籽粒苋AhNAD-ME的序列特征与表达

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白云凤, 聂江婷, 张忠梁, 李平, 张维锋, 闫建俊, 冯瑞云, 张耀. 籽粒苋AhNAD-ME的序列特征与表达. 作物学报, 40(12): 2192-2197
BAI Yun-Feng, NIE Jiang-Ting, ZHANG Zhong-Liang, LI Ping, ZHANG Wei-Feng, YAN Jian-Jun, FENG Rui-Yun, ZHANG Yao. Sequence Characteristics and Expression of NAD-malic enzyme inAmaranthus hypochondriacusL. . Acta Agronomica Sinica, 40(12): 2192-2197 复制到剪切板

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作物学报编辑部

籽粒苋AhNAD-ME的序列特征与表达

白云凤^1,^3,^*, 聂江婷², 张忠梁¹, 李平¹, 张维锋¹, 闫建俊¹, 冯瑞云¹, 张耀¹

¹山西省农业科学院作物科学研究所, 山西太原030032

²山西大学生物工程学院, 山西太原030006

³农业部黄土高原作物基因资源与种质创制重点实验室, 山西太原 030006

^*通讯作者(Corresponding author): 白云凤, E-mail:byfok@126.com

收稿日期:2014-09-16

基金:本研究由国家自然科学基金项目(30971838), 山西省科技攻关项目(201303110015-1)和山西省归国留学人员科研项目(2013-146)资助;

摘要

NAD(P)-苹果酸酶催化苹果酸氧化脱羧, 产生丙酮酸和CO₂, 伴随NAD(P)的还原。在C₄植物中, 苹果酸酶参与了C₄光合作用。本研究对克隆的双子叶C₄植物籽粒苋NAD-苹果酸酶基因(AhNAD-ME)编码的氨基酸序列进行了生物信息学分析, 结果表明, AhNAD-ME具有苹果酸酶的完整功能域, 包括苹果酸N端结构域和苹果酸酶的NAD结合结构域; 进化树表明, 该序列属于NAD-ME的α亚基, 该亚基定位于线粒体基质中。半定量RT-PCR分析表明, 该基因主要在叶片和茎中表达, 表达量随光照时间延长而增加。将AhNAD-ME基因重组到原核表达载体pEASY-E1中, 电击法转化到大肠杆菌Transette (DE3)菌株中, IPTG诱导其高效表达, 表达的融合蛋白的分子量与预期相符, 主要以包涵体形式存在。

关键词: 籽粒苋; AhNAD-ME; 序列特征; 表达模式; 原核表达

Sequence Characteristics and Expression of NAD-malic enzyme inAmaranthus hypochondriacusL.

BAI Yun-Feng^1,^3,^*, NIE Jiang-Ting², ZHANG Zhong-Liang¹, LI Ping¹, ZHANG Wei-Feng¹, YAN Jian-Jun¹, FENG Rui-Yun¹, ZHANG Yao¹

¹ Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan 030032, China

² College of Bioengineering, Shanxi University, Taiyuan 030006, China

³China Key Laboratory of Loess Plateau Crop Gene Resources and Germplasm Creation, Ministry of Agriculture, Taiyuan 030006, China

Abstract

The NAD(P)-malic enzyme (NAD(P)-ME) found in many metabolic pathways catalyzes the oxidative decarboxylation of L-malate, which results in producing pyruvate, CO₂ and NAD(P)H. In C₄ plants, NAD(P)-ME plays a key role in photosynthetic carbon fixation. This study was aimed to characterize the AhNAD-ME in dicotyledonous C₄Amaranthus hypochondriacus by sequence analysis, examine the expression patterns ofAhNAD-ME gene in different tissues and different durations of illumination time, and construct a recombinant plasmid pEASY-E1 harboring theAhNAD-ME cDNA and then transform the plasmid intoE. coli Transette (DE3) for prokaryotic expression after IPTG induction. The result showed that AhNAD-ME contains all of the motifs required for a complete and functional malic enzyme and is localized specifically to the mitochondrial matrix. Semi-quantitative RT-PCR results showed thatAhNAD-ME was constitutively expressed in all examined tissues, with different expression levels, and strongly up-regulated under light in the leaf and stem. Results of SDS-PAGE demonstrated that the specific fusion protein with an expected molecular weight was successfully expressed inE. coli transette (DE3) induced by IPTG.

Keyword: Amaranthus hypochondriacus; AhNAD-ME; Sequence characteristics; Expression pattern; Prokaryotic expression

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苹果酸是植物代谢过程中的重要中间产物。苹果酸酶(malic enzyme, ME) 在金属离子Mn²⁺或Mg²⁺的存在下, 催化苹果酸氧化脱羧生成丙酮酸和CO₂, 伴随产生还原性物质NAD(P)H, 调控植物体内的呼吸作用和脂类生成^{[ 1, 2]}等重要代谢途径, 对于保持植物细胞的渗透势、稳定细胞质的pH值^{[ 3]}、维持根系的离子吸收平衡、调节果实成熟和风味形成^{[ 4]}、应对生物和非生物逆境胁迫^{[ 5, 6]}等方面起重要作用。在C₄植物中, 苹果酸酶催化叶肉细胞的苹果酸脱羧释放出的CO₂被核酮糖-1,5二磷酸羧化酶/加氧酶(Rubisco)重新固定进入Calvin循环, 产生的丙酮酸再生为磷酸烯醇式丙酮酸(PEP), 使C₄途径得以循环。CO₂的释放提高了Rubisco周围的CO₂浓度, 抑制了Rubisco的加氧功能, 提高了CO₂固定效率^{[ 7, 8, 9]}。

根据辅酶因子的不同, 苹果酸酶分为NAD依赖型(NAD-malic enzyme, NAD-ME)和NADP依赖型 (NADP-malic enzyme, NADP-ME)。根据在细胞和组织中的分布, NAD(P)-ME分为细胞质型和质体型, 根据功能又可分为光合型和非光合型等类型。玉米NADP-ME有光合型(C₄)、非光合型(C₃)和根型3种类型^{[ 10, 11]}。光合型定位于绿色叶片, 参与C₄光合循环; 非光合型定位于黄化叶片, 具体作用尚不清楚; 根型定位在根细胞中, 主要参与防御反应^{[ 12]}。水稻和拟南芥的NADP-ME包含1个质体型和3个细胞质型^{[ 13]}, 具有组织和发育特异性表达。高等植物NAD(P)-ME家族成员催化同一个生化反应, 但代谢途径和表达模式不同^{[ 2, 14]}, 可能与其在细胞内的定位有关。

籽粒苋( A. hypochondriacus)粮饲兼用, 是双子叶C₄植物, 属于苋科( Amaranthus)苋属( Amaranth-ceae), 原产于中美洲和东南亚, 近年我国年种植面积在10万公顷以上, 从黑龙江到海南均有分布^{[ 15]}。籽粒苋能高效利用土壤养分^{[ 16]}和水分^{[ 17]}, 光合速率快, 生物学产量很高^{[ 18]}。在本课题组先前克隆到籽粒苋 NAD-ME ( AhNAD-ME)基因^{[ 19, 20]}的基础上, 分析其序列特征, 进行亚细胞定位, 探明时空表达特性, 构建原核表达载体获得原核表达蛋白, 将为进一步研究其酶学特性和转基因利用奠定基础。

1 材料与方法

1.1 试验材料

供试材料籽粒苋和克隆载体pSURE-AhNAD- ME由本实验室保存, 原核表达载体pEASY-E1和大肠杆菌菌株Trans1-T1和Transetta (DE3)的感受态细胞购自北京全式金生物技术有限公司。限制性内切酶、T₄ DNA连接酶、LA- Taq酶、M-MLV反转录酶、DNA Marker、Premixed Protein Marker、IPTG、考马斯亮蓝R-250为大连宝生物公司产品; 氨苄青霉素购自Sigma公司。DNA凝胶回收试剂盒、RNA提取试剂盒购自北京艾德来生物科技有限公司。

1.2 AhNAD-ME特征分析和亚细胞定位预测

以 AhNAD-ME基因推导的氨基酸序列作query, 利用BlastP在线工具分析其结构域和进化地位, PSORT (http://www.psort.org/)和SoftBerry ProtComp 9.0 (http://linux1.softberry.com/berry.phtml)在线预测AhDNA-ME的亚细胞定位, 用SignalP 4.1预测导肽^{[ 21]}。用Clustal 1.8.1和MEGA 4.1软件构建系统进化树。

1.3 时空表达模式分析

运用半定量RT-PCR检测 AhNAD-ME基因的表达量, 以 Actin为内标基因。将籽粒苋植株暗处理1 d后, 置光照下2、4、6和8 h, 分别提取暗处理和不同光照时段的根、茎、叶的总RNA, 以此为模板, 用Oligo dT Primer作引物, 通过反转录合成cDNA第1链。以cDNA第1链为模板, 扩增 AhNAD-ME基因和内标基因 Actin。扩增 AhNAD-ME的正向引物为5°-ACCCAC AAAGAGTGTTACCG-3°, 反向引物为5°-TTCCCT GATTAACGTGCC-3°, 扩增内标基因的正向引物为5°-CTACAATGAGCTTCGGGTTGC-3°, 反向引物为5°-GGACTTCTGGACAACGGAATCTC-3°。取RT-PCR产物20 μL经1%琼脂糖凝胶电泳, 然后成像, 采用软件BandScan进行灰度分析, 在Microsoft Excel中作图。

1.4 原核表达载体构建

以含有 AhNAD-ME全长基因的克隆载体pSURE-AhNAD-ME为模板, 利用引物P1 (5°-CAAG ATCTCAATGTTGTTGCTTTGTAGTC-3°)和P2 (5°-C GGTAACCTTCAGCGTTTCTTGTACAC-3°)进行PCR扩增, 琼脂糖凝胶电泳中可见大约1.8 kb的片段, 将其纯化回收, 取4 μL与1 μL原核表达载体pEASY-E1混匀, 25℃连接15 min。反应结束后, 转化大肠杆菌Trans1-T1感受态细胞。挑取单菌落至LB (含100 mg L^-1 Amp)液体培养基, 37℃过夜培养。提取质粒进行PCR检测, 以空载体pEASY-E1作为负对照, 引物为P1和P2。将PCR鉴定呈现阳性的单克隆提取质粒, 用 Xba I和 Sac I双酶切鉴定, 并送北京六合华大基因科技股份有限公司测序。

1.5 重组载体在大肠杆菌Transetta (DE3)的诱导表达和SDS-PAGE分析

将基因序列无变异、插入方向正确的重组质粒, 通过电击导入Transetta (DE3)感受态细胞。挑取单菌落至LB (含100 mg L^-1Amp)液体培养基, 37℃过夜培养。以菌液为模板进行PCR扩增, 以未转化的菌液为负对照, 引物为P1和P2。

将导入重组载体的6 mL单菌落菌液加入600 mL LB液体培养基(含100 mg L^-1 Amp), 于37℃, 200转 min^-1振荡培养。当菌液的OD₆₀₀值为0.4~0.6时, 加入IPTG, 终浓度为1 mmol L^-1。然后等量分成6管, 其中4管于30℃条件下分别诱导0、2、4和6 h。反应结束后, 从每管取菌液2 mL, 18 000× g离心1 min, 弃上清液, 加入SDS上样缓冲液, 悬浮混匀, 沸水浴5~10 min, 18 000× g离心1 min, 冰浴5 min, 进行SDS-PAGE电泳。另2管分别在30℃和16℃条件下诱导4 h, 离心收集菌体后用1×PBS将菌体悬浮, 用超声波破碎(20 mm的变幅杆, 400 W, 超声2 s, 间隔5 s, 重复60次), 9600× g离心10 min, 分离上清液和沉淀, 并分别加入SDS上样缓冲液, 混匀, 沸水浴5~10 min后上样, SDS-PAGE电泳。电泳完毕, 用考马斯亮蓝R-250染色并脱色至背景清晰。

2 结果与分析

2.1 籽粒苋AhNAD-ME特征分析和亚细胞定位预测

将 AhNAD-ME基因(GenBank登录号为HQ186303)编码的氨基酸序列提交NCBI, 进行BlastP。蛋白质保守结构域分析表明该序列含有完整的NAD-ME保守功能域, 包括苹果酸N端结构域和苹果酸酶的NAD结合结构域[含有6个NAD(P)结合位点]。系统进化树( 图1)显示, 该序列先与双子叶植物的NAD- ME的α亚基(62 kD)聚为一类, 再与单子叶植物的NAD-ME的α亚基(62 kD)聚为一类, 而单子叶植物与双子叶植物NAD-ME的β亚基(59 kD)聚为1类, 表明植物NAD-ME的α亚基和β亚基的分化在单、双子叶植物分化之前已经完成。

用SoftBerry ProtComp 9.0 (http://linux1.softberry. com/berry.phtml)在线预测AhNAD-ME的蛋白质亚细胞定位, 表明该蛋白位于线粒体。PSORT在线(http://www.psort.org/)分析进一步表明该蛋白质位于线粒体基质。SignaP4.1在线分析表明AhNAD-ME没有明显的导肽序列(结果未显示)。

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	图1 籽粒苋AhNAD-ME氨基酸序列与其他植物同源基因的系统进化树Fig. 1 Phylogenetic tree of AhNAD-ME and NAD-ME in other species based on amino acid sequence

2.2 AhNAD-ME基因的光诱导和组织特异性表达特性

在暗处理或光照条件下, AhNAD-ME基因在叶片中均表达, 并且随着光照时间的延长, 表达量相应增加。在光照0~4 h区间, 随光照时间延长, 表达量增加幅度大; 在光照4~8 h区间, 表达量增加幅度趋缓, 证实 AhNAD-ME基因具有光诱导表达特性。根、茎、叶表达模式不尽相同。在暗处理条件下, AhNAD-ME在根、茎、叶中均有低水平表达, 但在光照处理后, 叶片中的表达量急速增加, 茎中表达量也明显增加, 但根中表达量无明显变化( 图2和图3), 证实在无光条件下, 籽粒苋 AhNAD-ME基因呈现低水平的组成型表达, 在光照条件下则呈现绿色组织特异性表达。预示 AhNAD-ME基因低水平组成型表达可以满足苹果酸的基本代谢活动, 在光照条件下则可能参与了光合作用。

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	图2 AhNAD-ME的表达模式Fig. 2 Expression profile of AhNAD-ME

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	图3 AhNAD-ME表达模式灰度分析Fig. 3 Grayscale analysis of AhNAD-ME expression profile

2.3 AhNAD-ME的原核表达

用IPTG诱导含有重组质粒的Transetta (DE3)菌株表达重组蛋白, SDS-PAGE检测结果表明, 30℃诱导2 h就有特异的蛋白条带出现。随着诱导时间延长, 条带颜色进一步加深。诱导蛋白的分子量约66 kD ( 图4), 与预期的蛋白质分子量相符。

为了获得能够可溶性表达的目的蛋白, 用IPTG分别在30℃和16℃下诱导4 h, 对上清液和沉淀进行SDS-PAGE电泳检测。结果表明, 在2个温度条件下, 融合蛋白均能被诱导, 但30℃诱导的表达量高于16℃, 但2个温度诱导的融合蛋白主要以包涵体的形式存在( 图4)。

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图4 图 AhNAD-ME原核表达SDS-PAGE电泳M: 蛋白质分子量标准; 1: 不含重组质粒的菌株Transette (DE3) 在30℃诱导2 h后的表达产物; 2~9: 含有重组质粒的菌株Transette (DE3)的表达产物。其中2、3、4和5分别为30℃诱导0、2、4和6 h后的表达产物; 6和7为30℃诱导4 h后的沉淀和上清;
8和9为16℃诱导4 h后的沉淀和上清液。Fig. 4 SDS-PAGE analysis of the AhNAD-MEexpressionM: premixed protein marker; 1 refers to Transette (DE3) without recombined plasmid induced by IPTG under 30℃ for 2 h; 2, 3, 4, and 5 refer to Transette (DE3) with recombined plasmid induced by IPTG for 0, 2, 4, and 6 hours, respectively; 6 and 7 refer to precipitate and supernatant liquor of Transette (DE3) with recombined plasmid induced by IPTG under 30℃ for 4 hours, respectively; 8 and 9 refer to precipitate and supernatant liquor of Transette (DE3) with recombined plasmid induced by IPTG under 16℃ for 4 hours, respectively.

3 讨论

植物的多数蛋白质由核基因编码, 在细胞质内与内质网相结合的核糖体上合成, 之后在前导序列的指引下到达特定的细胞器, 部分蛋白质被分泌到细胞外或留在细胞质中。蛋白质只有转运到正确部位才能参与细胞的各种代谢活动, 协同保证生命活动的正常运行。前导序列一般位于前体蛋白质N-端, 长度约20~80个氨基酸。有些蛋白质则由内部定位信号指导它们在细胞内的转运和定位。内部定位信号序列隐藏在成熟的蛋白质中, 不会被删除, 可能对蛋白质的折叠和功能有重要作用^{[ 22]}。线粒体是重要的细胞器, 但线粒体的蛋白质合成能力有限。大量的线粒体蛋白质是在细胞质中合成的, 在前导序列, 也称之为导肽的指引下, 通过TOM复合体和TIM23转位酶分送到基质、内膜或膜间隙, 之后信号肽酶切除导肽, 前体蛋白变为成熟蛋白。导肽通常是疏水的, 这种特征性结构有利于穿过线粒体的双层膜。线粒体导肽有一些特征^{[ 23]}, 如富含丙氨酸、精氨酸、亮氨酸和丝氨酸, 很少含带有负电荷的谷氨酸、天冬氨酸以及异亮氨酸和赖氨酸, 在相对于切割位点-2位的氨基酸为精氨酸, 是protease-I的识别位点, 第8位为非极性氨基酸; 起始的2个氨基酸为Met-Leu。但同一种蛋白质的导肽在不同植物中差异较大, 不具有高度保守的序列^{[ 24]}。我们用SignalP在线分析了AhNAD-ME的氨基酸序列, 发现其不具备导肽序列, 而Long等^{[ 25]}微量测序结果表明成熟的AhNAD-ME的N-端缺少了31个氨基酸。分析AhNAD-ME N-端的31个氨基酸序列, 发现丙氨酸、精氨酸、亮氨酸和丝氨酸的含量占到54.8%, 不含有天冬氨酸以及异亮氨酸和赖氨酸等带负电荷的氨基酸, 符合线粒体导肽的序列特征。由于线粒体导肽的非保守性, 给判断蛋白质是否具有导肽序列带来困难, 需要生物信息学技术和亚细胞蛋白质组学技术相互印证。

植物的NAD-ME有不同的构成形式, 如三色苋( Amaranthus tricolor)、禾草龙爪稷( Eleusine coracana)^{[ 26]}和洋野黍( Panicum dichotomiflorum)^{[ 27]}叶片的NAD-ME是同源八聚体, 籽粒苋的NAD-ME是异源八聚体^{[ 22]}。本研究用AhNAD-ME作query进行BlastP, 表明双子叶植物的α亚基(62 kD)先与单子叶植物的α亚基聚合, 双子叶植物的β亚基(59 kD)则先与单子叶植物的β亚基聚合, 似乎表明在单、双子叶植物分化之前这2个亚基的编码基因已经分化。不同物种的α亚基均具有完整的苹果酸酶的结构域, 似乎β亚基与α亚基的聚合对于酶活性调节具有重要作用。深入研究不同植物的NAD-ME构成形式及其活性, 将有助于加深理解NAD-ME功能的自我调节和进化机制。

蛋白质的体外表达是功能鉴定和抗体制备的基础。原核表达系统具有培养方法简单, 产量高、速

度快、稳定性好等优点^{[ 28, 29]}。但真核基因在大肠杆菌中的正确表达受很多因子的影响, 如目的基因本身的特性、诱导温度和时间、密码子偏好性等。Transetta (DE3)菌株是BL21的衍生系, 该菌株补充了大肠杆菌缺乏的6种稀有密码子(AUA、AGG、AGA、CUA、CCC、GGA)对应的tRNA, 利于提高真核基因在原核系统中的表达水平。本研究将 AhNAD-ME基因重组到pEASY-T载体上, 电击转化到Transetta (DE3)菌株中, 在原核细胞中高效表达。籽粒苋 AhNAD-ME基因较大, 开放可读框长度为1872 bp, 编码623个氨基酸^{[ 20]}。原核表达蛋白的获得为基因功能鉴定奠定了基础, 也为制备抗体提供了抗原。

4 结论

籽粒苋AhNAD-ME具有苹果酸酶的完整功能域, 定位于线粒体基质中; 表达模式为光诱导、绿色组织特异性表达。构建了 AhNAD-ME基因的原核表达载体, 并在大肠杆菌中高效表达。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。

参考文献

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2004

3.37

0.0

2001

3.582

0.0

... 高等植物NAD(P)-ME家族成员催化同一个生化反应, 但代谢途径和表达模式不同^[2,14], 可能与其在细胞内的定位有关 ...

1986

6.555

0.0

... 苹果酸酶(malic enzyme, ME) 在金属离子Mn²⁺或Mg²⁺的存在下, 催化苹果酸氧化脱羧生成丙酮酸和CO₂, 伴随产生还原性物质NAD(P)H, 调控植物体内的呼吸作用和脂类生成^[1,2]等重要代谢途径, 对于保持植物细胞的渗透势、稳定细胞质的pH值^[3]、维持根系的离子吸收平衡、调节果实成熟和风味形成^[4]、应对生物和非生物逆境胁迫^[5,6]等方面起重要作用 ...

2013

0.0

1.889

董庆龙, 王海荣, 安淼, 余贤美, 王长君. 苹果NADP依赖的苹果酸酶基因克隆、序列和表达分析. 中国农业科学, 2013, 46: 1857-1866

Dong

Q L

, Wang

H R

, An

, Yu

X M

, Wang

C J.

Cloning, sequence and expression analysis of NADP-malic enzyme genes in apple. Sci Agric Sin, 2013, 46: 1857-1866 (in Chinese with English abstract)

【Objective】This study is aimed to characterize the genes of MdNADP-MEs involved in malic acid metabolism in Malus×domestica B. by gene cloning, sequence and expression of MdNADP-MEs in different tissues.【Method】 Three full-length cDNA sequences of MdNADP-ME1,2,3 were isolated by RT-PCR. The obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. qRT-PCR was used to assess the expression of MdNADP-MEs in different tissues.【Result】The sequencing results showed that three cDNAs (designated as MdNADP-ME1, 2 and 3; GenBank Accession No. JX971883, JX971884 and JX971885) were 1 512 bp, 1 782 bp and 1 926 bp. They contained an open reading frame (ORF) of 1 512 bp, 1 782 bp and 1 926 bp, respectively, and their ORFs encoded an protein with 503, 593 and 641 amino acids, respectively. Amino acid sequence and structure analysis indicated that MdNADP-MEs contained five conservative amino acid areas (motif I-V) and two functional structure domains: malic and NAD_bind_1_malic_enz. The result of phylogenetic analysis showed that MdNADP-ME1 and 2 belonged to cytosolic dicot NADP-ME (group I), MdNADP-ME3 belonged to plastidic dicot NADP-ME. qRT-PCR result showed that MdNADP-MEs were constitutively expressed in all examined tissues, but showed different expression levels.【Conclusion】MdNADP-ME1,2,3 belong to NADP-ME family, possess highly conserved structures, and show different expression patterns in different tissues of apple.

Shandong Institute of Pomology, Tai’an 271000，Shandong

【目的】克隆苹果（Malus×domestica B.）中苹果酸代谢的关键酶基因MdNADP-ME，进行序列特征分析，研究MdNADP-ME在苹果中组织表达的情况。【方法】利用RT-PCR技术获得苹果MdNADP-ME1,2,3全长的cDNA序列。对该序列进行生物信息学分析，采用荧光实时定量PCR技术研究MdNADP-ME1,2,3的组织表达。【结果】测序结果显示，获得的3个NADP-苹果酸酶基因（MdNADP-ME1、MdNADP-ME2 和 MdNADP-ME3； GenBank 登录号为JX971883、JX971884和JX971885），其ORF分别为1 512、1 782和1 926 bp，推测其分别编码503、593和641个氨基酸的多肽。氨基酸序列和结构分析显示，这3个基因均含有5个保守的氨基酸区域（motif I-V），含有2个功能结构域：malic和NAD_bind_1_malic_enz。进化树分析结果显示，MdNADP-ME1 和MdNADP-ME2属于双子叶植物细胞质NADP-ME型，MdNADP-ME3属于双子叶植物质体NADP-ME型。荧光实时定量PCR结果表明，MdNADP-ME1-3在被检测的组织中均有表达，但表达差异明显。【结论】MdNADP-ME1-3属于植物NADP-ME家族，结构高度保守，并且在苹果的不同组织中有不同表达模式。

1999

3.15

0.0

2009

1.804

0.0

2013

3.15

0.0

Berry

J O

, Yerramsetty

, Zielinski

A M

, Mure

C M.

Photosynthetic gene expression in higher plants. Photosynth Res, 2013, 117: 91-120

Abstract

Within the chloroplasts of higher plants and algae, photosynthesis converts light into biological energy, fueling the assimilation of atmospheric carbon dioxide into biologically useful molecules. Two major steps, photosynthetic electron transport and the Calvin-Benson cycle, require many gene products encoded from chloroplast as well as nuclear genomes. The expression of genes in both cellular compartments is highly dynamic and influenced by a diverse range of factors. Light is the primary environmental determinant of photosynthetic gene expression. Working through photoreceptors such as phytochrome, light regulates photosynthetic genes at transcriptional and posttranscriptional levels. Other processes that affect photosynthetic gene expression include photosynthetic activity, development, and biotic and abiotic stress. Anterograde (from nucleus to chloroplast) and retrograde (from chloroplast to nucleus) signaling insures the highly coordinated expression of the many photosynthetic genes between these different compartments. Anterograde signaling incorporates nuclear-encoded transcriptional and posttranscriptional regulators, such as sigma factors and RNA-binding proteins, respectively. Retrograde signaling utilizes photosynthetic processes such as photosynthetic electron transport and redox signaling to influence the expression of photosynthetic genes in the nucleus. The basic C₃ photosynthetic pathway serves as the default form used by most of the plant species on earth. High temperature and water stress associated with arid environments have led to the development of specialized C₄ and CAM photosynthesis, which evolved as modifications of the basic default expression program. The goal of this article is to explain and summarize the many gene expression and regulatory processes that work together to support photosynthetic function in plants.

<li>1.Department of Biological Sciences, University at Buffalo, Buffalo, NY, 14260, USA</li>

2013

3.15

0.0

Moreney

J V

, Jungnick

, Dimario

R J

, Longstreth

D J.

Photorespiration and carbon concentrating mechanisms: two adaptation to high O₂, low CO₂ conditions. Photosynth Res, 2013, 117: 121-131

Abstract

This review presents an overview of the two ways that cyanobacteria, algae, and plants have adapted to high O₂ and low CO₂ concentrations in the environment. First, the process of photorespiration enables photosynthetic organisms to recycle phosphoglycolate formed by the oxygenase reaction catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Second, there are a number of carbon concentrating mechanisms that increase the CO₂ concentration around Rubisco which increases the carboxylase reaction enhancing CO₂ fixation. This review also presents possibilities for the beneficial modification of these processes with the goal of improving future crop yields.

<li>1.Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, USA</li>

2011

9.251

0.0

2002

3.518

0.0

Tausta

, Coye

, Rothermel

, Stiefel

, Nelson

Maize C₄ and non-C₄ NADP-dependent malic enzymes are encoded by distinct genes derived from a plastid-localized ancestor. Plant Mol Biol, 2002, 50: 635-652

NADP-dependent malic enzymes (NADP-ME; EC1.1.1.40) have been implicated in a wide range of metabolic pathways in the plastids and cytosol of plant cells. In maize, an NADP-ME type C4 plant, the most abundant NADP-ME form is the chloroplastic leaf isoform that delivers CO₂ intracellularly to ribulose bisphosphate carboxylase (RuBPCase). A second NADP-ME isoform predominates in maize roots and exhibits distinct C3-like enzymatic characteristics. We show that the C3-like isoform is encoded by a pair of nearly identical genes that encode precursor proteins with functional chloroplast transit peptides. Using RT-PCR, we also show that the messages encoding the C4 and C3-like NADP-ME isoforms are differentially regulated with respect to the developmental stage of the leaf, light conditions, and tissue type. Based on these characteristics and on sequence comparison of ME families in other species, we propose a scheme for the origin of the C4-specific NADP-ME gene.

1.Yale University New Haven 2.Connecticut State Forensic Lab Meriden 3.Department of Biochemistry University of Texas Southwestern Medical Center Dallas 4.Department of Molecular Genetics CID-CSIC

2008

3.518

0.0

Detarsio

, Maurino

V G

, Alvarez

C E

, Mueller

G L.

Maize cytosolic NADP-malic enzyme (ZmCytNADP-ME): a phylogenetically distant isoform specifically expressed in embryo and emerging roots. Plant Mol Biol, 2008, 68: 355-367

Two maize plastidic NADP-malic enzyme isoforms have been characterized: the bundle sheath-located photosynthetic isoform (ZmC₄-NADP-ME) and a constitutively expressed one (Zm-nonC₄-NADP-ME). In this work, the characterization of the first maize cytosolic NADP-ME (ZmCytNADP-ME) is presented, which transcript is exclusively found in embryo and emerging roots. ZmCytNADP-ME expression in roots decreases with development, while Zm-nonC ₄ -NADP-ME increases concomitantly. On the other hand, ZmCytNADP-ME accumulation is differentially modulated by several stress conditions and shows coordination with that of Zm-nonC ₄ -NADP-ME in maize young roots. Recombinant ZmCytNADP-ME displays clearly distinct kinetic parameters and metabolic regulation than the plastidic isoforms. The particular properties and the specific-expression pattern of this novel isoform suggest that it may be involved in the control of cytosolic malate levels in emerging roots, e.g. during hypoxia. ZmCytNADP-ME is phylogenetically related to other cytosolic mono and dicot NADP-MEs, and data indicate that it belongs to an ancestral unique group among plant NADP-MEs.

1.Universidad Nacional de Rosario Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI) Suipacha 531 2000 Rosario Argentina 2.Universit?t zu K?ln Botanisches Institut Gyrhofstr. 15 50931 Cologne Germany

1997

5.242

0.0

... 根型定位在根细胞中, 主要参与防御反应^[12] ...

2004

0.0

... 水稻和拟南芥的NADP-ME包含1个质体型和3个细胞质型^[13], 具有组织和发育特异性表达 ...

2004

4.654

0.0

... 高等植物NAD(P)-ME家族成员催化同一个生化反应, 但代谢途径和表达模式不同^[2,14], 可能与其在细胞内的定位有关 ...

2005

0.0

张锡州, 李廷轩, 王昌金. 富钾植物籽粒览研究进展. 中国农学通报, 2005, 21(4): 230-235

Zhang

X Z

, Li

T X

, Wang

C J.

Progress of research on K-rich plant of grain amaranth. Chin Agric Sci Bull, 2005, 21(4): 230-235 (in Chinese with English abstract)

Introduction of health plant grain amaranth has the characteristics of K-rich, high biomass and food value. Here the physiological and biochemical characteristics, basis of molecular biology, K-rich mechanism, characteristics of mineral nutrition and other

(College of Resources and Environment, Sichuan Agricultural University, Ya’an Sichuan 625014)

籽粒苋是一种富钾植物，同时也是一种生物量很大的绿肥和营养价值较高的保健类植物。笔者综述了目前国内外对籽粒苋的生理生化特征、分子生物学基础、富钾机理、矿质营养特点及在食品、药用、饲料、绿肥等方面的研究进展，并对今后籽粒苋的研究提出了建议。

... hypochondriacus)粮饲兼用, 是双子叶C₄植物, 属于苋科(Amaranthus)苋属(Amaranth-ceae), 原产于中美洲和东南亚, 近年我国年种植面积在10万公顷以上, 从黑龙江到海南均有分布^[15] ...

2004

0.0

1.667

... 籽粒苋能高效利用土壤养分^[16]和水分^[17], 光合速率快, 生物学产量很高^[18] ...

2002

1.518

0.0

... 籽粒苋能高效利用土壤养分^[16]和水分^[17], 光合速率快, 生物学产量很高^[18] ...

2006

1.643

0.0

Shukla

, Bhargava

, Chatterjee

, Srivastava

, Singh

S P.

Genotypic variability in vegetable amaranth (Amaranthus tricolor L. ) for foliage yield and its contributing traits over successive cuttings and years. Euphytica, 2006, 151: 103-110

Twenty nine strains of vegetable amaranth (Amaranthus tricolor were grown for two successive seasons to study different selection parameters for foliage yield and its nine contributing morphological and quality traits. The strains AV-38 (5.06 kg/plot) and AV-31 (5.04 kg/plot) recorded highest foliage yield, followed by AV-30 (4.78 kg/plot) and AV-23 (4.70 kg/plot). The protein and carotenoid content averaged 1.24 ± 0.03 mg/100 mg and 0.83 ± 0.02 mg/g respectively. The leaves of A. tricolor also have considerable quantities of ascorbic acid (112.33 ± 5.00 mg/100 g) and fibre (8.39 ± 0.10%). The mean of individual cuttings for plant height, leaf size, stem diameter, foliage yield, protein, ascorbic acid and fibre content increased with successive cuttings till third cutting and thereafter showed a decline. Genotypic coefficient of variation (GCV) values ranged from 6.80 to 28.25%. However, the fibre content, branches/plant, leaves/plant, plant height and stem diameter showed lowest values of GCV. The values of heritability estimates were high for all the traits in all the cuttings as well as on pooled basis and ranged from 0.89 for branches/plant to 0.98 for foliage yield. Highest expected genetic advance was noticed for ascorbic acid (57.48%), followed by foliage yield (48.30%) and leaf size (29.51%).

1.Division of Genetics and Plant Breeding, National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226001 India

... 籽粒苋能高效利用土壤养分^[16]和水分^[17], 光合速率快, 生物学产量很高^[18] ...

2010

0.0

1.321

李平, 白云凤, 张维锋. 籽粒苋苹果酸酶基因克隆及分析. 西北植物学报, 2010, 30: 229-236

, Bai

Y F

, Zhang

W F.

Cloning and analysis of NAD-ME gene of Amaranthus hypochondriacus. Acta Bot Boreali-Occident Sin, 2010, 30: 229-236 (in Chinese with English abstract)

NAD/NADP-苹果酸酶(NAD-ME/NADP-ME)是C_4植物光合途径的关键酶.采用RT-PCR技术对籽粒苋NAD-ME基因进行克隆,获得了籽粒苋NAD-ME基因的cDNA序列.结果表明,该序列开放可读框长度为1 872 bp,编码623个氨基酸;多序列比对和进化树分析表明,该基因核苷酸序列与其他植物已报道的NAD-ME/NADP-ME基因的核苷酸序列一致性高达75.1%～80.6%,其氨基酸序列与其他植物的NAD-ME/NADP-ME蛋白一致性为73.2%～80.3%.对推断氨基酸序列的蛋白保守区、疏水性/亲水性、潜在跨膜片段、信号肽、蛋白固有无序化和蛋白二级结构分析表明,该蛋白具有苹果酸酶的保守区、兼具亲水性和疏水性,并且含有无序结构域,可能是一种跨膜的非分泌性蛋白.

Abstract：

NAD/NADP-malic enzyme is a key enzyme in C_4 photosynthetic pathway.The NAD-ME gene of Amaranthus hypochondriacus was cloned by RT-PCR.According to the analysis of the sequence,the cDNA included a 1 872 bp open reading frame that encoded 623 amino acids.Multi-sequence comparisons and phylogenetic analysis showed that the nucleotide sequence had 75.1%～80.6% identity with the reported NAD-ME/NADP-ME genes of other plants,and the amino acids sequence had 73.2%～80.3% identity.Furthermore,the physico-chemical property of NAD-ME of Amaranthus hypochondriacus,including the conservative domains,hydrophobicity/hydrophilicity,potential transmembrane segments,signal peptides,intrinsically disordered structure and secondary structure of the hypothetical protein were analyzed.The results suggested that the protein contained conservative domains of NAD-ME,both hydrophobic and hydrophilic regions,and also the disordered regions.Thus it seems a non-secreted transmembrane protein.

2011

0.0

0.867

... 籽粒苋AhNAD-ME基因较大, 开放可读框长度为1872 bp, 编码623个氨基酸^[20] ...

2011

23.565

0.0

... 1预测导肽^[21] ...

2003

9.494

0.0

... 内部定位信号序列隐藏在成熟的蛋白质中, 不会被删除, 可能对蛋白质的折叠和功能有重要作用^[22] ...

... 植物的NAD-ME有不同的构成形式, 如三色苋(Amaranthus tricolor)、禾草龙爪稷(Eleusine coracana)^[26]和洋野黍(Panicum dichotomiflorum)^[27]叶片的NAD-ME是同源八聚体, 籽粒苋的NAD-ME是异源八聚体^[22] ...

1989

0.0

... 线粒体导肽有一些特征^[23], 如富含丙氨酸、精氨酸、亮氨酸和丝氨酸, 很少含带有负电荷的谷氨酸、天冬氨酸以及异亮氨酸和赖氨酸, 在相对于切割位点-2位的氨基酸为精氨酸, 是protease-I的识别位点, 第8位为非极性氨基酸 ...

2008

9.737

0.0

... 但同一种蛋白质的导肽在不同植物中差异较大, 不具有高度保守的序列^[24] ...

1994

4.651

0.0

... 我们用SignalP在线分析了AhNAD-ME的氨基酸序列, 发现其不具备导肽序列, 而Long等^[25]微量测序结果表明成熟的AhNAD-ME的N-端缺少了31个氨基酸 ...

1989

6.555

0.0

1980

4.134

0.0

2006

0.0

Esposito

, Chatterjee

D K.

Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol, 2006, 17: 353-358

The soluble expression of heterologous proteins in Escherichia coli remains a serious bottleneck in protein production. Although alteration of expression conditions can sometimes solve the problem, the best available tools to date have been fusion tags that enhance the solubility of expressed proteins. However, a systematic analysis of the utility of these solubility fusions has been difficult, and it appears that many proteins react differently to the presence of different solubility tags. The advent of high-throughput structural genomics programs and advances in cloning and expression technology afford us a new way to compare the effectiveness of solubility tags. This data should allow us to better predict the effectiveness of tags currently in use, and might also provide the information needed to identify new fusion tags.

2005

9.66

0.0