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] | 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371. |
[2] | 秦璐, 韩配配, 常海滨, 顾炽明, 黄威, 李银水, 廖祥生, 谢立华, 廖星. 甘蓝型油菜耐低氮种质筛选及绿肥应用潜力评价[J]. 作物学报, 2022, 48(6): 1488-1501. |
[3] | 袁大双, 邓琬玉, 王珍, 彭茜, 张晓莉, 姚梦楠, 缪文杰, 朱冬鸣, 李加纳, 梁颖. 甘蓝型油菜BnMAPK2基因的克隆及功能分析[J]. 作物学报, 2022, 48(4): 840-850. |
[4] | 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607. |
[5] | 王瑞, 陈雪, 郭青青, 周蓉, 陈蕾, 李加纳. 甘蓝型油菜白花基因InDel连锁标记开发[J]. 作物学报, 2022, 48(3): 759-769. |
[6] | 余慧芳, 张卫娜, 康益晨, 范艳玲, 杨昕宇, 石铭福, 张茹艳, 张俊莲, 秦舒浩. 马铃薯CrRLK1Ls基因家族的鉴定及响应晚疫病菌信号的表达分析[J]. 作物学报, 2022, 48(1): 249-258. |
[7] | 王艳花, 刘景森, 李加纳. 整合GWAS和WGCNA筛选鉴定甘蓝型油菜生物产量候选基因[J]. 作物学报, 2021, 47(8): 1491-1510. |
[8] | 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798. |
[9] | 唐鑫, 李圆圆, 陆俊杏, 张涛. 甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究[J]. 作物学报, 2021, 47(5): 983-990. |
[10] | 周新桐, 郭青青, 陈雪, 李加纳, 王瑞. GBS高密度遗传连锁图谱定位甘蓝型油菜粉色花性状[J]. 作物学报, 2021, 47(4): 587-598. |
[11] | 李书宇, 黄杨, 熊洁, 丁戈, 陈伦林, 宋来强. 甘蓝型油菜早熟性状QTL定位及候选基因筛选[J]. 作物学报, 2021, 47(4): 626-637. |
[12] | 张春, 赵小珍, 庞承珂, 彭门路, 王晓东, 陈锋, 张维, 陈松, 彭琦, 易斌, 孙程明, 张洁夫, 傅廷栋. 甘蓝型油菜千粒重全基因组关联分析[J]. 作物学报, 2021, 47(4): 650-659. |
[13] | 唐婧泉, 王南, 高界, 刘婷婷, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. 甘蓝型油菜SnRK基因家族生物信息学分析及其与种子含油量的关系[J]. 作物学报, 2021, 47(3): 416-426. |
[14] | 蒙姜宇, 梁光伟, 贺亚军, 钱伟. 甘蓝型油菜耐盐和耐旱相关性状的QTL分析[J]. 作物学报, 2021, 47(3): 462-471. |
[15] | 魏丽娟, 申树林, 黄小虎, 马国强, 王曦彤, 杨怡玲, 李洹东, 王书贤, 朱美晨, 唐章林, 卢坤, 李加纳, 曲存民. 锌胁迫下甘蓝型油菜发芽期下胚轴长的全基因组关联分析[J]. 作物学报, 2021, 47(2): 262-274. |
|