作物学报 ›› 2015, Vol. 41 ›› Issue (11): 1663-1670.doi: 10.3724/SP.J.1006.2015.01663
刘芳**,刘睿洋**,彭烨,官春云*
LIU Fang**,LIU Rui-Yang**,PENG Ye,GUAN Chun-Yun*
摘要:
脂肪酸去饱和酶基因(FAD2)是控制植物体中油酸含量的关键基因,在甘蓝型油菜中有4个FAD2基因的拷贝,分别定位在A1、C1、A5、C5染色体上。参照前人研究方法,本文克隆了1个FAD2拷贝基因,依据油菜基因组数据库信息,将其定位到C1染色体上,命名为BnFAD2-C1,其开放阅读框为1155 bp。采用RACE (rapid-amplification of cDNA ends)技术获得了175 bp的5¢ UTR序列和212 bp的3¢ UTR序列。采用荧光定量PCR技术研究发现,BnFAD2-C1在根、花和角果皮中仅保持本底水平的表达,在种子发育中期呈现高效表达,具有种子特异性诱导表达的特征。根据甘蓝和油菜基因组数据库信息,同源克隆到BnFAD2-C1基因的启动子(promoter)和内含子(intron)序列,并通过PLACE和PlantCARE网站分析,初步预测到调控该基因转录的潜在顺式作用元件。通过茉莉酸诱导处理,BnFAD2-C1基因表达量发生变化,推断茉莉酸在BnFAD2-C1基因的表达过程中可能发挥一定的调控作用。
| [1]Okuley J, Lightner J, Feldmann K Yadav N, Lark E, Browseai J. Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell, 1994, 6: 147–158[2]Terés S, Barceló-Coblijn G, Benet M, lvarez R A, Bressani R, Halver J E, Escriba P V. Oleic acid content is responsible for the reduction in blood pressure induced by olive oil. Proc Natl Acad Sci USA, 2008, 105: 13811–13816[3]Wijesundera C, Ceccato C, Fagan P, Zhiping S, Burton W, Salisbury P. Canola quality Indian mustard oil (Brassica juncea) is more stable to oxidation than conventional Canola oil (Brassica napus). J Am Oil Chem Soc, 2008, 85: 693–699[4]Hu X, Sullivan-Gilbert M, Gupta M, Gupta M, and Thompson S A. Mapping of the loci controlling oleic and linolenic acid contents and development of FAD2 and fad3 allele-specific markers in canola (Brassica napus L.). Theor Appl Genet, 2006, 113: 497–507[5]Tang G Q, Novitzky W P, Carol Griffin H, Huber S C, Dewey R E. Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation. Plant J, 2005, 44: 433–446[6]Zhang D, Pirtle I L, Park S J, Nampaisansuk M, Neogi P, Wanjie S W, Pirtle R M, Kent D. C. Identification and expression of a new delta-12 fatty acid desaturase (FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants. Plant Physiol Biochem, 2009, 47: 462–471[7]Jung S, Powell G, Moore K. The high oleate trait in the cultivated peanut (Arachis hypogaea L.): II. Molecular basis and genetics of the trait. Mol Gen Genet, 2000, 263: 806–811[8]Jin U H, Lee J W, Chung Y S. Characterization and temporal expression of a ω-6 fatty acid desaturase cDNA from sesame (Sesamum indicum L.) seeds. Plant Sci, 2001, 161: 935–941[9]Jung J H, Hyojin K, Young S G, Saet B L, Cheol G H, Hyun U K, Mi C S. Identification of functional BrFAD2-1 gene encoding microsomal delta-12 fatty acid desaturase from Brassica rapa and development of Brassica napus containing high oleic acid contents. Plant Cell Rep, 2011, 30: 1881–1892[10]Xiao G, Zhang Z Q, Yin C F, Liu R Y, Wu X M, Tan T L, Chen S Y, Lu C M, Guan C Y. Characterization of the promoter and 5′-UTR intron of oleic acid desaturase (FAD2) gene in Brassica napus. Gene, 2014, 545: 45–55[11]Xiong Z, Gaeta R T, Pires J C. Homoeologous shuffling and chromosome compensation maintain genome balance in resynthesized allopolyploid Brassica napus. Proc Natl Acad Sci USA, 2011, 108: 7908–7913[12]Yang Q Y, FanC C, Guo Z H, Qin J, Wu J Z, Li Q Y, Fu T D, Zhou Y M. Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents. Theor Appl Genet, 2012, 125: 715–729[13]Lee K R, Sohn S I, Jung J H, Kima S H, Roha K H, Kima J B, Suhb M C, Kim H U. Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea. Gene, 2013, 531: 253–262[14]陈苇, 李劲峰, 董云松, 李根泽, 寸守铣, 王敬乔. 甘蓝型油菜Fad2基因的RNA干扰及无筛选标记高油酸含量转基因油菜新种质的获得. 植物生理与分子生物学学报, 2006, 32: 665–671Chen W, Li J F, Dong Y S, Li G Z, Cun S X, Wang J Q. Interferring of Fad2 gene using RNAi in Brassica napus and obtaining new germplasm of high oleic acid canola with no marker. Acta Phytophysiol Sin, 2006, 32: 665–671[15]肖钢, 张宏军, 彭琪, 官春云. 甘蓝型油菜油酸脱氢酶基因(FAD2)多个拷贝的发现及分析. 作物学报, 2008, 34: 1563–1568Xiao G, Zhang H J, Peng Q, Guan C Y. Screening and analysis of mutiple copy of oleate desaturase gene (FAD2) in Brassica napus. Acta Agron Sin, 2008, 34: 1563–1568[16]Kiefer E, Heller W, Ernst D. A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites. Plant Mol Biol Rep, 2000, 18: 33–39[17]高建芹, 浦惠明, 龙卫华, 胡茂龙, 戚存扣. 高油酸甘蓝型油菜油酸积累动态. 中国油料作物学报, 2012, 34: 359–365Gao J Q, Pu H M, Long W H, Hu M L, Qi C K. Dynamics of oleic acid conents in organs of high-oleic rapeseed lines. Chin J of Oil Crop Sci, 2012, 34: 359–365[18]Vicente-Carbajosa J, Moose S P, Parsons R L, and Robert J. A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2. Proc Natl Acad Sci USA, 1997, 94: 7685–7690[19]Ezcurra I, Wycliffe P, Nehlin L. Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis-elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G-box. Plant J, 2000, 24: 57–66[20]Parra G, Bradnam K, Rose A B. Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants. Nucl Acids Res, 2011, 39: 5328–5337[21]Wasternack C, Hause B. Jasmonates: an update on biosynthesis, perception, signal transduction and action in plant stress response, growth and development. Ann Bot, 2007,100: 681–697[22]Xu D, McElroy D, Thornburg R W, Ray W. Systemic induction of a potato pin2 promoter by wounding, methyl iasmonate, and abscisic acid in transgenic rice plants. Plant Mol Biol, 1993, 22: 573–588[23]Jusoh M, Loh S H, Chuah T S. Elucidating the role of jasmonic acid in oil accumulation, fatty acid composition and gene expression in Chlorella vulgaris (Trebouxiophyceae) during early stationary growth phase. Algal Res, 2015, 9: 14–20[24]Gundlach H, Müller M J, Kutchan T M, Zenk M H. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc Natl Acad Sci USA, 1992, 89: 2389–2393[25]Mangas S, Bonfill M, Osuna L, Moyano E, Tortoriello J, Cusido R M, Piñol M T, Palazón J. The effect of methyl jasmonate on triterpene and sterol metabolisms of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants. Phytochemistry, 2006, 67: 2041–2049[26]Ren C G, Dai C C. Jasmonic acid is involved in the signaling pathway for fungal endophyte-induced volatile oil accumulation of Atractylodes lancea plantlets. BMC Plant Biol, 2012, 12: 128[27]Rouster J, Leah R, Mundy J. Identification of a methyl jasmonate-responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain. Plant J, 1997, 11: 513–523 |
| [1] | 马毅娜, 吴晓明玉, 李藕琪, 王圆, 陈丽, 张盈川, 赵伦, 文静, 傅廷栋, 沈金雄. Bna-miR1040-EIF3A模块调控油菜开花时间的功能研究[J]. 作物学报, 2026, 52(2): 349-362. |
| [2] | 王彬, 蒙姜宇, 邱浩良, 贺亚军, 钱伟. 甘蓝型油菜BnaDUF579基因家族的鉴定与表达模式分析[J]. 作物学报, 2025, 51(8): 2100-2110. |
| [3] | 夏琦, 郭滢, 王坤美, 王思忆, 巨建业, 彭雅雯, 刘忠松, 夏石头. 甘蓝型油菜种子和种皮中水杨酸含量与原花色素积累的关系研究[J]. 作物学报, 2025, 51(5): 1189-1197. |
| [4] | 王晓琳, 刘忠松, 康雷, 杨柳. 甘蓝型油菜角果长度和每角粒数基因定位以及角果皮转录组动态分析[J]. 作物学报, 2025, 51(4): 888-899. |
| [5] | 张琴, 戴成, 马朝芝. 生长素响应报告基因转化甘蓝型油菜及各组织GUS动态信号分析[J]. 作物学报, 2025, 51(3): 667-675. |
| [6] | 孙程明, 周晓婴, 陈锋, 张维, 王晓东, 彭琦, 郭月, 高建芹, 胡茂龙, 付三雄, 张洁夫. 长链非编码RNA (lncRNA)在甘蓝型油菜分枝角度调控中的功能分析与预测[J]. 作物学报, 2025, 51(3): 559-567. |
| [7] | 黄绒, 周渠晨, 陈楚铭, 罗倩, 易东, 杜常欢, 黄祥宇, 盛锋, 杜雪竹. 过表达BnNRT2.3-like对油菜氮素吸收利用及产量的影响[J]. 作物学报, 2025, 51(12): 3184-3197. |
| [8] | 巨建业, 杨柳, 陈浩, 康雷, 夏石头, 刘忠松. 单细胞核转录组分析揭示油菜种皮分化过程和种子颜色差异原因[J]. 作物学报, 2025, 51(11): 2860-2874. |
| [9] | 张雯, 李玉, 王创, 石磊, 丁广大. 甘蓝型油菜磷转运蛋白BnaPT48的功能研究[J]. 作物学报, 2025, 51(11): 2983-2995. |
| [10] | 胡志康, 舒雨, 王会, 杨莹莹, 廖俊宇, 刘佳, 成洪涛, 郭晨, 张园园, 刘胜毅, 胡琼, 梅德圣, 李超. 甘蓝型油菜苗期耐碱性种质综合鉴定与评价[J]. 作物学报, 2025, 51(10): 2681-2692. |
| [11] | 王晨, 贺丹, 姚敏, 邱萍, 何昕, 熊兴华, 康雷, 刘忠松, 钱论文. 基于转录组分析鉴定甘蓝型油菜开花候选基因以及BnaCOR27功能验证[J]. 作物学报, 2025, 51(10): 2693-2704. |
| [12] | 徐林珊, 郜耿东, 王宇, 王家星, 杨吉招, 武亚瑞, 张宵寒, 常影, 李真, 谢雄泽, 龚德平, 王晶, 葛贤宏. 甘蓝型油菜漆酶基因家族成员表达模式及与茎秆抗折力的关联分析[J]. 作物学报, 2025, 51(1): 134-148. |
| [13] | 李嘉欣, 黄莹, 吴潞梅, 赵伦, 易斌, 马朝芝, 涂金星, 沈金雄, 傅廷栋, 文静. 甘蓝型油菜BnaSLY1基因进化分析及功能研究[J]. 作物学报, 2025, 51(1): 44-57. |
| [14] | 杨煜琛, 靳雅荣, 骆金婵, 祝鑫, 李葳航, 贾纪原, 王小珊, 黄德均, 黄琳凯. 珍珠粟WD40基因家族鉴定及表达特征分析[J]. 作物学报, 2024, 50(9): 2219-2236. |
| [15] | 钟元, 朱天宇, 戴成, 马朝芝. 耐亚磷酸盐除草剂转基因油菜的创建和抗性评价[J]. 作物学报, 2024, 50(5): 1158-1171. |
|
||