Cloning and Expression Analysis of DcPSY2 Gene Under Abiotic Stress in Carrot (Daucus carota)
DING Xu, WANG Ya-Hui, LI Tong, ZHANG Rong-Rong, XU Zhi-Sheng, XIONG Ai-Sheng*
College of Horticulture, Nanjing Agricultural University / State Key Laboratory of Crop Genetics and Germplasm Enhancement / Ministry of Agriculture and Rural Affairs Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Nanjing 210095, China
Abstract Carotenoids, as an important nutrient, are very common in carrot (Daucus carota). Lycopene, a kind of carotenoids, plays important roles in growth, development, and response to abiotic stress in higher plant. Phytoene synthase (PSY) is one of the key enzymes of lycopene biosynthesis. In this study, DcPSY2 (GenBank No. NM_001329167.1) was cloned from carrot cv. 'Junchuanhong' by RT-PCR (reverse transcription PCR). It contained an ORF with the length of 1 317 bp and encoded 438 amino acids. The amino acid sequences of DcPSY2 and the PSY proteins of 17 other plants were compared, the results showed that the overall similarity of PSY proteins was 82.22 %, which indicated that PSY proteins were conserved. Evolutionary relationship exhibited that DcPSY2 protein had the closest evolutionary relationship with the PSY protein in mahogany (Bixa orellana). DcPSY2 protein was a hydrophilic and non-secretory protein without signal peptide. It belonged to isoprenoid biosyn C1 superfamily and was distributed in various organelles. Secondary structure prediction demonstrated that DcPSY2 contained 53.65 % α-helix, 5.48 % β-fold, 12.79 % extended main chain and 28.08 % random curl. The results of qRT-PCR showed that DcPSY2 gene expression changed significantly in response to 4 kinds of abiotic stresses, including high temperature (38 ℃), low temperature (4 ℃), drought (200 g/L PEG6000) and high salt (200 mmol/L NaCl). Under high temperature or high salt treatments, the relative expression level of DcPSY2 reached the peak after 2 h. Whereas under low temperature or drought treatments, the expression level of DcPSY2 increased significantly at 1 h (P<0.05). The results provide a basic material for the functional study of DcPSY2 gene in carrot growth and its application study in stress resistance breeding.
DING Xu,WANG Ya-Hui,LI Tong, et al. Cloning and Expression Analysis of DcPSY2 Gene Under Abiotic Stress in Carrot (Daucus carota)[J]. 农业生物技术学报, 2019, 27(12): 2110-2119.
[1] 曹海燕. 2014. 过表达LcPSY提高洋桔梗耐盐性与LcAOC的克隆及表达[D]. 硕士学位论文, 天津大学, 导师: 季静, pp. 26-30.
(Cao H Y.2014. Overexpression of LcPSY improved eustoma grandiflorum salt tolerance and cloning and expression analysis of LcAOC[D]. Thesis for M.S., Tianjin University, Supervisor: Ji J, pp. 26-30.)
[2] 程洁. 2017. 甘薯IbPSY和IbSGR基因的克隆与功能分析[D]. 硕士学位论文, 西南大学, 导师: 廖志华, pp. 11-15.
(Cheng J.2017. Cloning and functional characterization of IbPSY and IbSGR from ipomoea batatas[D]. Thesis for M.S., Southwest University, Supervisor: Liao Z H, pp. 11-15.)
[3] 董新甜, 曹洪波, 张飞燕, 等. 2015. 遮光性套袋对黄肉桃类胡萝卜素合成及相关基因表达的影响[J]. 园艺学报, 42(04): 633-642.
(Dong X T, Cao H B, Zhang F Y, et al.2015. Effects of shading fruit with opaque paper bag on carotenogenesis and related gene expression in yellow-flesh peach[J]. Acta Horticulturae Sinica, 42(04): 633-642.)
[4] 费小红, 安保光, 赵宝华, 等. 2009. 类胡萝卜素参与水稻抗氧化胁迫的研究进展[J]. 现代农业科学, (1): 22-23.
(Fei X H, An B G, Zhao B H, et al. 2009. Carotenoid antioxidant coercion to participate in rice research[J]. Modern Agricultural Sciences, (1): 22-23.)
[5] 黄建峰, 秦于玲, 高爱平, 等. 2017. 芒果八氢番茄红素合成酶基因PSY的结构分析及功能预测[J]. 西南农业学报, 30(03):517-523.
(Huang J F, Qin Y L, Gao A P, et al.2017. Structural analysis and functional prediction of phytoene synthase gene PSY in mango[J]. Southwest China Journal of Agricultural Sciences, 30(03): 517-523.)
[6] 洪敏, 石丝, 何珊珊, 等. 2018. VIGS诱导PSY基因沉默对枇杷果实类胡萝卜素积累的影响[J]. 分子植物育种, 16(6): 1792-1797.
(Hong M, Shi S, He S S, et al.2018. Effects of VIGS-induced PSY gene silencing on carotenoid accumulation in fruit of Eriobotrya japonica lindl[J]. Molecular Plant Breeding, 16(6): 1792-1797.)
[7] 贾翠翠. 2015. 枸杞LcGGPS和LcGS基因的克隆及抗逆功能分析[D]. 硕士学位论文, 天津大学, 导师: 季静, pp. 48-49.
(Jia C C.2015. Isolation of LcGGPS and LcGS genes from Lycium chinense and functional analysis of the genes for abiotic stress tolerance[D]. Thesis for M.S., Tianjin University, Supervisor: Ji J, pp. 48-49.)
[8] 梁敏华, 杨震峰, 苏新国, 等. 2018. 桃果实八氢番茄红素合成酶基因的克隆与表达分析[J]. 浙江农业学报, 30(3): 399-405.
(Liang M H, Yang Z F, Su X G, et al.2018. Cloning and expression analysis of phytoene synthase gene from peach fruit[J]. Acta Agriculturae Zhejiangensis, 30(3): 399-405.)
[9] 刘歧茂, 丁永青, 迟瑞苹, 等. 2018. 设施胡萝卜产业化安全高效栽培技术[J]. 中国蔬菜, (1):92-94.
(Liu Q M, Ding Y Q, Chi R P, et al. 2018. Safe and efficient cultivation techniques for industrialization of facility carrots[J]. China Vegetables, (1): 92-94.)
[10] 孟超敏. 2008. 陆地棉逆境胁迫相关同源基因的克隆及其在非生物胁迫下的表达特性分析[D]. 博士学位论文, 南京农业大学, 导师: 郭旺珍, pp. 8-10.
(Meng C M.2008. Cloning of stress-related homolog genes and their responses to abiotic stresses in upland cotton[D]. Thesis for Ph.D., Nanjing Agricultural University, Supervisor: Guo W Z, pp. 8-10.)
[11] 阮婉贞. 2007. 胡萝卜的营养成分及保健功能[J]. 中国食物与营养, (6):51-53.
(Ruan W Z. 2007. Nutritional composition and health function of carrot[J]. Food and Nutrition in China, (6): 51-53.)
[12] 史馨怡, 刘丹, 陈黎黎,等. 2013. 逆境对红酵母发酵生产类胡萝卜素的促进作用[J]. 生物技术通报, (1):191-197. (Shi X Y, Liu D, Chen L L, et al. 2013. Enhanced carotenoids production by a Rhodotorula sp. under stress conditions[J]. Biotechnology Bulletin, (1): 191-197.)
[13] 王慧, 欧承刚, 庄飞云,等. 2014. 胡萝卜中类胡萝卜素积累与主要合成基因转录水平相关性分析[J]. 园艺学报, 41(12):2513-2520.
(Wang H, Ou C G, Zhuang F Y, et al.2014. Relationship of carotenoid accumulation and transcript of main genes in carotenoid biosynthesis in carrot[J]. Acta Horticulturae Sinica, 41(12): 2513-2520.)
[14] 吴焕章, 郭赵娟, 田朝辉. 2006. 我国胡萝卜生产中主要优良品种及其无公害高产栽培关键技术[J]. 当代蔬菜, (7):18-19.
(Wu H Z, Guo Z J, Tian Z H. 2006. Major excellent varieties in carrot production in China and key techniques for pollution-free high-yield cultivation[J]. Modern Vegetable, (7): 18-19.)
[15] 伍小松, 李军民, 吴家全,等. 2010. 胡萝卜栽培技术及其食用推广[J]. 中国果菜, (3): 24-25.
(Wu X S, Li J M, Wu J Q, et al. 2010. Carrot cultivation technology and its edible promotion[J]. China Fruit & Vegetable, (3): 24-25.)
[16] 薛蕾, 闫贵欣, 高桂珍,等. 2011. 白菜型油菜八氢番茄红素合酶基因BrPSY的克隆与序列分析[J]. 中国油料作物学报, (3): 210-215.
(Xue L, Yan G X, Gao G Z, et al.2011. Cloning and analyzing BrPSY gene of carotenoids biosynthesis in Brassica rapa L[J]. Chinese Journal of Oil Crop Sciences, 33(03): 210-215.)
[17] 肖政, 苏家乐, 孙晓波,等. 2018. 羊踯躅PSY基因的克隆及表达分析[J]. 中国农学通报, 34(30): 63-70.
(Xiao Z, Su J L, Sun X B, et al.2018. Cloning and expression of PSY gene from Rhododendron molle[J]. Chinese Agricultural Science Bulletin, 34(30): 63-70.)
[18] 尹航, 龚一富, 俞凯, 等. 2019. 绿色杜氏藻类胡萝卜素羟化酶基因家族分子特征及胁迫表达分析[J]. 农业生物技术学报, 27(02): 83-93.
(Yin H, Gong Y F, Yu K, et al.2019. Analysis of molecular characteristics and stress expression of carotenoid hydroxylase gene families in Dunaliella viridis[J]. Journal of Agricultural Biotechnology, 27(02): 83-93.)
[19] 尤丽佳, 郭新波, 付雪晴, 等. 2014. 干旱胁迫对转拟南芥Atpsy基因生菜的影响[J]. 上海农业学报, 30(5): 38-43.
(You L J, Guo X B, Fu X Q, et al.2014. Effects of drought stress on Atpsy transgenic lettuce[J]. Acta Agriculturae Shanghai, 30(5): 38-43.)
[20] 张建成, 王鹏飞, 郝燕燕, 等. 2014. 超量表达欧李ChPSY基因促进转基因番茄类胡萝卜素合成的研究[J]. 园艺学报, 41(08):1563-1572.
(Zhang J C, Wang P F, Hao Y Y, et al.2014. Overexpression of ChPSY gene from Cerasus humilis improved carotenoids synthesis in transgenic tomato[J]. Acta Horticulturae Sinica, 41(08): 1563-1572.)
[21] 郑阳霞, 季静, 王罡, 等. 2006. 宁夏枸杞八氢番茄红素合成酶基因的克隆与序列分析[J]. 林业科学, 42(5): 138-141.
(Zheng Y X, Ji J, Wang G, et al.2006. Cloning and sequence analysis of phytoene synthase cDNA from Lycium barbarum[J]. Scientia Silvae Sinicae, 42(5): 138-141.)
[22] Ahmad F T, Asenstorfer R E, Soriano I R, et al.2013. Effect of temperature on lutein esterification and lutein stability in wheat grain[J]. Journal of Cereal Science, 58(3): 408-413.
[23] Ahmadabadi M.2014. PSY is a rate-limiting enzyme in carotenoid biosynthesis pathway in maize (Zea mays) leaves[J]. New Cellular & Molecular Biotechnology Journal, 4(13): 71-75.
[24] Bang H, Pike L M, Patil B S, et al.2009. Structural gene expression in carotenoid biosynthetic pathway of carrot during root development[J]. Acta Horticulturae, 841(841): 475-478.
[25] Cárdenas P D, Gajardo H A, Huebert T, et al.2012. Retention of triplicated phytoene synthase (PSY) genes in Brassica napus L. and its diploid progenitors during the evolution of the Brassiceae[J]. Theoretical & Applied Genetics, 124(7): 1215-1228.
[26] Chamovitz D, Misawa N, Sandmann G, et al.2002. Molecular cloning and expression in Escherichia coli of a cyanobacterial gene coding for phytoene synthase, a carotenoid biosynthesis enzyme[J]. FEBS Letters, 296(3):305-310.
[27] Chavarriaga-Aguirre P, Prías M, López D, et al.2017. Molecular analysis of the expression of a crtB transgene and the endogenous psy2-y1 and psy2-y2 genes of cassava and their effect on root carotenoid content[J]. Transgenic Research, 26(5):639-651.
[28] Chikkappa G K, Tyagi N K, Venkatesh K, et al.2011. Analysis of transgene(s) (psy+crtⅠ) inheritance and its stability over generations in the genetic background of indica rice cultivar swarna[J]. Journal of Plant Biochemistry & Biotechnology, 20(1): 29-38.
[29] Campisi L, Fambrini M, Michelotti V, et al.2006. Phytoene accumulation in sunflower decreases the transcript levels of the phytoene synthase gene[J]. Plant Growth Regulation, 48(1): 79-87.
[30] Cidade L C, Oliveira T M D, Mendes A F S, et al.2012. Ectopic expression of a fruit phytoene synthase from Citrus paradisi macf promotes abiotic stress tolerance in transgenic tobacco[J]. Molecular Biology Reports, 39(12):10201-10209.
[31] Fu Z, Chai Y, Zhou Y, et al.2013. Natural variation in the sequence of PSY1 and frequency of favorable polymorphisms among tropical and temperate maize germplasm[J]. Theoretical and Applied Genetics, 126(4): 923-935.
[32] Han H, Li Y, Zhou S.2008. Overexpression of phytoene synthase gene from Salicornia europaeaalters response to reactive oxygen species under salt stress in transgenic Arabidopsis[J]. Biotechnology Letters, 30(8): 1501-1507.
[33] Kang B, Gu Q, Tian P, et al.2014. A chimeric transcript containing psy1 and a potential mRNA is associated with yellow flesh color in tomato accession PI 114490[J]. Planta, 240(5): 1011-1021.
[34] Li K, Xu J J, Wang L Y, et al.2016. Effects of acaulospora spinosa on plant growth and lycopene related genes (psyl and psy2) expression of tomato[J]. Chinese Journal of Applied Ecology, 27(02): 499-503.
[35] Liu Y J, Wang G L, Ma J, et al.2018. Transcript profiling of sucrose synthase genes involved in sucrose metabolism among four carrot (Daucus carota L.) cultivars reveals distinct patterns[J]. BMC Plant Biology, 18(1): 8.
[36] Ma J, Xu Z, Tan G, et al.2017. Distinct transcription profile of genes involved in carotenoid biosynthesis among six different color carrot (Daucus carota L.) cultivars[J]. Acta Biochimica Et Biophysica Sinica, 49(9): 817-826.
[37] Ma J, Li J, Xu Z, et al.2018. Transcriptome profiling of genes involving in carotenoid biosynthesis and accumulation between leaf and root of carrot (Daucus carota L.)[J]. Acta Biochimica Et Biophysica Sinica, 50(5): 481-490.
[38] Mahmood K, Kannangara R, Jørgensen K, et al.2014. Analysis of peptide PSY1 responding transcripts in the two Arabidopsis plant lines: Wild type and psy1r receptor mutant[J]. BMC Genomics, 15(1): 441.
[39] Mosher S, Kemmerling B.2013. PSKR1 and PSY1R-mediated regulation of plant defense responses[J]. Plant Signaling and Behavior, 8(5): e24119.
[40] Qin X, Coku A, Inoue K, et al.2011. Expression, subcellular localization, and regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.)[J]. Planta, 234(4): 737-748.
[41] Rohmer M.2003. Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis. Elucidation and distribution[J]. Pure & Applied Chemistry, 75(2-3): 375-388.
[42] Schäffer A A, Aravind L, Madden T L, et al.2001. Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements[J]. Nucleic Acids Research, 29(14): 2994-3005.
[43] Sellbom M, Bagby R M.2008. The validity and utility of the positive presentation management and negative presentation management scales for the revised NEO personality inventory[J]. Assessment, 15(2): 165-176.
[44] Tian C, Jiang Q, Wang F, et al.2015, Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves[J]. PLOS ONE, 10(2): e0117569.
[45] Xu Z S, Tan H W, Wang F, et al.2014. CarrotDB: A genomic and transcriptomic database for carrot[J]. Database: The Journal of Biological Databases and Curation, (bau096): 1-8.
[46] Yamaguchi-Shinozaki K, Shinozaki K.2006. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses[J]. Annual Review of Plant Biology, 57(1): 781-803.
[47] Young A J.1991. The photoprotective role of carotenoids in higher plants[J]. Physiologia plantarum, 83(4): 702-708.
