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作物学报 ›› 2013, Vol. 39 ›› Issue (01): 68-75.doi: 10.3724/SP.J.1006.2013.00068

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

GsPPCK1基因苜蓿植株的获得及其耐碱性分析

魏正巍,朱延明,化烨,才华*,纪巍,柏锡,王臻昱,文益东   

  1. 东北农业大学生命科学学院, 黑龙江哈尔滨 150030
  • 收稿日期:2012-02-13 修回日期:2012-09-05 出版日期:2013-01-12 网络出版日期:2012-11-14
  • 通讯作者: 才华, E-mail: caihuaneau@sohu.com
  • 基金资助:

    本研究由国家自然科学基金项目(31171578), 国家转基因生物新品种培育科技重大专项(2011ZX08004002-002), 黑龙江省高校科技创新团队建设计划(2011TD005)和黑龙江省博士后基金(LBH-Z12353)资助。

Transgenic Alfalfa with GsPPCK1 and Its Alkaline Tolerance Analysis

WEI Zheng-Wei,ZHU Yan-Ming,HUA Ye,CAI Hua*,JI Wei,BAI Xi,WANG Zhen-Yu,WEN Yi-Dong   

  1. College of Life Sciences, Northeast Agricultural University, Harbin 150030, China
  • Received:2012-02-13 Revised:2012-09-05 Published:2013-01-12 Published online:2012-11-14
  • Contact: 才华, E-mail: caihuaneau@sohu.com

摘要:

磷酸烯醇式丙酮酸羧化酶激酶(phosphoenolpyruvate carboxylase kinase, PPCK)是一种钙不依赖的丝氨酸/苏氨酸(Ser/Thr)类蛋白激酶, 参与碳氮代谢等多个生物学过程, 然而其在碱胁迫反应中的作用尚未见报道。本研究在前期野生大豆碱胁迫基因表达谱数据基础上, 采用同源克隆的方法分离野生大豆(Glycine soja)PPCK1基因, 该基因与大豆(Glycine max) PPCK1基因(AY374445)具有99%的相似性, 被命名为GsPPCK150 mmol L–1 NaHCO3 胁迫处理3 h, 根和叶中GsPPCK1基因上调表达, 属碱胁迫早期应答基因。通过农杆菌介导法对肇东苜蓿进行遗传转化, 并对RT-PCR阳性的超表达转基因株系进行耐碱性分析表明, 100 mmol L–1 NaHCO3处理15 d后转基因株系生长状态良好, 而非转基因对照株系明显萎蔫、失绿、甚至死亡; 转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05), 而叶绿素含量和根系活力显著高于非转基因对照(P<0.05), 说明超量表达GsPPCK1基因增强了苜蓿的耐碱能力。以上结果表明, GsPPCK1参于植物耐碱胁迫反应过程, 在碱胁迫基因工程研究领域具有良好的理论和实际应用意义。

关键词: 肇东苜蓿, GsPPCK1, 转基因株系, 耐碱性分析

Abstract:

Environmental stresses, such as drought, high salty and alkali, adversely affect plant growth and productivity. Plants adapt to these environmental stresses by inducing numerous genes at the transcriptional level and by protein phosphorylation. Phosphoenolpyruvate carboxylase kinase (PPCK) is a Ca2+ independent kinase in response to a range of signals in different plant tissues which plays a key role in the control of plant metabolism. As an important extension of our earlier studies summarized above on global transcriptome profiling of wild soybean under NaHCO3 treatment, an alkaline (NaHCO3) related gene GsPPCK1 was identified and subsequently cloned from Glycine soja, which has 99% similarity with PPCK1 of Glycine max (AY374445), named as GsPPCK1. Expression of GsPPCK1mRNA was induced by NaHCO3 stress in roots and leaves. GsPPCK1transcripts increased during 3 hour exposures to NaHCO3 stress. These results indicated that wild soybean PPCK1 was an early responded gene to alkaline stress. We transformed GsPPCK1 gene into alfalfa using a developed method, and transgenic alfalfa showed observably enhanced tolerance to NaHCO3 stress compared with wild-type plants. Transgenic alfalfa grew well in the conditions of 100 mmol L–1 NaHCO3, while wild type plants exhibited discoloration and stunted growth, or even death. There were significantly changes in malondialdehyde content and relative membrane permeability caused by saline-alkaline stress in non-transgenic lines compared to transgenic lines (P<0.05). Moreover, compared with non-transgenic, transgenic alfalfa had higher levels of chlorophyll content and root activity under alkali stress conditions. The result indicated that over-expression of GsPPCK1 in alfalfa could enhance alkaline tolerance. All results showed that GsPPCK1 gene could improve the tolerance of transgenic alfalfa to alkali stress; therefore, the study on this field is of significance not only in theory but also in practice.

Key words: Medicago sativa L. cv. Zhaodong, GsPPCK1, Transgenic alfalfa, Alkaline tolerance analysis

[1]Yin X-L(尹喜霖), Wang Y(王勇), Bai Y-C(柏钰春). Talk about alkalization of soil in Heilongjiang Province. Water Conserv Sci Technol Econ (水利科技与经济), 2004, 10(6): 361–363 (in Chinese with English Abstract)



[2]Besnard G, Pincon G, D’Hont A, Hoarau J Y, Cadet F, Offmann B. Characterisation of the phosphenolpyruvate carboxylase gene family in sugarcane (Saccharum spp.). Theor Appl Genet, 2003, 107: 470–478



[3]Feng R-Y(冯瑞云), Bai Y-F(白云凤), Li P(李平), Zhang W-F(张维锋), Wang Y-Y(王媛媛), Yang W-D(杨武德). Molecular cloning and expression analysis of C4 phosphoenolpyruvate carboxylase gene from A. hypochondriacus L. Acta Agron Sin (作物学报), 2011, 37(10): 1801–1808 (in Chinese with English Abstract)



[4]Du Z R, Aghoram K, Outlaw W H Jr. in vivo phosphorylation of phosphoenolpyruvate carboxylase and suppressed by abscisic acid. Archies Biochem Biophysics, 1997, 337: 345–350



[5]Wen X, Sato S J, Clemente T E, Chollet R. The PEP-carboxylase kinase gene family in Glycine max (GmPpcK1–4): an indepthmolecular analysiswith nodulated, non-transgenic and transgenic plants. Plant J, 2007, 49: 910–923



[6]Monreal J A, Lopez-Baena F J, Vidal J, Echevarria C, Garcia-Maurino S. Involvement of phospholipase D and phosphatidic acid in the light-dependent up-regulation of sorghum leaf phosphoenolpyruvate carboxylase-kinase. J Exp Bot, 2010, 61: 2819–2827



[7]Echevarria C, Garcia-Maurino S, Alvarez R, Soler A, Vidal J. Salt stress increases the Ca2+-independent phosphoenolpyruvate carboxylase kinase activity in Sorghum leaves. Planta, 2001, 214: 283–287



[8]Zhou B-Y(周宝元), Ding Z-S(丁在松), Zhao M(赵明). Alleviation of drought stress inhibition on photosynthesis by overexpression of PEPC gene in rice. Acta Agron Sin (作物学报), 2011, 37(1): 112–118 (in Chinese with English Abstract)



[9]Garcia-Maurino S, Monreal J A, Alvarez R, Vidal J, Echevarria C. Characterization of salt stress-enhanced phosphoenolpyruvate carboxylase kinase activity in leaves of Sorghum vulgare: independence from osmotic stress, involvement of ion toxicity and signi?cance of dark phosphorylation. Planta, 2003, 216: 648–655



[10]Antonio-Monreal J, Belen-Feria A, Vinardell J M, Vidal J, Echevarr?a C, Garc?a-Maurinoa S. ABA modulates the degradation of phosphoenolpyruvate carboxylase kinase in sorghum leaves. FEBS Lett, 2007, 581: 3468–3472



[11]Ge Y, Li Y, Zhu Y M, Bai X, Lv D K, Guo D J, Ji W, Cai H. Global transcriptome profiling of wild soybean (Glycine soja) roots under NaHCO3 treatment. BMC Plant Biol, 2010, 10: 153, http://www.biomedcentral.com/1471-2229/10/153



[12]Ge Y(葛瑛), Zhu Y-M(朱延明), Lü D-K(吕德康), Dong T-T(董婷婷), Wang W-S(王维世), Tan S-J(谭上进), Liu C-H(刘彩虹), Zou P(邹平). Research on responses of wild soybean to alkaline stress. Pratac Sci (草业科学), 2009, 26(2): 47–52 (in Chinese with English Abstract)



[13]Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohshi Y. Effcient Promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol, 1996, 37: 49–59



[14]Wang ZY, Song FB, Cai H, Zhu Y M, Bai X, Ji W, Li Y, Hua Y. Over-expressing GsGST14 from Glycine soja enhances alkaline tolerance of transgenic Medicago sativa. Biol Plant, 2011, 56: 516–520



[15]Wang Z-Y(王臻昱), Cai H(才华), Bai X(柏锡), Ji W(纪巍), Li Y(李勇), Wei Z-W(魏正巍), Zhu Y-M(朱延明), Isolation of GsGST19 from Glycine soja and analysis of saline-alkaline tolerance for transgenic Medicago sativa. Acta Agron Sin (作物学报), 2012, 38(6): 971–979 (in Chinese with English Abstract)



[16]Sheng H(盛慧), Zhu Y-M(朱延明), Li J(李杰), Bai X(柏锡), Cai H(才华). Genetic transformation of DREB2A gene into alfalfa. Pratac Sci (草业科学), 2007, 24(3): 40–45 (in Chinese with English Abstract)



[17]Bao A K, Wang S M, Wu G Q, Xi J J, Zhang J L, Wang C M Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). Plant Sci, 2009, 176: 232–240



[18]Hodges D M, DeLong J M, Forney C F, Prange R K. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 1999, 207: 604–611



[19]Zhang H X, Blumwald E. Transgenic salt-tolerance tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol, 2001, 19: 765–768



[20]Chen X-L(陈小龙), Chen C(陈灿), Zhou L(周莉). Determination and correlativity analysis of chlorophyll content at different developmental stages in rice. Mod Agric Sci Technol (现代农业科学), 2010, (17): 42–45 (in Chinese with English Abstract)



[21]Zhong X-H(钟旭华), Huang N-R(黄农荣). Preliminary study on the relationship between rice grain chalkiness and root activity at grain-filling stage. Chin J Rice Sci (中国水稻科学), 2005, 19(5): 471–474 (in Chinese with English Abstract)



[22]Chen S(陈硕), Chen J(陈珈). The structure and function of calcium-dependent protein kinases in plants. Chin Bull Bot (植物学通报), 2001, 18(2): 143–148 (in Chinese with English Abstract)



[23]Hodges D M, DeLong J M, Forney C F, Prange R K. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 1999, 207: 604−611



[24]Huang W(黄伟), Jia Z-K(贾志宽), Han Q-F(韩清芳). Effects of herbivore stress by Aphis medicaginis Koch on the Malondialdehyde contents and the activities of protective enzymes in different alfalfa varieties. Acta Ecol Sin (生态学报), 2007, 27(6): 2177–2183 (in Chinese with English Abstract)



[25]Ren L-H(任丽花), Yu H(余华), Luo T-Y(罗土炎), Huang M-M(黄敏敏), Cai N-T(蔡南通), Qiu Y-X(邱永祥). Effects of nitrogen in physiological and chloroplast characteristics of vegetable sweet potato. Fujian J Agric Sci (福建农业学报), 2011, 26(3): 360–364 (in Chinese with English Abstract)



[26]Fang L-F(方立锋), Ding Z-S(丁在松), Zhao M(赵明). Characteristics of drought tolerance in ppc overexpressed rice seedlings. Acta Agron Sin (作物学报), 2008, 34(7): 1220–1226 (in Chinese with English Abstract)



[27]Nakagawa T, Izumi T, Banba M, Umehara Y, Kouchi H, Izui K, Hata S. Characterization and expression analysis of genes encoding phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase of Lotus japonicus, a model legume. Mol Plant-Microbe Interact, 2003, 16: 281–288



[28]Zhu J K, Meinzer F C. Efficiency of C4 photosynthesis in Atriplex lentiformis under salinity stress. Functional Plant Biol, 1999, 26: 79–86



[29]Luo Y, Liu Y B, DongY X, Gao X Q, Zhang X S. Expression of a putative alfalfa helicase increases tolerance to abiotic stress in Arabidopsis by enhancing the capacities for ROS scavenging and osmotic adjustment. J Plant Physiol, 2009, 166: 385–394



[30]Zhu J K. Plant salt tolerance. Trends Plant Sci, 2001, 6: 66–71

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