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作物学报 ›› 2014, Vol. 40 ›› Issue (02): 253-263.doi: 10.3724/SP.J.1006.2014.00253

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗中性/碱性转化酶基因(SoNIN1)的克隆和表达分析

牛俊奇1,2,王爱勤1,2,黄静丽1,2,杨丽涛1,2,3*,李杨瑞2,3,4*   

  1. 1 广西大学农学院, 广西南宁 530005; 2 亚热带农业生物资源保护与利用国家重点实验室, 广西南宁 530005; 3 中国农业科学院甘蔗研究中心 / 广西农业科学院甘蔗研究所 / 农业部广西甘蔗生物技术与遗传改良重点实验室 / 广西甘蔗遗传改良重点实验室, 广西南宁 530007; 4 广西作物遗传改良生物技术重点开放实验室, 广西南宁 530007
  • 收稿日期:2013-03-01 修回日期:2013-09-16 出版日期:2014-02-12 网络出版日期:2013-10-22
  • 通讯作者: 杨丽涛, E-mail: liyr@gxu.edu.cn, Tel: 0771-3236407; 李杨瑞, E-mail: liyr@gxaas.net, Tel: 0771-3247689
  • 基金资助:

    本研究由国家高技术研究发展计划(863计划)项目(2013AA102604), 国家国际合作项目(2008DFA30600, 2009DFA30820, 2013DFA31600), 广西自然科学基金重点项目(2011GXNSFF018002), 广西科学研究与技术开发计划项目(桂科产1123008-1, 桂科攻1222009-1B, 桂科合13999002, 桂科合1347004-2)和广西农科院团队项目(桂农科2011YT01)资助。

Cloning and Expression Analysis of Sugarcane Alkaline/Neutral Invertase (SoNIN1) Gene

NIU Jun-Qi1,2,HUANG Jing-Li1,2,WANG Ai-Qing1,2,YANG Li-Tao1,2,3,*,LI Yang-Rui2,3,4,*   

  1. 1 Agricultural College, Guangxi University, Nanning 530005, China; 2 State Key Laboratory of Subtropical Bioresources Conservation and Utilization, Nanning 530005, China; 3 Sugarcane Research Center, Chinese Academy of Agricultural Sciences / Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences / Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture/ Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, China; 4 Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Nanning 530007, China
  • Received:2013-03-01 Revised:2013-09-16 Published:2014-02-12 Published online:2013-10-22
  • Contact: 杨丽涛, E-mail: liyr@gxu.edu.cn, Tel: 0771-3236407; 李杨瑞, E-mail: liyr@gxaas.net, Tel: 0771-3247689

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摘要：

中性/碱性转化酶(Alkaline/Neutral Invertase)能不可逆地催化蔗糖分解为葡萄糖和果糖, 在植物生长发育中起重要作用。本研究采用RT-PCR和RACE技术从甘蔗品种GT28心叶中克隆到甘蔗中性/碱性转化酶基因全长cDNA序列, 基因命名为SoNIN1。该基因全长2289 bp (GenBank登录号为JX109944), 开放阅读框为1812 bp, 编码603个氨基酸, 相对分子量为67.79 kD, 等电点为6.41。具有中性/碱性转化酶保守结构域, N-端不含跨膜结构和信号肽。其编码氨基酸序列与玉米、水稻和黑麦草的亲缘关系较近, 属于同一进化分支。利用染色体步移技术克隆到SoNIN1基因5¢侧翼启动子序列, 长度为1174 bp (GenBank登录号为KC854419)。启动子序列含有多个CAAT-box和TATA-box等基本作用元件, 参与分生组织特异性激活, 参与干旱诱导的MYB结合位点, 脱落酸、茉莉酸甲酯和赤霉素响应等作用元件。利用实时荧光定量PCR技术, 在生理成熟期甘蔗茎、叶、花序和芽中均能检测到SoNIN1的表达, 其表达量在叶中最高, 芽中次之, 而在+1节间最低。苗期根和叶中SoNIN1基因都受PEG和NaCl诱导表达。

关键词: 甘蔗, 中性/碱性转化酶, 启动子, 克隆, 基因表达

Abstract:

Neutral/alkaline invertase irreversibly catalyzes sucrose into glucose and fructose, and plays an important regulating role in plant growth and development. In the present study, the whole length of neutral/alkaline invertase gene cDNA was cloned and sequenced from leaf of sugarcane variety GT28 with RT-PCR (reverse transcription-polymerase chain reaction) and RACE (rapid amplification of cDNA ends), and named as SoNIN1. The gene consists of 2289 bp with an ORF (open reading frame) of 1812 bp encoding a polypeptide of 603 amino acids with calculated protein molecular weight of 67.79 kD, and an isoelectric point of pH 6.41. SoNIN1 gene encodes a protein that is close to that of Zea mays, Oryza sativa, andLolium perenne, belonging to the same evolutionary branch. The putative protein contains the conserved domain of alkaline/neutral invertase, and no membrane-spanning domain and signal peptide at N-end. The gene promoter sequence was cloned by using genome walking, which contains the basic components of CAAT-boxes and TATA-boxes, as well as specific acting elements such as cis-regulatory element involved in meristem activation, methyl jasmonate response and circadian rhythm, plus drought-induced MYB binding sites, abscisic acid response element, gibberellin response element, etc. Real-time PCR results showed that expression of SoNIN1 could be detected in the roots, stalks, leaves, flowers and buds, among which the highest was found in leaves, the medium in buds, and the lowestin internode +1 (enrolled by the top visible dewlap leaf). The expression of SoNIN1 gene in roots and leaves of sugarcane seedling was induced by PEG and NaCl.

Key words: Sugarcane, Alkaline/neutral invertase, Promoter, Cloning, Gene expression

[1]蓝基贤, 唐朝荣. 高等植物中转化酶生理生化特性的研究进展. 热带作物学报, 2012, 33: 1702–1707



Lan J X, Tang C R. Advance on the studies of physiological and biochemical characteristics of invertase in higher plants. Chin J Trop Crops, 2012, 33: 1702–1707 (in Chinese with English abstract)



[2]李肖蕖, 王建设, 张根发. 植物蔗糖转化酶及其基因表达调控研究进展. 园艺学报, 2008, 35: 1384–1392



Li X Q, Wang J S, Zhang G F. Advanced in plant invertase and regulation of gene expression. Acta Hortic Sin, 2008, 35: 1384–1392 (in Chinese with English abstract)



[3]Sturm A. Invertases: primary structures, functions and roles in plant development and sucrose partitioning. Plant Physiol, 1999, 121: 1–7



[4]Zhu Y J, Albert H H, Moore P H. Differential expression of soluble acid invertase genes in the shoots of high-sucrose and low-sucrose species of Saccharum and their hybrids. Aust J Plant Physiol, 2000, 27: 193–199



[5]郭家文,刘少春, 张跃彬. 李文凤成熟期不同基因型甘蔗糖分和叶片酶活性的变化. 中国糖料, 2006, (3): 16–21



Guo J W, Liu S C, Zhang Y B, Li W F. Studies on sucrose content and foliar enzyme activity of sugarcane during mature period. Sugar Crops China, 2006, (3): 16–21 (in Chinese with English abstract)



[6]Pan Y Q, Lou H L, Li Y R. Soluble acid invertase and sucrose phosphate synthase: key enzymes in regulating sucrose accumulation in sugarcane stalk. Sugar Technol, 2009, 11: 28–33



[7]Sachdeva M, Mann A P S, Batta S K. Sucrose metabolism and expression enzyme activities in low and high storing sugarcane  genotypes of key sucrose. Sugar Technol, 2003, 5: 265–271



[8]Barratt D H P, Derbyshire P, Findlay K, Pike M, Wellner N, Lunn J, Feil R, Simpson R, Maule A J, Smith A M. Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase. Proc Natl Acad Sci USA, 2009, 106: 13124–13129



[9]Gallagher J A, Pollock C J. Isolation and characterization of a cDNA clone from Lolium temulentum L. encoding for a sucrose hydrolytic enzyme which shows alkaline/neutral invertase activity. J Exp Bot, 1998, 49: 789–795



[10]Vargas W A, Pontis H G, Salerno G L. Differential expression of alkaline and neutral invertases in response to environmental stresses: characterization of an alkaline isoform as a stress-response enzyme in wheat leaves. Planta, 2007, 226: 1535–1545



[11]刘术金, 李旖璠, 唐朝荣. 橡胶树2个胶乳转化酶基因的原核表达. 热带作物学报, 2010, 31: 1091–1097



Liu S J, Li Y F, Tang C R. Construction of prokaryotic expression vectors for two invertases from latex and optimization of their expression conditions. Chin J Trop Crops, 2010, 31: 1091–1097 (in Chinese with English abstract)



[12]Bocock P N, Morse A M, Dervinis C, Davis J M. Evolution and diversity of invertase genes in Populus trichocarpa. Planta, 2008, 227: 565–576



[13]姜立智, 林长发, 梁宗锁, 卫春, 杨金水. 水稻蔗糖转化酶基因的克隆及其功能的初步探讨. 复旦大学学报, 2003, 42: 588–592



Jiang L Z, Lin C F, Liang Z S, Wei C, Yang J S. Cloning and characterization of a cDNA encoding putative neutral/ alkaline invertase from rice (Oryza sativa L.). J Fudan Univ, 2003, 42: 588–592 (in Chinese with English abstract)



[14]Murayama S J, Handa H. Genes for alkaline/neutral invertase in rice: alkaline/neutral invertases are located in plant mitochondria and also in plastids. Planta, 2007, 225: 1193–1203



[15]Xiang L, Roy L K, Bolouri-Moghaddam M R, Vanhaecke M, Lammens W, Rolland F, Ende W V D. Exploring the neutral invertase–oxidative stress defense connection in Arabidopsis thaliana. J Exp Bot, 2011, 62: 3849–3862



[16]Szarka A, Horemans N, Passarella S, Tarcsay Á, Örsi F, Salgó A, Bánhegyi G. Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (Helianthus tuberosus L.) tubers. Planta, 2008, 228: 765–775



[17]Vargas W A, Pontis H G, Salerno G. New insights on sucrose metabolism: evidence for an active A/N-Inv in chloroplasts uncovers a novel component of the intracellular carbon trafficking. Planta, 2008, 227: 795–807



[18]Jia L Q, Zhang B T, Mao C Z, Li J H, Wu Y R, Wu P, Wu Z C. OsCYT-INV1 for alkaline/neutral invertase is involved in root cell evelopment and reproductivity in rice (Oryza sativa L.). Planta, 2008, 228: 51–59



[19]Yao S G, Mushika J, Taketa S, Ichii M, Masahiko Ichii. The short-root mutation srt5 de?nes a sugar-mediated root growth in rice (Oryza sativa L.). Plant Sci, 2004, 167: 49–54



[20]Yao S G, Kodama R, Wang H, Ichii M, Taketa S, Yoshida H. Analysis of the rice SHORT-ROOT5 gene revealed functional diversi?cation of plant neutral/alkaline invertase family. Plant Sci, 2009, 176: 627–634



[21]Welham T, Pike J, Horst1 I, Flemetakis E, Katinakis P, Kaneko T, Sato S, Tabata S, Perry J, Parniske M, Wang T L. A cytosolic invertase is required for normal growth and cell development in the model legume, Lotus japonicus. J Exp Bot, 2009, 60: 3353–3365



[22]Qi X P, Wu Z C, Li J H, Mo X R, Wu S H, Chu J, Wu P. AtCYT-INV1, a neutral invertase, is involved in osmotic stress-induced inhibition on lateral root growth in Arabidopsis. Plant Mol Biol, 2007, 64: 575–587



[23]刘海斌, 何红, 邓展云, 张革民, 李杨瑞, 方锋学, 贤武, 刘晓静, 方位宽, 闭少玲, 容凤玉, 唐红琴. 特高糖抗寒甘蔗新品种桂糖28的选育. 中国糖料, 2010, (1): 13–15



Liu H B, He H, Deng Z Y, Zhang G M, Li Y R, Fang F X, Xian W, Liu X J, Fang W K, Bi S L, Rong F Y, Tang H Q. Breeding of new sugarcane variety Guitang 28. Sugar Crops China, 2010, (1): 13–15 (in Chinese with English abstract)



[24]应雄美, 蔡青, 毕艳, 刘新龙, 马丽, 毛钧, 陆鑫. 两种不同甘蔗基因组DNA提取方法的比较. 中国糖料, 2009, (4): 22–23



Ying X M, Cai Q, Bi Y, Liu X L, Ma L, Mao J, Lu X. Comparison between two sugarcane genome DNA extraction method. Sugar Crops China, 2009, (4): 22–23 (in Chinese with English abstract)



[25]阙友雄, 许莉萍, 徐景升, 张积森, 张木清, 陈如凯. 甘蔗基因表达定量PCR分析中内参基因的选择. 热带作物学报, 2009, 30: 274–278



Que Y X, Xu L P, Xu J S, Zhang J S, Zhang M Q, Chen R K. Selection of control genes in real-time qPCR analysis of gene expression in sugarcane. Chin J Trop Crops, 2009, 30: 274–278 (in Chinese with English abstract)



[26]Iskandar H M, Simpson R S, Casu R E, Bonnett G D, Maclean D J, Manners J M. Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Mol Biol Rep, 2004, 22: 325–337



[27]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods, 2001, 25: 402–408



[28]Bosch S, Grof C P L, Botha F C. Expression of neutral invertase in sugarcane. Plant Sci, 2000, 166: 1125–1133



[29]Rose S, Botha F C. Distribution patterns of neutral invertase and sugar content in sugarcane internodal tissues. Plant Physiol Biochem, 2000, 38: 819−824



[30]Rossouw D, Bosch S, Kossmann J, Botha F C, Groenewald J H. Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation. Funct Plant Biol, 2007, 34: 490–498



[31]Rossouw D, Kossmann J, Botha F C, Groenewald J H. Reduced neutral invertase activity in the culm tissues of transgenic sugarcane plants results in a decrease in respiration and sucrose cycling and an increase in the sucrose to hexose ratio. Funct Plant Biol, 2010, 37: 22–31



[32]聂丽娜, 夏兰琴, 徐兆师, 高东尧, 李琳, 于卓, 陈明, 李连城, 马有志. 植物基因启动子的克隆及其功能研究进展. 植物遗传资源学报, 2008, 9: 385–391



Nie L N, Xia L Q, Xu Z S, Gao D Y, Li L, Yu Z, Chen M, Li L C, Ma Y Z. Progress on cloning and functional study of plant gene promoters. J P1ant Genet Resour, 2008, 9: 385–391 (in Chinese with English abstract)
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