Cloning and Expression Analysis of mtACP3 Genes in Peanut

doi:10.11924/j.issn.1000-6850.casb16100110

Abstract

Abstract: To investigate the mechanism of acyl carrier protein (ACPs) in the pathway of plant fatty acid synthesis, one ACP gene (AhmtACP3) was isolated from peanut by traditional PCR. The expression characteristics of the gene were analyzed by quantitative real- time RT- PCR (qRT- PCR). The length of AhmtACP3 gene was 859 bp. The length of open reading flame (ORF) was 390 bp, and its genomic sequence was 543 bp with 2 exons and 1 intron. It encoded a protein of 129 deduced amino acid residues. The molecular weight was 14.5345 kD and its isoelectric point was 5.22. The deduced protein was predicted to locate in mitochondria. Phylogenetic analysis indicated that peanut mitochondrial ACPs fell into two clusters. AhmtACP1 was grouped with AhmtACP2, which was distinct from AhmtACP3. qRT- PCR indicated that AhmtACP3 transcripts were more abundant in flowers than in any other tissues tested, followed by cotyledons and roots. The expression levels ofAhmtACP3 were the highest at the initial stage of seed development, but gradually decreased in abundance during the later stages.

References

[1] 李孟军,史占良,郭进考,何明琦,王彦霞. 植物酰基载体蛋白基因家族序列分析 [J]. 华北农学报,2010,25(增刊)：1-6.
[2] 单雷,唐桂英,徐平丽,赵学彬,柳展基. 花生质体型酰基载体蛋白基因5’侧翼调控序列的克隆与分析 [J]. 作物学报,2014,40(3)：381-389.
[3] Branen J K, Chiou T J, Engeseth N J. Overexpression of acyl carrier protein-1 alters fatty acid composition of leaf tissue in Arabidopsis [J]. Plant Physiology, 2001, 127(1): 222-229.
[4] 江璐玎,王云霄,王迎春,张颖,秦小波,徐莺,陈放. 麻疯树酰基载体蛋白的基因克隆、表达分析及原核表达 [J]. 应用与环境生物学报,2008,14 (6): 769-773.
[5] Ramelot T A, Smola M J, Lee H W, Ciccosanti C, Hamilton K, Acton T B, Xiao R, Everett J K, Prestegard J H, Montelione G T, Kennedy M A. Solution structure of 4’-phosphopantetheine - GmACP3 from Geobacter metallireducens: a specialized acyl carrier protein with atypical structural features and a putative role in lipopolysaccharide biosynthesis [J]. Biochemistry, 2011, 50(9): 1442-1453.
[6] Ramelot T A, Rossi P, Forouhar F, Lee H W, Yang Y H, Ni S S, Unser S, Lew S, Seetharaman J, Xiao R, Acton T B, Everett J K, Prestegard J H, Hunt J F, Montelione G T, Kennedy M A. Structure of a specialized acyl carrier protein essential for lipid A biosynthesis with very long chain fatty acids in open and closed conformations [J]. Biochemistry, 2012, 51(37): 7239-7249.
[7] 谭晓风,王威浩,刘卓明,张党权,陈鸿鹏,胡芳名. 油茶ACP基因的全长cDNA克隆及序列分析 [J]. 中南林业科技大学学报,2008, 28 (4)：8-14.
[8] Beattie, Emily A, Lowther, Patrick J, Campopiano, James D. Investigating an unusual bacterial acyl carrier protein: a direct link between fatty acid and sphingolipid biosynthesis? [J]. Faseb Journal, 2011, 25(1): 109-112.
[9] Cultrera N G M, Alagna F, Mariotti R, Marchis F D, Pompa A, Bellucci M, Baldoni L. Isolation and molecular characterization of three acyl carrier protein genes in olive (Olea europaea L.) [J]. Tree Genetics Genomes, 2014, 10(4): 895-909.
[10] Zhu L, Cronan J E. The conserved modular elements of the acyl carrier proteins of lipid synthesis are only partially interchangeable [J]. The Journal of Biological Chemistry, 2015, 290 (22): 13791-13799.
[11] Aznar-Moreno J A, Venegas-Calerón M, Martínez-Force E, Garcés R, Salas J. Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities [J]. Planta, 2016, 244(2): 479-490.
[12] Bonaventure G, Ohlrogge J B. Differential regulation of mRNA levels of acyl-carrier protein isoforms in Arabidopsis [J]. Plant Physiology, 2002, 128: 223-235.
[13] Aluru M R, Mazourek M, Landry L G, Curry J, Jahn M, Mary A. O'Connell M A. Differential expression of fatty acid synthase genes, Ac1, Fat and Kas, in Capsicum fruit [J]. Journal of Experimental Botany, 2003, 54(388): 1655-1664.
[14] Li M J, Wang X J, Su L, Bi Y P, Wan S B. Characterization of five putative acyl carrier protein (ACP) isoforms from developing seeds of Arachis hypogaea L [J]. Plant Molecular Biology Reporter, 2010, 28(3): 365-372.
[15] 魏丽奇. 花生酰基载体蛋白基因的克隆与功能分析 [D]. 济南：山东师范大学,2009.
[16] 陈娜,胡东青,潘丽娟,迟晓元,陈明娜,王通,王冕,杨珍,禹山林. 花生中UDP-葡萄糖基转移酶基因的克隆及在非生物胁迫下的表达研究 [J]. 中国油料作物学报,,2014,36(3)：308-315.
[17] 陈娜,潘丽娟,迟晓元,陈明娜,王通,王冕,杨珍,胡冬青,王道远,禹山林. 花生果糖-1,6-二磷酸醛缩酶基因AhFBA1的克隆与表达 [J]. 作物学报,2014,40(5): 934-941.
[18] Guo AYS,SZhu Q HS,SChen XS,SLuo J C. GSDS: a gene structure display server [J]. Yi Chuan, 2007, 29 (8): 1023-1026.
[19] 迟晓元,雷永,陈娜,王通,王冕,陈明娜,潘丽娟,杨珍,禹山林. 三个花生二酰甘油酰基转移酶基因的克隆与序列分析 [J]. 花生学报. 2014,43(4)：1-12.
[20] 孙娇娇. 花生油脂代谢关键基因在酵母和聚球藻中表达及功能验证S[D]. 威海：哈尔滨工业大学,S2014.
[21] Thompson J D, Higgins D G, Gibson T J. CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice [J]. Nucleic Acids Res, 1994, 22: 4673-4680.
[22] Tamura K,Dudley J,Nei M,Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0 [J]. Molecular Biology and Evolution, 2007, 24(8): 1596-1599.
[23] Mofid M R, Finking R, Marahiel M A. Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4’-phosphopantetheinyl transferases acpS and sfp [J]. Journal of Biological Chemistry, 2002, 277(19): 17023-17031.
[24] Zhang Y M,Marrakchi H,White S W,Rock C O. The application of computational methods to explore the diversity and structure of bacterial fatty acid synthase [J]. Journal of Lipid Research, 2003, 44(1): 1-10.
[25] 潘丽娟,刘风珍,万勇善,迟晓元,陈娜,陈明娜,王通,王冕,杨珍,禹山林. 花生FUSCA3基因的克隆与表达分析 [J]. 核农学报. 2015,29(6)：1044-1051.
[26] Liu X L, Hicks W M, Silver P A, Way J C. Engineering acyl carrier protein to enhance production of shortened fatty acids [J]. Biotechnology for Biofuels, 2016, 9(1): 1-7.
[27] Song P, Allen A D. Identification of a cotton fiber specific acyl carrier protein cDNA by differential display [J]. Biochimica Et Biophysica Acta Gene Structure Expression, 1997, 1351(3): 305-312.
[28] Branen J K, Shintani D K, Engeseth N J. Expression of antisense acyl carrier protein-4 reduces lipid content in Arabidopsis leaf tissue [J]. Plant Physiology, 2003, 132(2): 748-756.
[29] Suh M C, Schultz D J, Ohlrogge J B. Isoforms of acyl carrier protein involved in seed specific fatty acid synthesis [J]. Plant Physiology, 1999, 17: 679-688.
[30] Battey J F, Ohlrogge J B. Evolutionary and tissue-specific control of expression of multiple acyl-carrier protein isoforms in plants and bacteria [J]. Planta, 1990, 180: 352-360.
[31] Wang J, Tóth K,STanaka K,SNguyen C T,SYan Z,SBrechenmacher L,SDahmen J,SChen M,SThelen J J,SQiu L,SStacey G. A soybean acyl carrier protein, GmACP, is important for root nodule symbiosis [J]. Molecular Plant-Microbe Interactions, 2014, 27(5): 415-423.
[32] Marchis F D, Valeri M C, Pompa A, Bouveret E, Alagna F, Grisan S, Stanzione V, Mariotti R, Cultrera N, Baldoni L, Bellucci M. Overexpression of the olive acyl carrier protein gene (OeACP1) produces alterations in fatty acid composition of tobacco leave [J]. Transgenic Research, 2016, 25: 45-61.
[33] Kim M J, Shin J S, Kim J K, Suh M C. Genomic structures and characterization of the 5’-flanking regions of acyl carrier protein and delta4-palmitoyl-acp desaturase genes from Coriandrum sativum [J]. Biochimica Et Biophysica Acta, 2005, 1730(3): 235-244.