欢迎访问作物学报,今天是

作物学报 ›› 2015, Vol. 41 ›› Issue (03): 507-514.

• 研究简报 • 上一篇    

稗草(Echinochloa crusgalli)根型ppc 基因对水稻的遗传转化及其对光合速率的调节效应

张桂芳1,2,丁在松1,赵明1,*   

  1. 1 中国农业科学院作物科学研究所 / 农业部作物生理生态重点实验室, 北京 100081; 2 北京师范大学生命科学学院 / 北京师范大学学报(自然科学版)编辑部, 北京 100875
  • 收稿日期:2014-05-09 修回日期:2014-12-19 出版日期:2015-03-12 网络出版日期:2015-01-28
  • 通讯作者: 赵明, E-mail: zhaomingcau@vip.tom.com
  • 基金资助:

    本研究由中国农业科学院创新工程项目资助。

Transformation of Barnyardgrass (Echinochloa crusgalli) Root Type Phosphoenolpyruvate Carboxylase Gene into Rice (Oryza sativa) Plants and Their Effects on Photosynthesitic Gas Exchange

ZHANG Gui-Fang1,2,DING Zai-Song1,ZHAO Ming1,*   

  1. 1 Institute of Crop Science, Chinese Academy of Agricultural Sciences / Key Laboratory of the Ministry of Agriculture Crop Physiology and Ecology,
    Beijing 100081,China; 2 College of Life Science / Editorial Department of Journal of Beijing Normal University (Natural Science), Beijing 100875, China
  • Received:2014-05-09 Revised:2014-12-19 Published:2015-03-12 Published online:2015-01-28
  • Contact: 赵明, E-mail: zhaomingcau@vip.tom.com

摘要:

稗草(Echinochloa crusgalli)是稻田中的C4 光合型杂草, 为了探索稗草ppc 基因(Eppc)对水稻遗传转化的可行性及其对光合速率的调节效应, 首次将含有稗草根型磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)基因ppc cDNA 的2 个植物表达载体pUbi-Eppc、pRbcS-Eppc 通过农杆菌介导法对水稻进行了遗传转化。对分化植株进行的PCR、RT-PCR、克隆测序和Western 杂交等结果均表明稗草ppc 基因已经整合到了水稻基因组中, 并且在转录和翻译水平都得到了表达。转基因水稻PEPC 活性和气体交换参数测定结果表明T0 代多数植株的PEPC 活性高于对照, 最高达到了对照的5.85 倍; T0 代大多数转基因植株叶片的净光合速率(Pn)比对照提高了20.00%, 最大地提高了47.16%, 同时叶片水分利用效率(WUE)也得到增强; T6 代大部分转化植株的PEPC 活性及Pn 仍保持高于对照, 本研究表明C3 根型ppc 基因过量表达也可以提高水稻的Pn, 且证明稗草PEPC 对光合作用具有较强的调节作用。

关键词: 稗草, 根型Eppc 基因, 水稻, PEPC 活性, 净光合速率, 水分利用效率

Abstract:

Barnyardgrass (Echinochloa crusgalli) is a C4 weed commonly found in rice field. To fully utilize the photosynthestic potential of Barnyardgrass C4 gene, we transformed Barnyardgrass root Phosphoenolpyruvate Carboxylase gene into rice plant with vectors contained promoters of Ubiqitin gene and Rubisco small unit gene by Agrobactirium- mediated transformation. Both marker genes Hygr and ppc were detected by PCR in regenerated plants. RT-PCR and Western blot analysis confirmed that the ppc gene was incorporated into rice plant and expressed with stable transcripts and proteins. PEPC activity as measured in most of the transgenic rice plants was higher than that in control, being up to 5.85-fold of that in untransformed rice. At T0 generation, net photosynthetic rate (Pn) in most of transgenic rice plants was 20.00% higher than that in untransformed rice, with the highest increase of 47.16%. Water utilization efficiency (WUE) in transgenic rice was also improved. At T6 generation, PEPC activity and Pn of transgenic lines remained higher than those of the wild type. These indicate that over-expressing C3 Eppc gene also can improve rice photosynthesis.

Key words: Echinochloa crusgalli, Root phosphoenolpyruvate carboxylase gene, Rice, PEPC activities, Net photosynthetic efficiency, Water use efficiency

[1]Ku M S B, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, Miyao M, Matsuoka M. High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotechnol, 1999, 17: 76–80

[2]Fukayama H, Imanari E, Tsuchida H, Izui K, Matsuoka M. In vivo activity of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Plant Cell Physiol, 2000, 41: S112

[3]Matsuoka M, Fukayama H, Tsuchida H, Nomura M, Agari S, Ku M S B, Miyao M. How to express some C4 photosynthesis genes at high levels in rice. In: Sheehy J E, Mitchell P L, Hardy B, eds. Redesigning Rice Photosynthesis to Increase Yield. Proceedings of the Workshop on the Quest to Reduce Hunger: Redesigning Rice Photosynthesis, 30 November to 3 December 1999, Los Banos, Philippines. International Rice Research Institute and Amsterdam: Elsevier Science BV, 2000. pp 167–175

[4]Agarie S, Miura A, Sumikura R, Tsukamoto S, Nose A, Arima S, Matsuoka M, Miyao-Tokutomi M. Overexpression of C4 PEPC caused O2-insensitive photosynthesis in transgenic rice plant. Plant Sci, 2002, 162: 257–265

[5]Fukayama H, Hatch M D, Tamai T, Tsuchida H, Sudoh S, Furbank R T, Miyao M. Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants. Photosynth Res, 2003, 77: 227–239

[6]Ding Z S, Huang S H, Zhou B Y, Sun X F, Zhao M. Over-expression of phosphoenolpyruvate carboxylase cDNA from C4 millet (Seteria italica) increase rice photosynthesis and yield under upland condition but not in wetland fields. Plant Biotechnol Rep, 2013, 7: 155–163

[7]O’Leary B, Park J, Plaxton W C. The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem J, 2011, 436: 15–34

[8]Setién I, Vega-Mas I,Celestino N, Calleja-Cervantes M E,González-Murua C, Estavillo J M, González-Moro M B. Root phosphoenolpyruvate carboxylase and NAD-malic enzymes activity increase the ammonium-assimilating capacity in tomato. J Plant Physiol, 2014, 171: 49–63

[9]张桂芳, 赵明, 丁在松, 张丽, 肖俊涛. 稗草磷酸烯醇式丙酮酸羧化酶(PEPCase)基因的克隆与分析. 作物学报, 2005, 31: 1365–1369

Zhang G F, Zhao M, Ding Z S, Zhang L, Xiao J T. Cloning and characterization of phosphoenolpyruvate carboxylase gene from Echinochloa crusgalli. Acta Agron Sin, 2005, 31: 1365–1369 (in Chinese with English abstract)

[10]Kawamura T, Shigesada K, Yanagisawa S, Izui K. Phosphoenolpyruvate carboxylase prevalent in maize roots: Isolation of cDNA clone and its use for analysis of the gene and the gene expression. J Biochem (Tokyo), 1990, 107: 165–168

[11]Kawamura T, Shigesada K, Toh H, Okumura S, Yanagisawa S, Izui K. Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function. J Biochem (Tokyo), 1992, 112: 147–154

[12]Hudspeth R L, Grula J W. Structure and expression of the maize gene encoding the phosphoenolpyruvte carboxylse isozyme involved in C4 photosynthesis. Plant Mol Biol, 1989, 12: 579–589

[13]Westhoff P, Svensson P, Ernst K, Blasing O, Bruscheidt J, Stockhaus J, von Caemmerer S, Furvank R T. Molecular evolution of C4 phosphoenolphyruvate carboxylase in the genus Flaveria. Aust J Plant Physiol, 1997, 24: 429–436

[14]Gowik U, Westhoff P. C4-phosphoenolpyruvate carboxylase. In: Raghavendra A S, Sage R F. C4 Photosynthesis and Related CO2 Concentrating Mechanisms. Advances in Photosynthesis and Respiration. Dordrecht: Springer, 2011, 32: 257−275

[15]Guillet C, Just D, Bénard N, Destrac-Irvine A, Baldet P, Hernould M, Causse M, Raymond P, Rothan C. A fruit-specific phosphoenolpyruvate carboxylase is related to rapid growth of tomato fruit. Planta, 2002, 214: 717−726

[16]Rolletschek H, Borisjuk L, Radchuk R, Miranda M, Heim U, Wobus U, Weber H. Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy. Plant Biotech J, 2004, 2: 211−219

[17]张占琴, 王金梅, 王学军, 汪凯华, 袁春新, 麻浩. 油菜籽粒发育过程中PEPCase活性与油脂、蛋白及亚基积累的特点. 中国油料作物学报, 2009, 31: 14–18

Zhang Z Q, Wang J M, Wang X J, Wang K H, Yuan C X, Ma H. The characteristics of PEPCase activity and accumulation of oil, protein and major protein subunits during seed development of rape (Brassica napus). Chin J Oil Crop Sci, 2009, 31: 14–18 (in Chinese with English abstract)

[18]Pan L J, Yang Q L, Chi X Y, Chen M N, Yang Z, Chen N, Wang T, Wang M, He Y N, Yu S L. Functional analysis of the phosphoenolpyruvate carboxylase on the lipid accumulation of peanut (Arachis hypogaea L.) seeds. J Integr Agric, 2013, 12: 36–44

[19]凌丽俐, 林宏辉, 焦德茂. 转PEPC基因水稻种质的稳定光合生理特性. 作物学报, 2006, 32: 527–531

Ling L L, Lin H H, Jiao D M. The stable photosynthetic characteristics of a PEPC transgenic rice germplasm. Acta Agron Sin, 2006, 32: 527–531 (in Chinese with English abstract)

[20]Jiao D M, Huang X Q, Li X. Characteristics of carbon assimilation and tolerance to photooxidation in transgenic rice expressing C4 photosynthesis enzymes. In: PS2001 Proceedings, 12th International Congress on Photosynthesis. Brisbane: CSIRO Publishing, 2001, S33-004, 1–6

[21]焦德茂, 李霞, 黄雪清, 迟伟, 匡廷云, 古森本. 转PEPC基因水稻的光合CO2同化和叶绿素荧光特性. 科学通报, 2001, 46: 414–418

Jiao D M, Li X, Huang X Q, Chi W, Kuang T Y, Gu S B. The characteristics of CO2 assimilation of photosynthesis and chlorophyll fluorescence in transgenic PEPC rice. Chin Sci Bull, 2001, 46: 414–418

[22]焦德茂, 匡廷云, 李霞, 戈巧英, 黄雪清, 郝乃斌, 白克智. 转PEPC基因水稻具有初级CO2浓缩机制的生理特点. 中国科学, 2003, 33: 33–39

Jiao D M, Kuang T Y, Li X, Ge Q Y, Huang X Q, Hao N W, Bai K Z. Physiological characteristics of the primitive CO2 concentrating mechanism in PEPC transgenic rice. Sci China, 2003, 33: 33–39

[23]Huang X Q, Jiao D M, Chi W, Ku M S B. Characteristics of CO2 exchange and chlorophyll fluorescence of transgenic rice with C4 genes. Acta Bot Sin, 2002, 44(4): 405–412

[24]张边江, 华春, 周峰, 周泉澄, 陈全战, 王荣富, 焦德茂. 转PEPC+PPDK双基因水稻的光合特性. 中国农业科学, 2008, 41: 3008–3014 (in Chinese with English abstract)

Zhang B J, Hua C, Zhou F, Zhou Q C, Chen Q Z,Wang R F, Jiao D M. Photosynthetic characteristics of transgenic rice with PEPC+PPDK gene. Sci Agric Sin, 2008, 41: 3008–3014 (in Chinese with English abstract)

[25]Jeanneau M, Gerentes D, Foueillassar X, Zivy M, Vidal J, Toppan A, Perez P. Improvement of drought tolerance in maize: towards the functional validation of the Zm-Asr1 gene and increase of water use efficiency by over-expressing C4-PEPC. Biochimie, 2002, 84: 1127–1135

[26]丁在松, 赵明, 荆玉祥, 李良璧, 匡廷云. 玉米ppc基因过表达对转基因水稻光合速率的影响. 作物学报, 2007, 33: 717–722

Ding Z S, Zhao M, Jing Y X, Li L B, Kuang T Y. Effect of overexpression of maize ppc gene on photosynthesis in transgenic rice plants. Acta Agron Sin, 2007, 33: 717 – 722 (in Chinese with English abstract)

[27]方立锋, 丁在松, 赵明. 转ppc基因水稻苗期抗旱特性研究. 作物学报, 2008, 34: 1220−1226

Fang L F, Ding Z S, Zhao M. Characteristics of drought tolerance in ppc overexpressed rice seedlings. Acta Agron Sin, 2008, 34: 1220−1226 (in Chinese with English abstract)

[28]周宝元, 丁在松, 赵明. PEPC过表达可以减轻干旱胁迫对水稻光合的抑制作用. 作物学报, 2011, 37: 112–118

Zhou B Y, Ding Z S, Zhao M. Alleviation of drought stress inhibition on photosynthesis by overexpression of PEPC gene in rice. Acta Agron Sin, 2011, 37: 112–118 (in Chinese with English abstract)

[29]Scheibe R. Malate valves to balance cellular energy supply. Physiol Plant, 2004, 120: 21–26

[30]Andreo C S, Gonzalez D H, Iglesias A A. Higher plant phosphoenolpyruvate carboxylase: Structure and regulation. FEBS Lett, 1987, 213: 1–8

[1] 田甜, 陈丽娟, 何华勤. 基于Meta-QTL和RNA-seq的整合分析挖掘水稻抗稻瘟病候选基因[J]. 作物学报, 2022, 48(6): 1372-1388.
[2] 郑崇珂, 周冠华, 牛淑琳, 和亚男, 孙伟, 谢先芝. 水稻早衰突变体esl-H5的表型鉴定与基因定位[J]. 作物学报, 2022, 48(6): 1389-1400.
[3] 周文期, 强晓霞, 王森, 江静雯, 卫万荣. 水稻OsLPL2/PIR基因抗旱耐盐机制研究[J]. 作物学报, 2022, 48(6): 1401-1415.
[4] 郑小龙, 周菁清, 白杨, 邵雅芳, 章林平, 胡培松, 魏祥进. 粳稻不同穗部籽粒的淀粉与垩白品质差异及分子机制[J]. 作物学报, 2022, 48(6): 1425-1436.
[5] 颜佳倩, 顾逸彪, 薛张逸, 周天阳, 葛芊芊, 张耗, 刘立军, 王志琴, 顾骏飞, 杨建昌, 周振玲, 徐大勇. 耐盐性不同水稻品种对盐胁迫的响应差异及其机制[J]. 作物学报, 2022, 48(6): 1463-1475.
[6] 杨建昌, 李超卿, 江贻. 稻米氨基酸含量和组分及其调控[J]. 作物学报, 2022, 48(5): 1037-1050.
[7] 杨德卫, 王勋, 郑星星, 项信权, 崔海涛, 李生平, 唐定中. OsSAMS1在水稻稻瘟病抗性中的功能研究[J]. 作物学报, 2022, 48(5): 1119-1128.
[8] 朱峥, 王田幸子, 陈悦, 刘玉晴, 燕高伟, 徐珊, 马金姣, 窦世娟, 李莉云, 刘国振. 水稻转录因子WRKY68在Xa21介导的抗白叶枯病反应中发挥正调控作用[J]. 作物学报, 2022, 48(5): 1129-1140.
[9] 王小雷, 李炜星, 欧阳林娟, 徐杰, 陈小荣, 边建民, 胡丽芳, 彭小松, 贺晓鹏, 傅军如, 周大虎, 贺浩华, 孙晓棠, 朱昌兰. 基于染色体片段置换系群体检测水稻株型性状QTL[J]. 作物学报, 2022, 48(5): 1141-1151.
[10] 王泽, 周钦阳, 刘聪, 穆悦, 郭威, 丁艳锋, 二宫正士. 基于无人机和地面图像的田间水稻冠层参数估测与评价[J]. 作物学报, 2022, 48(5): 1248-1261.
[11] 陈悦, 孙明哲, 贾博为, 冷月, 孙晓丽. 水稻AP2/ERF转录因子参与逆境胁迫应答的分子机制研究进展[J]. 作物学报, 2022, 48(4): 781-790.
[12] 王吕, 崔月贞, 吴玉红, 郝兴顺, 张春辉, 王俊义, 刘怡欣, 李小刚, 秦宇航. 绿肥稻秆协同还田下氮肥减量的增产和培肥短期效应[J]. 作物学报, 2022, 48(4): 952-961.
[13] 巫燕飞, 胡琴, 周棋, 杜雪竹, 盛锋. 水稻延伸因子复合体家族基因鉴定及非生物胁迫诱导表达模式分析[J]. 作物学报, 2022, 48(3): 644-655.
[14] 陈云, 李思宇, 朱安, 刘昆, 张亚军, 张耗, 顾骏飞, 张伟杨, 刘立军, 杨建昌. 播种量和穗肥施氮量对优质食味直播水稻产量和品质的影响[J]. 作物学报, 2022, 48(3): 656-666.
[15] 王琰, 陈志雄, 姜大刚, 张灿奎, 查满荣. 增强叶片氮素输出对水稻分蘖和碳代谢的影响[J]. 作物学报, 2022, 48(3): 739-746.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!