspected (Fig.1b, c). Based on the sequences of two 5' RACE and 3' RACE, A DNA band of about 7.3 kb containing the complete ORF has been obtained from both foxtail millet (Fig.1d). After assembling the sequences from RACE and RTPCR, we obtained the full length cDNA sequence of ACCase gene from sethoxydim-resistant S. italica, designated as foxACC-R, which has 7 446 bp and includes an ORF of 6 966 bp, 58 nucleotides of 5' untranslated sequence (UTR) and 422 nucleotides of 3' UTR (accession number: AY219175). The full-length cDNA sequence from sethoxydim-sensitive S. italica, designated as foxACC-S, has 7 271 bp and includes an ORF of 6 966 bp, 26 bp of 5' UTR and 279 nt of 3' UTR (accession number: AY219174). Both genes are deduced to encode a protein of 2 321 amino acids with an pI of 5.89 and molecular weight ca. 256 kD. There are eight amino acids difference between foxACC-R and foxACC-S. Table 1 Primers of RACE and full-length cDNA amplification Experiment Thefirst5' RACE The second 5' RACE 3' RACE Full-length amplification Primer GSP1 GSP2 GSP3 GSP4 GSP5 FLL FLR Sequence (5' to 3') CCCACAGCCTTAGCAAGCCT GATCACAAAGCAACTGAACTTCAAG TTGCTGCTGCCATTCCATTG GTCTGCCATTATGTGCTTCATTTAC CCAGGTCAGCTTGATTCCCATGAG TGTGCTGTCTGGGCTACGGAACGAC CAGAATTGAACCGCTGGTTACATCACATAACTA ZHAO Hu-Ji et al.: Cloning of Plastid Acetyl-CoA Carboxylase cDNA from Setaria italica and Sequence Analysis of Graminicide Target Site 753 Fig.1. Product of 5' RACE/3' RACE and full-length cDNA amplification. a. The first 5' RACE. b. The second 5' RACE. c. 3' RACE. d. Full-length cDNA amplification. M, DNA marker; R, sethoxydim-resistant foxtail; S, sethoxydim-sensitive foxtail. 2.2 Comparison of the cloned cDNA with reported plant ACCase According to our BLAST results, it is confirmed that the cloned cDNA from foxtail millet encoded plastid ACCase. The amino acid sequence encoded by foxACC-R showed 91% and 84% identity with the plastid ACCase from Zea mays and Triticum aestivum, respectively, but only 64%- 69% identity with the cytosolic ACCase from Triticum aestivum and other plants (Table 2). Table 2 Comparison of the amino acid sequence deduced from foxACC-R with reported eukaryotic-type ACCase of plants Species Location Zea mays Triticum aestivum Plastid Plastid Cytosolic Cytosolic Cytosolic Cytosolic Cytosolic Arabidopsis thaliana Brassica napus Medicago sativa Glycine max Identity (%) 91 84 69 67 64 67 68 Accession number U19183 AF029895 U39321 L27074 Y10301 L25042 L42814 It is found that the foxtail ACCase has about 100 amino acids in N-terminal sequences that are rich in hydroxylated amino acids Ser and Thr, containing multiple small hydrophobic residues such as Ala and Val, and positively charged amino acids such as Arg and Lys. These features are characteristic of many transit sequences (Schleiff and Soll, 2000). The transit peptides also have a characteristic Met-Ala sequence at their N termini. Analysis of the protein-sorting signals (http://www.cbs.dtu.dk) predicted that the probability of chloroplast transit peptide in the foxtail ACCase was 94.3%, and that of mitochondrial targeting peptide was only 33.1%. Another software also predicted the likelihood of 83.9% for the cloned foxtail ACCase locating in plastids (www.inra.fr/predotar/). There are four conservative domains among the foxtail ACCase (Fig.2), which are commonly existed in eukaryotic ACCase (Gornicki et al., 1994; Egli et al., 1995). The biotin carboxylase domain is located at amino acid position 132- 634. Within the domain, there are three most conserved regions, carbamoyl-phosphate synthase L chain N-terminal region(132-254), ATP binding region(256-487) and biotin carboxylase C-terminal region(527-634). The biotin carboxyl carrier domain is found at amino acid position 770-838 and the highly conservative biotinylation site EVEVMKM is located at amino acid position 801-807 within this domain. It has been shown previously that approximately 30 amino acid residues on each side of the consensus sequence are important for biotinylation in vivo (Elborouga et al., 1996). The β- and α-domains of the CT are located at amino acid positions 1 638-1 707 and 1 936- 2 191, respectively, which is the most conserved domains among the four domains. The highly conserved carboxybiotin binding site(1 658-1 707)and the acetyl-CoA-binding site(1 946-1 965)play an important role in this domain. 2.3 Analysis of graminicide target site There are eight amino acid differences between sethoxydim-resistant and sensitive foxtail ACCase. Compared with amino acid sequence of other eukaryotic ACCases, we could find an interesting correlation (Fig.3). At amino position 1 780, sethoxydim-resistant foxtail is Leu (codon TTA), but sethoxydim-sensitive Ile (codon ATA). Most graminaes sensitive to APPs and CHDs are also found 754 Acta Botanica Sinica 植物学报 Vol.46 No.6 2004 Fig.2. The conserved amino acid domains and regions of foxACC. A, carbamoyl-phosphate synthase L chain N-terminal region; B, ATP binding region; C, biotin carboxylase C-terminal region; D, biotin carboxyl carrier domain; E, Ctβ domain; F, Ctα domain. Ile in the site in their plastidic eukaryotic ACCase. All dicots and nongraminaceous monocots, which are resistant to the herbicides, have Leu at the position of prokaryotic ACCases and cytosolic, eukaryotic ACCase. Graminaes cytosolic ACCase, animals and yeast ACCase are eukaryotic and have Leu in this position, which are not sensitive to these herbicides. From these results and analysis, we concluded that the change of Leu from Ile residue is critical for the interaction of ACCase with APPs and CHDs. The result is consistant with Zagnitko et al. (2001) and Délye et al. (2002). 2.4 Southern blotting/hybridization analysis of foxtail millet genomic DNA To investigate the copy number of foxACC, genomic DNA of foxtail millet was digested with EcoRⅠ, SacⅠ, EcoRV, SphⅠ, Bgl Ⅱ, SpeⅠand KpnⅠ respectively, separated on 0.8% agarose gel and transferred to nylon membrane (Hybond-N + ). Two probes were used for the hybridization. One probe was a 400 bp fragment from 5' coding region of plastid ACCase cDNA, which encoded chloroplast transit peptide sequence and was lack in cytosolic-ACCase cDNA. Another probe was a 1.4 kb fragment from biotin carboxylase domain, which has the highest identity (79%) with cytosolic ACCase cDNA. The hybridization bands for the above two probes were almost identical (Fig.4). These results support the view that there is only one copy for the foxACC in foxtail millet. The identity between plastid and cytosolic ACCase cDNA is not so high Fig.3. Alignment of the amino acid sequence of the CT domain of eukaryotic-type ACCase where an isoleucine/leucine substitution was found in resistant sethoxydim Setaria italica. The isoleucine residue is indicted in bold. ACCase sequences were obtained from GenBank, accession No. from top to bottom: AY219175, AY219174, U19183, AF359513, AF359514, U10187, AF029895, X99102, AF072737, L27074, X77576, L25042, L42814, AF330145, Z46886, L20784, P78820, M92156, Y15996, S41121, J03541, J03808, Q28559, AJ132890. *, the isoleucine residue. ZHAO Hu-Ji et al.: Cloning of Plastid Acetyl-CoA Carboxylase cDNA from Setaria italica and Sequence Analysis of Graminicide Target Site 755 ing characteristics of the two isoforms of maize ACCase from plastid and cytosol, which have Leu and Ile respectively at the herbicide binding site. The result showed that their molecular masses, native conformations and Michaelis constants for three substrates were all rather similar. Moreover, the reaction characteristics were close to an ordered mechanism in both cases. However, there is a small stereochemical change at the herbicide-binding site, including the critical Leu/Ile residue, which may alter the enzymeinhibitor interaction. Therefore, perhaps other residues might also play a role in defining the binding site of ACCase with herbicides (Zagnitko et al., 2001). References: Alban C, Baldewt P, Douce R. 1994. Localization and characterFig.4. Southern hybridization analysis of foxtail millet genomic DNA. The probe was from 5' coding region of plastid ACCase cDNA, which encoded chloroplast transit peptide sequence. Lanes 1 to 7, foxtail millet genomic DNA digested with EcoRⅠ, SacⅠ, EcoR V, SphⅠ, Bgl Ⅱ, SpeⅠ and KpnⅠ, respectively. as having cross hybridization. 3 Discussion ACCase has been identified as an important regulatory enzyme for plant fatty acid synthesis by three diverse in vivo approaches. Analysis of substrate and product pool sizes implicated the role of ACCase in the light/dark regulation of fatty acid synthesis in spinach leaves (PostBeittenmiller et al., 1992) and chloroplasts (PostBeittenmiller et al., 1991; Akiko et al., 2000). Herbicide inhibition of ACCase was used to determine flux control coefficients, and led to the conclusion that ACCase exerts major control over fatty acid synthesis rates in barley and maize (Page et al., 1994; Herbert et al., 1997). ACCase was also the apparent site of feedback inhibition of fatty acid synthesis in tobacco suspension cells supplemented with exogenous fatty acids (Shintani and Ohlrogge, 1995). But, the molecular mechanism of this regulation is unknown until now. Cloning the full-length cDNA of foxtail plastid ACCase will lay a basis for the understanding of the molecular mechanism in this process. Moreover, the full-length cDNA of the foxACC-R can be introduced into graminaceous crops to confer herbicide resistance, the overexpression of the foxACC-R is also likely to lead to an increase in the oil content of the plants and seeds. Although the isoleucine/leucine residue substitution is critical for the interaction of ACCase with APPs, leucine and isoleucine have similar chemical structures. Herbert et al. (1996) studied the reaction kinetics and herbicide bind- ization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides. Biochem J, 300: 557- 565 Brownsey R W, Zhande R, Boone A N. 1997. Isoforms of acetylCoA carboxylase: structures, regulatory properties and metabolic functions. Biochem Soc T, 25: 1232-1238. Christoffers M J, Berg M L, Messersmith C G. 2002. An isoleucine to leucine mutation in acetyl-CoA carboxylase confers herbicide resistance in wild oat. Genome, 45: 1049-1056. Délye C, Wang T, Darmency H. 2002. An isoleucine-leucine substitution in plastid acetyl-CoA carboxylase from green foxtail (Setaria viridis (L.) Beauv.) is responsible for resistance to the cyclohexanedione herbicide sethoxydim. Planta, 214: 421-427. Egli M A, Lutz S M, Somers D A, Gegenbach B G. 1995. A maize acetyl-coenzyme A carboxylase cDNA sequence. Plant Physiol, 108: 1299-1300. Elborouga K M, Winz R, Deka R K, Markham J E, White A J, Rawsthorne S, Slabas A R. 1996. Biotin carboxyl carrier Protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassion napus: cloning and analysis of expression during oilseed rape embryogenesis. Biochem J, 315: 103-112. Fu R-Z, Sun Y-R, Jia S-R. 1994. Plant Genetic Transformation. Beijing: China Science and Technology Press. 131-137. ( in Chinese) Gornicki P, Podkowinshi J, Scappino L A, DiMaio J, Ward E, Haselkorn R. 1994. Wheat acetyl-coenzyme A carboxylase cDNA and protein structure. Proc Natl Acad Sci USA, 91: 6860-6864. Gornicki P, Faris J, King I, Podkowinshi J, Gill B, Haselkorn R. 1997. Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral 756 chromosome sets. Proc Natl Acad Sci USA, 94: 14179-14184. Herbert D, Price L J, Alban C, Dehaye L, Job J, Cole D J, Pallett Acta Botanica Sinica 植物学报 Vol.46 No.6 2004 Biol Chem, 266: 1858-1865. Post-Beittenmiller D, Roughan G, Ohlrogge J B. 1992. Regula- K E, Harwood J L. 1996. Kinetic studies on two isoforms of tion of plant fatty acid biosynthesis analysis of acyl-coen- acetyi-CoA carboxylase from maize leaves. Biochem J, 318: zyme A and acyl-acyl carrier protein substrate pools in spin- 997-1006. Herbert D, Walker K A, Price L J, Cole D J, Pallett K E, Ridley S M, Harwood J L. 1997. Acetyl-CoA carboxylase — a graminicide target site. Pestic Sci, 50: 67-71. Konishi T, Sasaki Y. 1994. Compartmentalization of two forms of acetyl-CoA carboxylase in plants and the origin of their tolerance toward herbicides. Proc Natl Acad Sci USA, 91: 3598-3601. Kozaki A, Kamada K, Nagano Y, Iguchi H, Sasaki Y. 2000. Recombinant carboxyltransferase responsive to redox of pea ach and pea chloroplast. Plant Physiol, 100: 923-930. Schleiff E, Soll J. 2000. Travelling of proteins through membranes: translocation into chloroplasts. Planta, 211: 449-456. Shintani D K, Ohlrogge J B. 1995. Feedback inhibition of fatty acid synthesis in tobacco suspension cells. Plant J, 7: 577- 587. Wang T, Darmency H. 1997. Inheritance of sethoxydim resistance in foxtail millet, Setaria italica (L.) Beauv. Euphytica, 94: 69-73. Zagnitko O, Jelenska J, Tevzadze G, Haselkorn R, Gornicki P. plastidic Acety-CoA carboxylase. J Biol Chem, 275: 10702- 2001. An isoleucine/leucine residue in the carboxyltransferase 10708. domain of acetyl-CoA carboxylase is critical for interaction Page R A, Okada S, Harwood J L. 1994. Acetyl-CoA carboxylase exerts strong flux control over lipid synthesis in plants. Biochim Biophys Acta, 1210: 369-372. Post-Beittenmiller D, Jaworski J G, Ohlrogge J B. 1991. In vivo pools of free and acylated acyl carrier proteins in spinach: with aryloxyphenoxypropionate and cyclohexanedione inhibitors. Proc Natl Acad Sci USA, 98: 6617-6622. Zhao H-J, Wang G-Y. 2003. Molecular biology and gene engineering of plant acetyl-CoA carboxylase. J Chin Biotechnol, 23: 12-16. (in Chinese with English abstract) evidence for sites of regulation of fatty acid biosynthesis. J (Managing editor: ZHAO Li-Hui)