甘蔗中性/碱性转化酶基因<italic>SoNIN1</italic>的克隆和表达分析

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牛俊奇, 王爱勤, 黄静丽, 杨丽涛, 李杨瑞. 甘蔗中性/碱性转化酶基因SoNIN1的克隆和表达分析 . 作物学报, 2014, 40(2): 253-263[NIU Jun-Qi, WANG Ai-Qin, HUANG Jing-Li, YANG Li-Tao, LI Yang-Rui. Cloning and Expression Analysis of Sugarcane Alkaline/Neutral Invertase GeneSoNIN1 . 作物学报, 2014, 40(2): 253-263] 复制到剪切板

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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

^* 通讯作者(Corresponding authors): 杨丽涛, E-mail:liyr@gxu.edu.cn, Tel: 0771-3236407; 李杨瑞, E-mail:liyr@gxaas.net, Tel: 0771-3247689

收稿日期:2013-09-16

基金: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(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), 广西出八桂学者、特聘专家专项经费和广西农业科学院团队项目(桂农科2011YT01)资助。

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

Cloning and Expression Analysis of Sugarcane Alkaline/Neutral Invertase GeneSoNIN1

NIU Jun-Qi^1,², WANG Ai-Qin^1,², HUANG Jing-Li^1,², YANG Li-Tao^1,^2,^3,^*, LI Yang-Rui^2,^3,^4,^*

¹Agricultural College, Guangxi University, Nanning 530005, China

²State 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

⁴Guangxi Crop Genetic Improvement and Biotechnology Key Laboratory, Nanning 530007, China

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 using RT-PCR (reverse transcription-polymerase chain reaction) and RACE (rapid amplification of cDNA ends), and named asSoNIN1. 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.SoNIN1gene encodes a protein that is close to that ofZea 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 ascis-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 ofSoNIN1 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 lowest in internode +1 (enrolled by the top visible dewlap leaf). The expression ofSoNIN1 gene in roots and leaves of sugarcane seedling was induced by PEG and NaCl.

Keyword: Sugarcane; Alkaline/neutral invertase; Promoter; Cloning; Gene expression

Show Figures

转化酶是蔗糖代谢的关键酶, 能不可逆地催化蔗糖水解成葡萄糖和果糖, 在作物经济产量形成与果实品质改良中发挥重要作用^{[ 1, 2]}。植物转化酶依据亚细胞定位、酶活性最适pH值不同可分为酸性转化酶和中性/碱性转化酶两个亚基因家族^{[ 3]}。转化酶活性的缺乏是蔗糖积累的先决条件, 酶活性高于临界阈值时将不再积累高浓度的蔗糖, 主要起分解蔗糖作用^{[ 4]}。在甘蔗成熟期, 叶片的酸性转化酶活性随甘蔗的成熟而降低; 而中性/碱性转化酶活性随甘蔗的成熟而升高, 酶活性提高有利于蔗糖的积累^{[ 5]}。酸性转化酶是调节甘蔗蔗茎发育过程中不同节间蔗糖含量的关键酶, 是节间蔗糖积累负调节因子, 成熟茎中酸性转化酶酶活性降低有利于蔗糖的积累^{[ 6, 7]}。转化酶在植物正常生长中的作用远远大于蔗糖合成酶^{[ 8]}。

目前, 已从黑麦草^{[ 9]}、小麦^{[ 10]}、橡胶^{[ 11]}、杨树^{[ 12]}、水稻^{[ 13]}等植物中克隆到中性/碱性转化酶基因。该酶是一种胞质酶, 主要存在于细胞质中, 也存在于线粒体和质体中^{[ 14, 15]}。在线粒体中, 该酶与线粒体膜上蔗糖转运蛋白酶活性密切相关, 在调节细胞渗透压和中间产物代谢方面起着重要作用^{[ 16]}。敲除拟南芥叶绿体 A/N-InvE基因后, 叶片中 A/N-InvE基因表达量降低, 淀粉积累量减少, 推测 A/N-InvE基因可能参与叶绿体和线粒体之间的碳循环调节^{[ 17]}。中性/碱性转化酶基因突变体表型为短根、开花延迟、部分不育, 说明其对植物根系细胞发育和生殖发育起着重要作用^{[ 18, 19, 20, 21]}。拟南芥 Atcyt-inv1突变体证实, Atcyt-inv1抑制侧根发育是通过控制细胞内己糖浓度实现的^{[ 22]}

本研究采用RT-PCR和RACE-PCR技术, 从甘蔗心叶中克隆到一个全长的中性/碱性转化酶基因。同时采用染色体步移技术, 克隆该基因的上游启动子序列。用实时荧光定量PCR分析 SoNIN1在花期甘蔗不同组织部位的表达情况, 以及在盐胁迫和干旱胁迫下该基因在甘蔗根和叶中的表达情况, 以期为研究 SoNIN1在不同胁迫处理下基因的表达调控规律提供参考依据。

1 材料与方法

1.1 试验材料

甘蔗品种桂糖28^{[ 23]}(GT28)由广西农业科学研究院甘蔗研究所提供。生理成熟期的甘蔗取材于广西大学农学院甘蔗资源圃新植蔗, 取材时间为2013年1月22日。采样时以蔗茎完全露出地面的节间为+1节, 依次向上类推到+31节。以顶端第一片完全展开叶为+1叶, 处于顶端中心位置还没有展开的叶为心叶。

把蔗茎砍成单芽, 在广西大学农学院甘蔗智能温室大棚进行沙土育苗的桶栽实验。当甘蔗长到2~3片真叶时, 移植于5 L的塑料桶, 每桶3株苗。采用Hoagland营养液水培, 每隔1 d换一次营养液。当甘蔗长到6~7片真叶时, 把甘蔗分成3组, 每组6桶。一组为同期对照, 另外两组分别为模拟干旱(15% PEG-6000)和盐(100 mmol L^-1 NaCl)胁迫处理, 并分别于处理后0、3、6、12和24 h取样。分别取甘蔗幼根和+1叶, 用锡箔纸包好后迅速用液氮冷冻, 于-80℃保存。

1.2 试验试剂

RNA提取TRIzol试剂盒购于TIANGEN; RNA反转录试剂盒购于Fermentas; 5°RACE试剂盒购于Invitrogen公司; Ex Taq酶、dNTPs、pMD18-T载体、PrimeScript RT reagent Kit With gDNA Eraser、Genome Walking Kit、SYBR Premix Ex Taq II购自TaKaRa (大连), 其余试剂均为国产分析纯。本实验所用特异引物由上海生工生物工程技术服务有限公司合成。采用ABI Stepone Plus型荧光定量PCR仪进行实时荧光定量PCR。

1.3 RNA提取和cDNA合成

参照TRIzol试剂盒说明书, 以甘蔗心叶为材料提取总RNA。利用琼脂糖凝胶电泳检测总RNA完整性, 利用Thermo Scientific NaNoDrop 2000c检测总RNA浓度及纯度。按照ReverAid First Strand cDNA Synthesis Kit试剂盒说明书反转录成cDNA第1链。

1.4 甘蔗 SoNIN1基因的克隆

1.4.1 甘蔗 SoNIN1基因中间片段的克隆以已报道的玉米中性/碱性转化酶基因(GenBank登录号为EU955523)核苷酸序列, 运用NCBI网站上Blast程序在GenBank数据库中进行同源核苷酸序列搜索, 选取与甘蔗亲缘关系较近植物的核苷酸序列在Vector NTI Advance 11.0进行同源性比对分析, 在核苷酸序列保守性较高的地方设计1对特异引物NIZJF1和NIZJR1 (表1)。扩增体系为25 µL, 含Ex Taq酶0.25 µL、2×GC buffer II 12.5 µL、2.5 mmol L^-1 dNTPs 1.5 µL、10 mmol L^-1上下游引物各1 µL, 模板cDNA 1 µL、ddH₂O 7.75 µL。PCR反应程序为94℃

预变性5 min; 94℃变性50 s, 56.5℃退火30 s, 72℃延伸2 min, 35个循环; 最后72℃延伸10 min。PCR产物经胶回收纯化后克隆到T载体, 转入感受态细胞DH5α中, 经PCR菌液检测后, 把含有目的片段的菌液送深圳华大基因研究院测序。

1.4.2 甘蔗 SoNIN1基因5°-RACE克隆根据1.4.1测序获得的 SoNIN1基因中间片段核苷酸序列, 设计下游特异引物NISYF1、NISYF2和NISYF3 (表1)。由5′-Full RACE Kit (TaKaRa)试剂盒提供上游引物Outer Primer: 5′-CATGGCTACATGCTGACAGCCT A-3′和Inner Primer: 5′-CGCGGATCCACAGCCTACTG ATGATCAGTCGATG-3′。

表1 SoNIN1基因克隆与表达分析中使用的引物 Table 1 Primers used in cloning and expression analysis of SoNIN1

按照5′-Full RACE Kit (TaKaRa)说明书合成cDNA模板, 做巣式PCR扩增。以外引物NISYF1和Outer Primer进行第1轮PCR扩增。PCR反应体系94℃ 5 min; 94℃ 50 s, 56℃ 30 s, 72℃ 2 min, 35个循环; 72℃延伸10 min。取第1轮PCR产物2 µL, 以内引物1 (NISYF3和Inner Primer)和内引物2 (NISYF2和Inner Primer)分别进行第2轮PCR扩增, PCR反应体系为94℃预变性5 min; 94℃变性50 s, 58.5℃退火30 s, 72℃延伸1.5 min, 35个循环; 72℃ 10 min。PCR产物经胶回收纯化后克隆到T载体, 转入感受态细胞DH5α中, 经菌液PCR检测正确后送华大测序。

1.4.3 甘蔗 SoNIN1基因3′-RACE克隆根据1.4.1测序获得的 SoNIN1基因中间片段核苷酸序列, 设计用于3′端扩增的2条特异上游引物NIXYF1和NIXYF2、1条下游引物NIXYR1和1条反转录特异引物GZS (表1)。以提取的甘蔗心叶总RNA为模板, 按照ReverAid First Strand cDNA Synthesis Kit试剂盒说明书, 以GZS核苷酸序列作为反转录引物, 反转录合成cDNA第一链。用NIXYF1和NIXYR1引物进行第1轮PCR扩增, 将PCR产物稀释50倍后作为引物NIXYF2和NIXYR1的模板, 进行第2轮PCR扩增。PCR反应程序94℃ 5 min; 94℃ 50 s, 57℃ 30 s, 72℃ 1.30 min, 35个循环; 72℃ 10 min。产物经胶回收纯化后克隆到T载体并测序。

1.5 甘蔗 SoNIN1全长cDNA拼接验证

将1.4.1、1.4.2和1.4.3中所克隆的3个序列, 利用软件ContigExpress拼接, 获得 SoNIN1基因的全长cDNA序列, 而后在该基因的编码区设计一对引物NIF11和NIR11 (表1), 对3部分拼接结果进行PCR扩增验证。PCR扩增程序为94℃ 5 min; 94℃ 50 s, 59℃ 30 s, 72℃ 2 min, 35个循环; 72℃ 10 min。

1.6 甘蔗 SoNIN1基因启动子序列克隆

在 SoNIN1 5′上游核苷酸序列区设计3条下游引物NINSP1、NINSP2和NINSP3 (表1), 与Genome Walking Kit试剂盒提供的上游引物AP2作巢式PCR扩增。采用改良的SDS法提取甘蔗基因组DNA^{[ 24]}。PCR扩增体系50 μL含DNA 2 μL、2.5 mmol L^-1dNTPs混合物, 8 μL、10×LA PCR buffer II (Mg²⁺ plus) 5 μL、5 U μL^-1TaKaRa LA Taq0.5 μL、100 pmol μL^-1AP2 Primer 1 μL、10 pmol μL^-1 NINSP1 Primer 1 μL、ddH₂O 32.5 μL。按试剂盒说明书进行PCR, 优化后第1步移PCR扩增NINSP2和AP2引物退火温度为63℃, 第2步移PCR扩增NINSP1和AP2引物温度为63.5℃, 第3步移PCR扩增NINSP3和AP2引物退火温度为65℃。

1.7 SoNIN1基因的生物信息学分析

利用http://web.expasy.org/cgi-bin/protparam/protparam网站在线分析基因的氨基酸组成、理论分子量和等电点; 利用http://smart.embl-heidelberg.de/网站进行基因编码蛋白质的结构功能域分析; 利用MEGA4软件进行基因氨基酸序列同源比较及进化

树分析; 在http://www.cbs.dtu.dk/services/SignalP网站进行蛋白质信号肽分析; 在http://genome.cbs.dtu. dk/services/TargetP预测基因的亚细胞定位; 用TMHMM Server v.2.0预测编码蛋白的跨膜结构域。用PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html)和PLACE (http://www.dna.affrc.go.jp/PLACE/signalup.html)对 SoNIN1启动子顺式作用元件进行在线预测。

1.8 SoNIN1基因的实时荧光定量表达分析

根据 SoNIN1基因全长序列设计用于实时荧光定量PCR的引物NI-F和NI-R (表1)。甘蔗内参基因选择, 以基因表达稳定性好的甘油醛-3-磷酸脱氢酶基因( GAPDH)为内参基因^{[ 25, 26]}, 根据GenBank数据库中甘蔗 GAPDH (EF189713), 设计内参引物GAPDH-Fn和GAPDH-Rn (表1)。利用荧光染料法进行实时荧光定量表达分析, 反应参数95℃ 30 s, 一个循环; 95℃ 5 s, 60℃ 20 s, 共45个循环。采用2^-ΔΔCT法^{[ 27]}, 分析基因的相对表达量。

2 结果与分析

2.1 甘蔗 SoNIN1基因全长序列的拼接和验证

经华大测序中间片段PCR产物长度1131 bp (图1-A); 5′片段测序结果内引物1的PCR产物长度为769 bp (图1-B), 内引物2的PCR产物长度826 bp (图1-B), 两者经ContigExpress拼接后长度为894 bp; 3′片段扩增产物561 bp (图1-C)。三部分核苷酸序列经ContigExpress拼接后, 其cDNA序列长度2289 bp。该核苷酸序列与玉米(EU955523)、水稻(AK121301)和黑麦草(AJ003114)的中性/碱性转化酶基因核苷酸序列同源性较高, 分别是95%、86%和86%, 说明本研究克隆到的是甘蔗中性/碱性转化酶基因。

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	图1 甘蔗 SoNIN1基因序列扩增结果M: DL2000; A: 中间片段扩增结果; B: 5′端片段扩增结果(1: 内引物1; 2: 内引物2); C: 3′端片段扩增结果; D: ORF序列扩增结果。Fig. 1 PCR product of SoNIN1 gene from sugarcaneM: DL2000; A: intermediate fragment; B: 5′ RACE result (1: Inner primer 1; 2: Inner primer 2); C: 3′ RACE result; D: PCR product of ORF.

参照Murayama和Handa^{[ 14]}对水稻中性/碱性转化酶基因的命名, 把该基因命名为 SoNIN1。在NCBI上比对分析发现已经克隆到该基因的全长cDNA序列, 包括一个1812 bp (在287~2098 bp间)的完整开放阅读框(open reading frame, ORF), 编码603个氨基酸(图2), 其中5°非编码区长度为286 bp, 3′非编码区长度为191 bp, 该基因编码蛋白与玉米(EU955523)、水稻(AK121301)、毒麦(AJ003114)、黑麦草(AM489692)、甜菜(AJ422050)、拟南芥(NM122156)中编码中性/碱

性转化酶基因编码的蛋白质同源性分别为96%、90%、87%、86%、69%和68%, 但与Bosch等^{[ 28]}报道的甘蔗SNI蛋白质同源性仅为46%, 说明 SoNIN1是甘蔗中性/碱性转化酶基因家族的新成员。

为了验证拼接结果, 根据拼接所获得的全长cDNA序列, 在编码区两端设计一对引物NIF11和NIR11。PCR扩增产物经1%琼脂糖凝胶电泳检测, 获得1.8 kb左右的单一条带(图1-D)。该片段经华大公司测序, 获得1812 bp核苷酸片段, 该片段与拼接的编码区cDNA同源性为99.7%, 证明拼接结果正确。该基因已在DDBJ/EMBL/GenBank基因数据库注册, 注册号为JX109944。

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	图2 甘蔗 SoNIN1全长核苷酸序列及推测编码的氨基酸序列方框代表起始密码子; 代表终止密码子; 下画线代表poly (A)。Fig. 2* Nucleotide sequence and putative amino acid sequence of the full-length of SoNIN1The panes show the positions of ORF. Frame: initiation codon; * termination codon; underline: poly (A).

2.2 甘蔗 SoNIN1基因启动子的克隆与顺式作用元件分析

第1轮、第2轮和第3轮染色体步移PCR扩增产物, 经1%琼脂糖凝胶电泳检测, 结果如图3所示。回收第3轮的PCR产物胶。连接pMD18 T-vector后, 经菌液PCR检测正确, 序列测序结果为1182 bp。用Blastn进行同源性比对分析, 与玉米中性/碱性转化酶基因(EU955523)核苷酸序列同源性为94%, 说明克隆到的序列是甘蔗中性/碱性转化酶基因的启动子序列, 在GenBank登录号为KC854419。用 SoNIN1cDNA的5′非编码区序列与启动子序列进行同源性比对分析, 发现二者在其同源的282个核苷酸中仅有5个碱基的差异, 说明我们克隆的序列为 SoNIN1的上游未知启动子序列, 图4黑色阴影部分为二者之间的同源序列区, 其中位于启动子ATG之前的序列为1174 bp。该启动子序列在48~ 316 bp间存在一个268 bp的非转录区, 二者的交界处符合GT-AG法, 该区域可能起诱导和增强 SoNIN1基因转录的作用。

用PlantCARE在线预测的 SoNIN1启动子区顺式调控作用元件, 将其翻译起始密码子ATG中的A

定义为+1位, SoNIN1启动子序列含有6个CAAT-box和8个TATA-box。同时还有脱落酸(motif IIb)和赤霉素(P-box)响应元件、茉莉酸甲酯(CGTCA-motif)顺式调控元件, 参与干旱诱导的MYB结合位点, 分生组织特异性激活顺式调控元件, 以及光应答元件等(表2), 表明甘蔗 SoNIN1基因的表达可能受光、脱落酸、赤霉素、茉莉酸和干旱等调控。

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	图3 甘蔗 SoNIN1基因启动子序列扩增产物M: DL2000 marker; A: 第1轮步移产物; B: 第2轮步移产物; C: 第3轮步移产物。Fig. 3 PCR product of the promoter sequence of SoNIN1 geneM: DL2000 marker; A: the first amplification of genomic walking; B: the second amplification of genomic walking; C: the third amplification of genomic walking.

表2 SoNIN1基因启动子顺式作用元件预测 Table 2 Predicting cis-acting elements of SoNIN1 promoter

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	图4 甘蔗 SoNIN1基因启动子区域序列及预测转录因子结合位点阴影部分为 SoNIN1cDNA的5′非编区。Fig. 4 Nucleotide sequence (part) and in silico prediction of TBFs of the SoNIN1 promoter regionThe shading parts are cDNA non-coding region of SoNIN1.

2.3 甘蔗 SoNIN1基因生物信息学和聚类分析

用SMART分析SoNIN1蛋白在116~587位氨基酸具有植物中性/碱性转化酶基因保守结构域Pfam: Invertase_neut, 属于Bac_rhamnosid亚基因家族。推测的SoNIN1蛋白质的分子量为67.79 kD、等电点为6.41。SignalP信号肽预测显示该基因不含信号肽序列。用TMHMM Server v.2.0预测该基因不含跨膜结构。TargetP 1.1 Server预测该基因可能定位于线粒体中。

利用MEGA4.0软件对甘蔗中性/碱性转化酶基因编码的蛋白与其他物种编码的蛋白进行进化树分析, 结果植物中性/碱性转化酶可分为I和II类。SoNIN1蛋白首先与单子叶禾本科植物黑麦草聚类, 其次与玉米(ACG27641.1)和水稻(BAH00419.1)聚类, 最后与拟南芥、甜菜和木薯等聚类, 在进化分支上属于I类。

2.4 甘蔗 SoNIN1基因在甘蔗植株不同部位的表达

以甘蔗 GAPDH为内参基因, 实时荧光定量PCR检测 SoNIN1在甘蔗植株生理成熟期不同部位的相对表达量。图6表明, SoNIN1在茎、叶、花序、花序轴和芽中都能检测到表达, 基因表达量为叶>芽>花序>茎, 而在叶中为+3叶>心叶> +6叶> +1叶; 在甘蔗不同节间的基因表达, 表现为从+1节间到+16节间基因的表达量逐渐上升, +16节间最大, 之后降低, 而在+16、+21、+31节间基因的表达量相差不大, 都略高于+1节。在花序中基因的表达量高于花序轴, 大约为其2.54倍。

2.5 NaCl和PEG胁迫处理对甘蔗 SoNIN1基因表达的影响

在NaC胁迫处理下, SoNIN1在根、叶中的表达呈现出“低-高-低”趋势(图7), 短期内表现出先抑制后促进的趋势, 处理3 h该基因在根和叶中的表达均受到抑制, 之后逐渐升高, 处理12 h表达量达到最高; 但随着胁迫时间的延长, 处理24 h该基因在根、叶中的表达迅速降到最低水平(图7)。盐胁迫下, 该基因在根部表达较叶部高(图7)。

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	图5 SoNIN1蛋白质与其他22个不同来源的中性/碱性转化酶的进化关系分析Fig. 5 Phylogenetic tree of SoNIN1 protein and 22 NINs from other species

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图6 SoNIN1在甘蔗不同器官中的表达1~7: +1、+6、+11、+16、+21、+26、+31节间; 8~11: 心叶、+1、+3、+6叶; 12: 花序轴; 13: 花序; 14: 芽。Fig. 6 Expression of SoNIN1 gene in different sugarcane organs assayed1-7: internodes +1, +6, +11, +16, +21, +26, +31, respectively; 8-11: top leaf roll, leaf +1, leaf +3, leaf +6, respectively; 12: rachis; 13: inflorescence; 14: bud.

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	图7 NaCl胁迫对甘蔗 SoNIN1在叶和根中表达的影响Fig. 7 Effects of NaCl stress on the expression of SoNIN1in leaf and root of sugarcane

在15%PEG模拟干旱胁迫处理下, SoNIN1在甘蔗叶中基因的表达量在胁迫处理6 h降到最低, 12 h达到最大, 24 h降到较低水平, 但仍高于0 h 。而根中 SoNIN1基因表达量则持续地上升, 24 h维持在较高的水平, 是0 h的2.92倍。说明PEG胁迫诱导 SoNIN1基因在根、叶的表达。

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	图8 15% PEG胁迫对甘蔗 SoNIN1在叶和根中表达的影响Fig. 8 Effects of 15% PEG stress on the expression of SoNIN1 in leaf and root of sugarcane

3 讨论

本研究采用RT-PCR和RACE技术克隆到甘蔗一个中性/碱性转化酶基因, 它具有中性/碱性转化酶保守结构域Pfam: Invertase_neut。序列分析表明该基因具有完整的ORF, 所编码的蛋白与其他禾本科物种具有很高的同源性, 而与Bosch等^{[ 28]}报道的甘蔗中性/碱性转化酶基因同源性很低, 氨基酸聚类分析表明二者都属于I类, 但在I类中又属于不同的亚类群, 说明它们是甘蔗中性/碱性转化酶家族的不同成员。

甘蔗是重要的糖料作物, 节间蔗糖含量与中性/碱性转化酶活性成负相关, 而不同节间的中性/碱性转化酶活性不同^{[ 29]}。用荧光定量PCR法检测到 SoNIN1在花期蔗茎中基因表达量低于叶、花序和芽中的表达, 茎中 SoNIN1在转录水平上基因的表达量低, 可能有利于蔗茎中的蔗糖积累。对转甘蔗反义 SNI基因植株的研究表明, 中性/碱性转化酶酶活性受到抑制, 导致细胞内己糖含量减小而蔗糖含量增加^{[ 30]}。通过转反义基因技术抑制中性/碱性转化酶基因的表达, 使中性/碱性转化酶蛋白合成减少, 酶活性降低, 蔗茎中己糖含量减少, 而蔗糖含量增加^{[ 31]}。

启动子是基因表达所必需的重要DNA信号序列, 是细胞内控制基因转录与表达的时空特异性的重要因子, 可以调控基因转录表达的方向和效率, 是转录调控的中心^{[ 32]}。本研究采用基于热不对称交错PCR原理的染色体步移技术克隆到甘蔗 SoNIN1基因启动子序列, 从转录水平上定向调控中性/碱性转化酶基因表达方向和效率, 进而探讨蔗糖代谢途径的调控。在15% PEG胁迫处理12 h后, 甘蔗根和叶中 SoNIN1表达都被诱导提高, 这与启动子序列上参与干旱诱导的MYB结合位点, 以及在受到脱落酸、赤霉素和茉莉酸甲酯等外源激素诱导时, 与启动子上相应的作用元件之间有无直接联系及其作用模式, 还需要进一步的验证。下一步我们将构建启动子序列瞬时表达载体, 在拟南芥中验证 SoNIN1基因的启动子序列的功能。

NaCl和PEG胁迫提高甘蔗 SoNIN1在根叶中的差异表达, 但对贮藏蔗糖的器官—茎的蔗糖代谢的影响和关系, 有待进一步研究。本研究中, 甘蔗中性/碱性转化酶基因的克隆及其表达分析, 不仅为中性/碱性转化酶基因家族增添了新的成员, 同时为进一步研究该基因在甘蔗蔗糖积累中作用及其功能鉴定打下了良好的基础。

4 结论

从甘蔗中克隆到一个中性/碱性转化酶基因, 基因全长2289 bp, 编码603个氨基酸。具有中性/碱性转化酶基因保守结构域Pfam: Invertase_neut, N-端不含跨膜结构和信号肽序列。 SoNIN1 5°端侧翼启动子序列长1174 bp, 除了具有基因启动子的特定结构TATA-box和CAAT-b box, 还含有赤霉素、脱落酸、茉莉酸甲酯响应元件以及与干旱诱导的MYB结合位点等特异的作用元件。 SoNIN1为组成型表达, 生理成熟期表达量在甘蔗叶中最大, 芽中次之, 而在+1节间中最低。PEG胁迫和NaCl能诱导该基因在根和叶中的表达。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

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2012

0.0

... 转化酶是蔗糖代谢的关键酶, 能不可逆地催化蔗糖水解成葡萄糖和果糖, 在作物经济产量形成与果实品质改良中发挥重要作用^[1,2] ...

2008

0.0

1.194

... 转化酶是蔗糖代谢的关键酶, 能不可逆地催化蔗糖水解成葡萄糖和果糖, 在作物经济产量形成与果实品质改良中发挥重要作用^[1,2] ...

1999

6.555

0.0

... 植物转化酶依据亚细胞定位、酶活性最适pH值不同可分为酸性转化酶和中性/碱性转化酶两个亚基因家族^[3] ...

2000

0.0

... 转化酶活性的缺乏是蔗糖积累的先决条件, 酶活性高于临界阈值时将不再积累高浓度的蔗糖, 主要起分解蔗糖作用^[4] ...

2006

0.0

0.5408

Sugar Crop China. 2006, (3):16 - 18

Studies on Sucrose Content and Foliar Enzyme Activity of Sugarcane during Mature Period

成熟期不同基因型甘蔗糖分和叶片酶活性的变化

GUOJia-wen;LIUShao-chun;ZHANGYue-bin;LIWen-feng

郭家文;刘少春;张跃彬;李文凤

研究了9个甘蔗品种叶片的酸性转化酶、中性转化酶、淀粉酶活性在甘蔗成熟期的变化规律.结果表明,在甘蔗成熟期,叶片的酸性转化酶活性随着甘蔗的成熟而降低,中性转化酶活性随着甘蔗的成熟而升高,淀粉酶活性在成熟期都呈下降趋势.转化酶和淀粉酶的基因型效应差异较大,不同的供试材料具有不同的酶活性变化,材料间的差异较大.

... 而中性/碱性转化酶活性随甘蔗的成熟而升高, 酶活性提高有利于蔗糖的积累^[5] ...

2009

0.0

Sugar Tech. 2009, 11:28-

Soluble acid invertase and sucrose phosphate synthase: key enzymes in regulating sucrose accumulation in sugarcane stalk

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

... 酸性转化酶是调节甘蔗蔗茎发育过程中不同节间蔗糖含量的关键酶, 是节间蔗糖积累负调节因子, 成熟茎中酸性转化酶酶活性降低有利于蔗糖的积累^[6,7] ...

2003

0.0

Sugar Tech. 2003, 5:265-

Sucrose metabolism and expression enzyme activities in low and high storing sugarcane genotypes of key sucrose

Sachdeva

,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

2009

9.737

0.0

Proc. Natl. Acad. Sci. U.S.A.. 2009, 106(31):13124-9 DOI:10.1073/pnas.0900689106

Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase.

Barratt DH, Derbyshire P, Findlay K, Pike M, Wellner N, Lunn J, Feil R, Simpson C, Maule AJ, Smith AM

John Innes Centre and Institute of Food Research, Norwich Research Park, Norwich NR4 7UH, United Kingdom.

The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.

PMID: 19470642

... 转化酶在植物正常生长中的作用远远大于蔗糖合成酶^[8] ...

1998

5.242

0.0

... 目前, 已从黑麦草^[9]、小麦^[10]、橡胶^[11]、杨树^[12]、水稻^[13]等植物中克隆到中性/碱性转化酶基因 ...

2007

3.347

0.0

Planta. 2007, 226(6):1535 - 1545 DOI:10.1007/s00425-007-0590-3

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

Walter A. Vargas(12) Horacio G. Pontis(1) Graciela L. Salerno(1)

1.Fundación para Investigaciones Biológicas Aplicadas (FIBA) Centro de Investigaciones Biológicas C.C. 1348 7600 Mar del Plata Argentina
2.Texas A&M University Department of Plant Pathology and Microbiology College Station TX 77843 USA

It is well accepted that sucrose (Suc) metabolism is involved in responses to environmental stresses in many plant species. In the present study we showed that alkaline invertase (A-Inv) expression is up-regulated in wheat leaves after an osmotic stress or a low-temperature treatment. We demonstrated that the increase of total alkaline/neutral Inv activity in wheat leaves after a stress could be due to the induction of an A-Inv isoform. Also, we identified and functionally characterized the first wheat cDNA sequence that codes for an A-Inv. The wheat leaf full-length sequence encoded a protein 70% similar to a neutral Inv ofLolium temulentum; however, after functional characterization, it resulted to encode a protein that hydrolyzed Suc to hexoses with an optimum pH of 8, and, consequently, the encoding sequence was namedTa-A-Inv. By RT-PCR assays we demonstrated thatTa-A-Inv expression is induced in response to osmotic and cold stress in mature primary wheat leaves. We propose that Ta-A-Inv activity could play an important role associated with a more efficient cytosolic Suc hydrolysis during environmental stresses.

... 目前, 已从黑麦草^[9]、小麦^[10]、橡胶^[11]、杨树^[12]、水稻^[13]等植物中克隆到中性/碱性转化酶基因 ...

2010

0.0

... 目前, 已从黑麦草^[9]、小麦^[10]、橡胶^[11]、杨树^[12]、水稻^[13]等植物中克隆到中性/碱性转化酶基因 ...

2008

3.347

0.0

Planta. 2008, 227(3):565 - 576 DOI:10.1007/s00425-007-0639-3

Evolution and diversity of invertase genes inPopulus trichocarpa

Philip N. Bocock(1) Alison M. Morse(2) Christopher Dervinis(2) John M. Davis(12)

1.University of Florida Plant Molecular and Cellular Biology Program PO Box 110690 Gainesville FL 32611 USA
2.University of Florida School of Forest Resources and Conservation PO Box 110410 Gainesville FL 32611 USA

Invertase (EC 3.2.1.26) plays a key role in carbon utilization as it catalyzes the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families thought to have distinct evolutionary origins and can be distinguished by their pH optima for activity: acid invertases and neutral/alkaline invertases. The acid invertases apparently originated in eubacteria and are targeted to the cell wall and vacuole, while neutral/alkaline invertases apparently originated in cyanobacteria and function in the cytosol. The recently sequenced genome ofPopulus trichocharpa (Torr. and Gray) allowed us to identify the genes encoding invertase in this woody perennial. Here we describe the identification of eight acid invertase genes; three of which belong to the vacuolar targeted group (PtVIN1–3), and five of which belong to the cell wall targeted group (PtCIN1–5). Similarly, we report the identification of 16 neutral/alkaline invertase genes (PtNIN1–16). Expression analyses using whole genome microarrays and RT-PCR reveal evidence for expression of all invertase family members. An examination of the micro-syntenic regions surrounding the poplar invertase genes reveals extensive colinearity withArabidopsis invertases. We also find evidence for expression of a novel intronless vacuolar invertase (PtVIN1), which apparently arose from a processedPtVIN2 transcript that re-inserted into the genome. To our knowledge, this is the first intronless invertase found in plants. This work increases the understanding of the role this family plays in carbon allocation and partitioning in forest trees as well as its evolutionary development.

... 目前, 已从黑麦草^[9]、小麦^[10]、橡胶^[11]、杨树^[12]、水稻^[13]等植物中克隆到中性/碱性转化酶基因 ...

2003

0.0

... 目前, 已从黑麦草^[9]、小麦^[10]、橡胶^[11]、杨树^[12]、水稻^[13]等植物中克隆到中性/碱性转化酶基因 ...

2007

3.347

0.0

Planta. 2007, 225(5):1193 - 1203 DOI:10.1007/s00425-006-0430-x

Genes for alkaline/neutral invertase in rice: alkaline/neutral invertases are located in plant mitochondria and also in plastids

Seiji Murayama(1) Hirokazu Handa(23)

1.National Agricultural Research Center for Hokkaido Region Research Team for Crop Cold Tolerance 1 Hitsuji-ga-oka, Toyohira-ku Sapporo 062-8555 Japan
2.National Institute of Agrobiological Sciences Plant Genome Research Unit 2-1-2 Kan-non-dai Tsukuba 305-8602 Japan
3.University of Tsukuba Graduate School of Life and Environmental Sciences 1-1-1 Ten-noh-dai Tsukuba 305-8572 Japan

Two cDNA clones (OsNIN1 andOsNIN3) encoding an alkaline/neutral invertase localized in organelles were identified from rice. The deduced amino acid sequences of these cDNA clones showed high homology to other plant alkaline/neutral invertases. Semi-quantitative reverse transcription polymerase chain reaction revealed that the expression ofOsNIN1 was constitutive and independent of organ difference, although its expression level was low. Analyses using five types of web software for the prediction of protein localization in the cell, Predotar, PSORT, Mitoprot, TargetP, and ChloroP, strongly supported the possibility that OsNIN1 is transported into the mitochondria and that OsNIN3 is transported into plastids. Transient expression of fusion proteins combining the amino terminal region of these two proteins with sGFP demonstrated that N-OsNIN1::GFP and N-OsNIN3::GFP fusion proteins were transported into the mitochondria and plastids, respectively. We expressed the OsNIN1 protein in vitro and revealed that the translated protein had an invertase activity. These results clearly indicate that some of alkaline/neutral invertases are located in plant organelles, mitochondria and plastids, and that they might have a novel physiological function in plant organelles.

... 该酶是一种胞质酶, 主要存在于细胞质中, 也存在于线粒体和质体中^[14,15] ...

... 参照Murayama和Handa^[14]对水稻中性/碱性转化酶基因的命名, 把该基因命名为SoNIN1 ...

2011

5.242

0.0

J. Exp. Bot.. 2011, 62(11):3849-62 DOI:10.1093/jxb/err069

Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana.

Xiang L, Le Roy K, Bolouri-Moghaddam MR, Vanhaecke M, Lammens W, Rolland F, Van den Ende W

KULeuven, Laboratory of Molecular Plant Physiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium.

Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.

PMID: 21441406

... 该酶是一种胞质酶, 主要存在于细胞质中, 也存在于线粒体和质体中^[14,15] ...

2008

3.347

0.0

Planta. 2008, 228(5):765 - 775 DOI:10.1007/s00425-008-0778-1

Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (Helianthus tuberosus L.) tubers

András Szarka(12) Nele Horemans(3) Salvatore Passarella(4) ákos Tarcsay(1) Ferenc ?rsi(1) András Salgó(1) Gábor Bánhegyi(2)

1.Budapest University of Technology and Economics Department of Applied Biotechnology and Food Science, Laboratory of Biochemistry and Molecular Biology M?egyetem rakpart 3 1111 Budapest Hungary
2.Semmelweis University, Pathobiochemistry Research Group of The Hungarian Academy of Sciences Department of Medical Chemistry, Molecular Biology and Pathobiochemistry P.O. Box?260 1444 Budapest Hungary
3.University of Antwerp Research Group of Plant Physiology, Department of Biology Groenenborgerlaan 171 2020 Antwerp Belgium
4.Università del Molise Dipartimento di Scienze per la Salute Via De Sanctis 86100 Campobasso Italy

Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix ofHelianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism.

... 在线粒体中, 该酶与线粒体膜上蔗糖转运蛋白酶活性密切相关, 在调节细胞渗透压和中间产物代谢方面起着重要作用^[16] ...

2008

3.347

0.0

Planta. 2008, 227(4):795 - 807 DOI:10.1007/s00425-007-0657-1

New insights on sucrose metabolism: evidence for an active A/N-Inv in chloroplasts uncovers a novel component of the intracellular carbon trafficking

Walter A. Vargas(12) Horacio G. Pontis(1) Graciela L. Salerno(1)

The presence of sucrose (Suc) in plastids was questioned for several decades. Although it was reported some decades ago, neither Suc transporters nor Suc metabolizing enzymes were demonstrated to be active in those organelles. By biochemical, immunological, molecular and genetic approaches we show that alkaline/neutral invertases (A/N-Invs) are also localized in chloroplasts of spinach andArabidopsis. A/N-Inv activity and polypeptide content were shown in protein extracts from intact chloroplasts. Moreover, we functionally characterized theArabidopsisAt-A/N-InvE gene coding for a chloroplast-targeted A/N-Inv. TheAt-A/N-InvE knockout plants displayed a lower total A/N-Inv activity in comparison with wild-type plants. Furthermore, neither A/N-Inv activity nor A/N-Inv polypeptides were detected in protein extracts prepared from chloroplasts of mutant plants. Also, the measurement of carbohydrate content, in leaves harvested either at the end of the day or at the end of the night period, revealed that the knockout plants showed a decrease in starch accumulation but no alteration in Suc levels. These are the first results demonstrating the presence of a functional A/N-Inv inside chloroplasts and its relation with carbon storage inArabidopsis leaves. Taken together our data and recent reports, we conclude that the participation of A/N-Invs in the carbon flux between the cytosol and the plastids may be a general phenomenon in plants.

... 敲除拟南芥叶绿体A/N-InvE基因后, 叶片中A/N-InvE基因表达量降低, 淀粉积累量减少, 推测A/N-InvE基因可能参与叶绿体和线粒体之间的碳循环调节^[17] ...

2008

3.347

0.0

Planta. 2008, 228(1):51 - 59 DOI:10.1007/s00425-008-0718-0

OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (Oryza sativa L.)

Liqiang Jia(1) Botao Zhang(1) Chuanzao Mao(1) Jinhui Li(1) Yunrong Wu(1) Ping Wu(1) Zhongchang Wu(1)

1.Zhejiang University The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science Zi Jin Gang Campus 310058 Hangzhou China

A short root mutant was isolated from an EMS-generated rice mutant library. Under normal growth conditions, the mutant exhibited short root, delayed flowering, and partial sterility. Some sections of the roots revealed that the cell length along the longitudinal axis was reduced and the cell shape in the root elongation zone shrank. Genetic analysis indicated that the short root phenotype was controlled by a recessive gene. Map-based cloning revealed that a nucleotide substitution causing an amino acid change from Gly to Arg occurred in the predicted rice gene (Os02g0550600). It coded an alkaline/neutral invertase and was homologous to Arabidopsis geneAtCyt-inv1. This gene was designated asOsCyt-inv1. The results of carbohydrate analysis showed an accumulation of sucrose and reduction of hexose in theOscyt-inv1 mutant. Exogenously supplying glucose could rescue the root growth defects of theOscyt-inv1 mutant. These results indicated thatOsCyt-inv1 played important roles in root cell development and reproductivity in rice.

... 中性/碱性转化酶基因突变体表型为短根、开花延迟、部分不育, 说明其对植物根系细胞发育和生殖发育起着重要作用^{[18,19,20,21]} ...

2004

2.922

0.0

... 中性/碱性转化酶基因突变体表型为短根、开花延迟、部分不育, 说明其对植物根系细胞发育和生殖发育起着重要作用^{[18,19,20,21]} ...

2009

2.922

0.0

... 中性/碱性转化酶基因突变体表型为短根、开花延迟、部分不育, 说明其对植物根系细胞发育和生殖发育起着重要作用^{[18,19,20,21]} ...

2009

5.242

0.0

J. Exp. Bot.. 2009, 60(12):3353-65 DOI:10.1093/jxb/erp169

A cytosolic invertase is required for normal growth and cell development in the model legume, Lotus japonicus.

Welham T, Pike J, Horst I, Flemetakis E, Katinakis P, Kaneko T, Sato S, Tabata S, Perry J, Parniske M, Wang TL

Metabolic Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.

Neutral/alkaline invertases are a subgroup, confined to plants and cyanobacteria, of a diverse family of enzymes. A family of seven closely-related genes, LjINV1-LjINV7, is described here and their expression in the model legume, Lotus japonicus, is examined. LjINV1 previously identified as encoding a nodule-enhanced isoform is the predominant isoform present in all parts of the plant. Mutants for two isoforms, LjINV1 and LjINV2, were isolated using TILLING. A premature stop codon allele of LjINV2 had no effect on enzyme activity nor did it show a visible phenotype. For LjINV1, premature stop codon and missense mutations were obtained and the phenotype of the mutants examined. Recovery of homozygous mutants was problematic, but their phenotype showed a severe reduction in growth of the root and the shoot, a change in cellular development, and impaired flowering. The cellular organization of both roots and leaves was altered; leaves were smaller and thicker with extra layers of cells and roots showed an extended and broader zone of cell division. Moreover, anthers contained no pollen. Both heterozygotes and homozygous mutants showed decreased amounts of enzyme activity in nodules and shoot tips. Shoot tips also contained up to a 9-fold increased level of sucrose. However, mutants were capable of forming functional root nodules. LjINV1 is therefore crucial to whole plant development, but is clearly not essential for nodule formation or function.

PMID: 19474088

... 中性/碱性转化酶基因突变体表型为短根、开花延迟、部分不育, 说明其对植物根系细胞发育和生殖发育起着重要作用^{[18,19,20,21]} ...

2007

3.518

0.0

Plant Mol Biol. 2007, 64(5):575 - 587 DOI:10.1007/s11103-007-9177-4

AtCYT-INV1, a neutral invertase, is involved in osmotic stress-induced inhibition on lateral root growth inArabidopsis

Xiaopeng Qi(1) Zhongchang Wu(1) Jinhui Li(1) Xiaorong Mo(1) Shihua Wu(1) Jun Chu(1) Ping Wu(1)

1.The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058 PR China

Neutral/Alkaline invertases are unique to plant and photosynthetic bacteria. The function of Neutral/Alkaline invertases in plant development is not clear so far. In this study, we isolated anArabidopsis (Col-0) mutant insensitive to osmotic stress-induced inhibition on lateral root growth. Map-based cloning reveals that a neutral invertase gene (AtCYT-INV1) was point-mutated. The mutantAtcyt-inv1 showed short primary root, smaller size of leaves and siliques, and promotion of the reproductive compared to the wild type (WT). Carbohydrate measurement showed that sucrose is accumulated and glucose is reduced in the mutantAtcyt-inv1 under normal and 3% mannitol treatments. Taken together, AtCYT-INV1 plays multiple roles in plant development and is involved in osmotic stress-induced inhibition on lateral root growth by controlling the concentration of hexose in cells.

... 拟南芥Atcyt-inv1突变体证实, Atcyt-inv1抑制侧根发育是通过控制细胞内己糖浓度实现的^[22] ...

2010

0.0

0.5408

Sugar Crop China. 2010, (1):13 - 15

Breeding of New Sugarcane Variety Guitang28

特高糖抗寒甘蔗新品种桂糖28的选育

(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

刘海斌^[1];何红^[2];邓展云^[1];张革民^[1];李杨瑞^[2];方锋学^[1];贤武^[3];刘晓静^[1];方位宽^[1];闭少玲^[1];容凤玉^[1];唐红琴^[1]

为培育高产、高糖、多抗的甘蔗新品种,不断满足糖料生产发展的需要,2000～2008年,采用"五圃制"的常规杂交育种的技术路线进行选择与培育,育成了特高糖高产抗旱抗寒的甘蔗新品种桂糖28.桂糖28(原名桂糖00-122),其亲系为CP80-1018×CP88-2032.该品种中至中大茎,特早熟、特高糖、高产、宿根性好,抗逆性强,适应性广,极易脱叶.甘蔗蔗糖分(16.60%)较ROC16(15.07%)高1.53%,最高甘蔗蔗糖分达19.19%:产蔗量(86.2 t/hm2)较ROC16(83.1 t/hm2)增产3.73%,最高产蔗量达159.2 t/hm2;含糖量(14.31 t/hm2)较ROC16(12.52 t/hm2)增产14.30%.适应于广西、云南、广东、福建等省蔗区种植,桂糖28的育成是广西甘蔗高糖育种取得的突破性进展,对丰富我国甘蔗品种材料,改良目前甘蔗单一化的品种结构具有重要现实意义.

... 1 试验材料甘蔗品种桂糖28^[23](GT28)由广西农业科学研究院甘蔗研究所提供 ...

2009

0.0

0.5408

Sugar Crop China. 2009, (4):22 - 23

Comparision between Two Sugarcane Genome DNA Extraction Methods

两种不同甘蔗基因组DNA提取方法的比较

(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

应雄美^[1];蔡青^[2];毕艳^[1];刘新龙^[1];马丽^[1];毛钧^[1];陆鑫^[1]

采用改良CTAB法和SDS法提取甘蔗嫩叶与正一叶基因组DNA,通过琼脂糖凝胶电泳与紫外光吸收检测,证明SDS法能获得高质量的DNA,适合分子生物学操作的要求,为后续甘蔗遗传多样性分析、遗传图谱构建等研究奠定基础.

... 采用改良的SDS法提取甘蔗基因组DNA^[24] ...

2009

0.0

... 甘蔗内参基因选择, 以基因表达稳定性好的甘油醛-3-磷酸脱氢酶基因(GAPDH)为内参基因^[25,26], 根据GenBank数据库中甘蔗GAPDH (EF189713), 设计内参引物GAPDH-Fn和GAPDH-Rn (表1) ...

2004

5.319

0.0

Plant Mol Biol Rep. 2004, 22(4):325 - 337 DOI:10.1007/BF02772676

Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane

Hayati M. Iskandar(1234) Robert S. Simpson(1) Rosanne E. Casu(23) Graham D. Bonnett(23) Donald J. Maclean(1) John M. Manners(23)

1.School of Molecular and Microbial Sciences, University of Queensland, 4067 St Lucia, Queensland Australia
2.CSIRO, Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, 4067 St Lucia, Queensland Australia
3.Cooperative Research Centre for Sugar Industry Innovation through Biotechnology, University of Queensland, 4072 St Lucia, Queensland Australia
4.Indonesian Biotechnology Research Institute for Estate Crops, J1. Taman Kencana, No. 1, 16151 Bogor, Indonesia

A protocol for reverse transcription followed by real-time quantitative PCR (RT-qPCR) analysis of tissue-specific and genotype-variable gene expression in sugarcane (Saccharum sp.) was developed. A key requirement for this analysis was the identification of a housekeeping gene with transcript levels that were relatively stable across tissues and genotypes, suitable for use as a reference. Primers for β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes and 25S ribosomal RNA were designed and tested by RT-qPCR, and formation of product in the reactions was measured with the SYBR green I dye system. Ribosomal RNA was the most sensitive and consistent as a reference gene. Determination of the expression levels of β-actin, β-tubulin, and GAPDH transcripts relative to that of 25S rRNA showed that GAPDH had the most consistent mRNA expression of protein-coding genes across different tissues. GAPDH also showed low variation in expression in maturing stem internodes when compared across 2 cultivars and 3 otherSaccharum species. GAPDH therefore appears to be a suitable “housekeeping gene” in addition to 25S rRNA as a reference for measuring the relative expression of other genes in sugarcane. With use of GAPDH as a reference, the relative expression of the sugarcane sugar transporter genePst2a was assessed in a range of tissues. The result obtained was similar to our previously published Northern blot analysis. The protocol described here, using GAPDH as a reference gene, is recommended for studying the expression of other genes of interest in diverse tissues and genotypes of sugarcane.

2001

3.641

0.0

... CT法^[27], 分析基因的相对表达量 ...

2000

2.922

0.0

... 性转化酶基因编码的蛋白质同源性分别为96%、90%、87%、86%、69%和68%, 但与Bosch等^[28]报道的甘蔗SNI蛋白质同源性仅为46%, 说明SoNIN1是甘蔗中性/碱性转化酶基因家族的新成员 ...

... 序列分析表明该基因具有完整的ORF, 所编码的蛋白与其他禾本科物种具有很高的同源性, 而与Bosch等^[28]报道的甘蔗中性/碱性转化酶基因同源性很低, 氨基酸聚类分析表明二者都属于I类, 但在I类中又属于不同的亚类群, 说明它们是甘蔗中性/碱性转化酶家族的不同成员 ...

2000

0.0

... 甘蔗是重要的糖料作物, 节间蔗糖含量与中性/碱性转化酶活性成负相关, 而不同节间的中性/碱性转化酶活性不同^[29] ...

2007

2.471

0.0

... 对转甘蔗反义SNI基因植株的研究表明, 中性/碱性转化酶酶活性受到抑制, 导致细胞内己糖含量减小而蔗糖含量增加^[30] ...

2010

0.0

... 通过转反义基因技术抑制中性/碱性转化酶基因的表达, 使中性/碱性转化酶蛋白合成减少, 酶活性降低, 蔗茎中己糖含量减少, 而蔗糖含量增加^[31] ...

2008

0.0

1.1628

... 启动子是基因表达所必需的重要DNA信号序列, 是细胞内控制基因转录与表达的时空特异性的重要因子, 可以调控基因转录表达的方向和效率, 是转录调控的中心^[32] ...