甘蔗<italic>S-</italic>腺苷甲硫氨酸合成酶基因(<italic>ScSAM</italic>)的克隆及表达

引用本文

宋修鹏, 张保青, 黄杏, 杨丽涛, 李杨瑞. 甘蔗S-腺苷甲硫氨酸合成酶基因(ScSAM)的克隆及表达 . 作物学报, 2014, 40(6): 1002-1010[SONG Xiu-Peng, ZHANG Bao-Qing, HUANG Xing, YANG Li-Tao, LI Yang-Rui. Cloning and Expression of SugarcaneS-adenosylmethionine Synthetase GeneScSAM . Acta Agron Sin, 2014, 40(6): 1002-1010] 复制到剪切板

Permissions

作物学报编辑部

甘蔗S-腺苷甲硫氨酸合成酶基因(ScSAM)的克隆及表达

宋修鹏¹, 张保青², 黄杏², 杨丽涛^1,^2,^*, 李杨瑞^1,^2,^*

¹广西大学农学院 / 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530004

²广西农业科学院 / 中国农业科学院甘蔗研究中心 / 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

^* 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

第一作者联系方式: E-mail:xiupengsong@163.com

收稿日期:2014-03-04

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(2013DFA31600); 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1347004-2); 广西八桂学者、特聘专家专项基金, 广西自然科学基金项目(2011GXNSFF018002、2013NXNSFAA019073); 广西农业科学院团队项目(桂农科2011YT01)资助;

摘要

利用RT-PCR和RACE技术从甘蔗品种新台糖22 (ROC 22)中克隆获得SAM基因的cDNA序列, 命名为ScSAM, GenBank登录号为KC172558。用生物信息学方法预测分析其序列, cDNA全长1466 bp, 含有1个1191 bp的完整开放阅读框(ORF), 编码396个氨基酸, 与高粱和玉米等植物的SAM蛋白有很高的相似性。系统进化树分析显示, 甘蔗ScSAM与高粱的SAM蛋白亲缘关系较近。Real-time PCR分析表明ScSAM为组成型表达, 在根中的表达量最高, 是叶中表达量的3.6倍。其在黑穗病菌胁迫和低温(4℃)、聚乙二醇(PEG)、NaCl非生物胁迫下均被诱导表达, 但表达模式不同; 在H₂O₂胁迫下其表达被抑制。推测其可能参与甘蔗抗黑穗病过程, 且在甘蔗抗寒、抗旱、抗盐和抗氧化等胁迫过程中也起某种作用。

关键词: 甘蔗; S-腺苷甲硫氨酸合成酶; 克隆; 表达分析

Cloning and Expression of SugarcaneS-adenosylmethionine Synthetase GeneScSAM

SONG Xiu-Peng¹, ZHANG Bao-Qing², HUANG Xing², YANG Li-Tao^1,^2,^*, LI Yang-Rui^1,^2,^*

¹Agricultural College / State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China

²Guangxi Academy of Agricultural Sciences / Sugarcane Research Center, Chinese 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

Fund:

Abstract

The full-length sequence cDNA ofScSAM (GenBank accession number: KC172558) was cloned from sugarcane variety ROC 22 using RT-PCR combined with RACE techniques. This sequence consists of 1466 bp with an intact open reading frame of 1191 bp, encoding a polypeptide of 396 amino acids. Homology analysis showed that the deduced ScSAM protein was highly homologous to SAM proteins from different species. Phylogenetic analysis indicated thatScSAM was closely related to theSAM of sorghum. Real-time PCR results showed that theScSAM gene constitutively expressed in plant, with different expression levels in root, stalk and leaf. The transcript ofScSAMin root was the highest among the three organs, which was 3.6 times higher than that in leaf. Furthermore,ScSAM transcription was induced by biotic (smut infection) and abiotic (low temperature, PEG and NaCl) stresses, but the expression patterns were different. Under oxidative stress (H₂O₂), the expression ofScSAM was inhibited. We suggested thatScSAM might participate in smut-resistant activities in sugarcane, and also play a role in sugarcane resistances to chill, drought, salt and oxidation stresses.

Keyword: Sugarcane; S-adenosylmethionine synthetase (SAM); Clone; Expression analysis

Show Figures

0 引言

甘蔗( Saccharum officenarum L.)是我国最主要的糖料和能源作物^{[ 1]}。甘蔗安全生产受到干旱、盐渍、极端温度和病虫害的威胁, 利用基因工程的方法可以有效提高甘蔗的抗逆性^{[ 2]}, 而挖掘抗逆基因是首要基础。 S-腺苷甲硫氨酸合成酶( S-adenosyl-methionine synthetase, SAM)是植物代谢过程中的一个关键酶, 它是生物合成多胺和乙烯等的前体, 参与了植物的转氨丙基、转甲基和转硫等多种重要的生化反应过程, 还能催化ATP与甲硫氨酸反应生成 S-腺苷甲硫氨酸^{[ 3, 4, 5]}。此外, SAM还可与RNA结合参与体内基因表达的调控^{[ 6, 7]}。因此, 对 S -腺苷甲硫氨酸合成酶基因的研究在植物逆境生理、衰老生理、植物生物代谢及其调控研究上均具有重要的意义。现已证实多胺和乙烯均积极地参与了植物的抗逆反应, 因而推测SAM在植物抵御逆境的过程中也发挥重要的作用^{[ 8, 9]}。目前已从番茄^{[ 10]}、盐地碱蓬^{[ 11]}、玉米^{[ 12]}、石蒜^{[ 13]}和大豆^{[ 14]}等植物中克隆获得 S-腺苷甲硫氨酸合成酶基因的cDNA序列, 但迄今尚未见有甘蔗 SAM基因克隆及其在不同组织和不同胁迫条件下表达特性研究的报道。

本研究在前期甘蔗幼苗响应黑穗病菌侵染的蛋白质组学研究的基础上(尚未发表), 通过RT-PCR和RACE技术克隆了甘蔗 S-腺苷甲硫氨酸合成酶基因的全长, 用生物信息学分析该基因的序列特征, 最后利用Real-time PCR技术研究其在不同组织和不同胁迫下的表达特性, 以期为该基因在甘蔗抗逆特别是抗病育种中的应用提供理论依据。

1 材料与方法

1.1 试验材料

以甘蔗品种ROC 22为材料, 经过炼苗, 选取生长健壮一致的组培苗种植在沙土比例为1∶1的黑色塑料桶(上径40 cm, 下径30 cm, 高40 cm)中, 置广西大学甘蔗智能温室大棚, 水分含量控制在田间持水量的70%左右, 待其长到6~7片真叶时分组处理。一组针刺接种黑穗病菌^{[ 15]}, 将采集的萌发率在95%以上的黑穗病病原菌孢子用灭菌的ddH₂O稀释成5×10⁶个mL^-1的悬浮液, 用灭菌的一次性注射器在甘蔗的生长点针刺4次, 然后沿着叶鞘滴4滴孢子悬浮液, 对照组以ddH₂O代替孢子悬浮液, 0、1、2、3和4 d后从针刺位点下2 cm取样; 二组在4℃处理室(白昼16 h/8 h, 湿度65%)低温处理; 三组用10 mmol L^-1 H₂O₂喷洒叶面; 另两组分别用100 mmol L^-1 NaCl和15% PEG浇灌; 上述处理均于0、6、12、24、48和72 h后取样。另取不处理的健康甘蔗ROC 22根、茎、叶样品作对照。各样品经液氮速冻, 于-80℃保存备用。

1.2 试验所用试剂

胶回收试剂盒购自Bioflux公司, Dream Taq酶购自Fermentas公司, pMD18-T载体、Reverse Transcriptase M-MLV试剂盒、Primescript RT Reagent Kit和SYBR Premix Ex Taq均购自TaKaRa公司, TRIzol试剂购自北京康为试剂公司, 其他试剂均为国产分析纯。

1.3 蛋白质点的质谱鉴定

差异蛋白质点经胰蛋白酶酶解后, 取0.5 μL点入串联飞行时间质谱仪(4700 Proteomics Analyzer)的MALDI-TOF靶中, 在校准点内加入外标(已知质量数的肽混合物)。利用反射模式采集蛋白质的一级质谱信息(MS), 质量范围在800~4000 Da, 激光强度为4000。一级质谱扫描完成后, 取信号强度最高的母离子进行二级质谱分析(MS/MS), 用2 kV的高电压加速, 用CID碰撞裂解母离子后获取每个母离子的离子碎片指纹谱。随后将一级和二级质谱数据导入GPS软件, 利用MASCOT搜索算法对质谱数据综合分析, 搜索Uniprot蛋白质数据库(全部生物种类)和NCBI (植物)中与试验材料相匹配的相关蛋白质。

1.4 总RNA的提取及cDNA的合成

选用TRIzol试剂提取甘蔗幼苗总RNA, 利用琼脂糖凝胶电泳和Thermo Scientific NanoDrop 2000/ 2000C检测RNA的完整性和浓度。参照Reverse Transcriptase M-MLV试剂盒的说明书稍作修改合成基因克隆所用cDNA模板, 逆转录引物为RT-P: 5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTT-3'。按照Primescript RT Reagent Kit说明书合成荧光定量PCR所用cDNA模板, 将样品稀释至50 ng μL^-1保存于-20℃备用。

1.5 ScSAM的克隆及生物信息学分析

以质谱鉴定的SAM蛋白氨基酸序列为参照, 通过Blast分析, 选取同源性较高的核酸序列, 利用Vector NTI 11.0设计兼并引物SAM-F: 5′-ATGGC(A,C) G(G,C)(G,A,T)(G,C)T(C,T)GA(C,T)ACCTTCCTCTT-3′, 下游用逆转录加尾引物3'端: 5'-GGCCACGCG TCGACTAGTAC-3'。反应体系为cDNA 1.0 μL、2.5 mmol L^-1dNTPs 2.0 μL、10 μmol L^-1上下游引物各1.0 μL、10×buffer 2.5 μL、Dream Taq酶0.15 μL和ddH₂O 17.35 μL。反应程序为95℃ 5 min; 94℃ 35 s, 58℃ 35 s, 72℃ 1.5 min, 35个循环; 72℃ 10 min。RT-PCR扩增产物经1%琼脂糖凝胶电泳, 切胶回收目标条带, 与pMD18-T载体连接后转化大肠杆菌感受态细胞DH5α, 37℃过夜培养, 挑取阳性克隆, 经菌体PCR验证后送深圳华大基因科技服务有限公司测序。结果经Blast确认为 SAM基因, 设计引物SAM-ORF-F: 5′-ATGGCCGGTCTCGACACCTTCCT CTT-3′和SAM-ORF-R: 5′-TTAGGCAGAAGGTTTC TCCCACTTGAG-3′扩增基因的完整开放阅读框(open reading frame, ORF)。反应体系及反应程序同3'RACE。

用BioXM 2.6软件预测 ScSAM编码的氨基酸序列; 用NCBI-Protein Blast (http://blast.ncbi.nlm.nih. gov/)在线分析 ScSAM与其他物种的同源性; 用ExPASy Proteomics Server的ProtScale程序预测分析 ScSAM的亲水性和跨膜结构; 在线http://isoelectric. ovh.org/预测 ScSAM编码氨基酸的等电点和蛋白质分子量; 用SOSUIsignal软件预测 S cSAM的信号肽; 用SOPMA软件预测其二级结构; 用SMART和Motif Scan软件分析其蛋白质功能结构域; 用Mega 5.0软件构建SAM氨基酸序列进化树。

1.6 ScSAM的实时荧光定量PCR分析

根据获得的 ScSAM序列设计荧光定量PCR引物Y-SAM-F: 5′-GAGACAGTCACCAATGATGA-3′, Y-SAM-R: 5′-ATGGGTTAAGGTGGAAGATG-3′; 以甘蔗GAPDH (NCBI登录号为EF189713)为内参基因, 内参引物为Y-GAPDH-F: 5'-AAGGGTGGTGCC AAGAAGG-3'和Y-GAPDH-R: 5'-CAAGGGGAGCA AGGCAGTT-3'^{[ 16]}。利用LightCycle480荧光定量PCR仪分析, 反应体系20 μL, 含2.0 μL的50 ng μL^-1 cDNA模板、10 μmol L^-1各0.8 μL上下游引物、 10.0 μL SYBR Premix Ex Taq II、6.4 μL ddH₂O, 每个样品3个重复。反应程序为95℃ 30 s; 95℃ 5 s, 60℃ 20 s, 共45个循环; 95℃ 1 s, 65℃ 15 s, 95℃延续, 溶解曲线测定; 40℃ 30 s。利用2^-ΔΔCT法计算基因的相对表达量。

2 结果与分析

2.1 质谱鉴定

本课题组前期利用蛋白质双向电泳技术研究甘蔗幼苗响应黑穗病菌侵染时发现蛋白点8在接菌处理和对照样品中其相对表达量分别为10.34和5.10 (图1)。质谱鉴定分析(表1)发现, 其与牛奶子和野生稻的 S-腺苷甲硫氨酸合成酶匹配度最高, 蛋白得分和蛋白得分的统计学可靠程度显著高于质谱成功鉴定的阈值60和95%, 初步推断此差异蛋白为 S-腺苷甲硫氨酸合成酶(SAM)。

	Figure Option View Download New Window
	图1 差异蛋白质点8相对表达量变化Fig. 1 Relative expressions of the differentially expressed protein spot 8

表1 质谱分析结果 Table 1 Mass spectrometry results

2.2 ScSAM基因全长cDNA克隆

以逆转录合成的cDNA为模板, 利用引物SAM-F和3'端进行PCR扩增, 获得一条约1.5 kb的目标条带(图2-A), 经胶回收纯化、连接转化后挑取阳性克隆测序表明, 该片段长度为1466 bp, 经Blast分析发现其与其他作物 S-腺苷甲硫氨酸合成酶基因的相似性为89%~94%, 命名为 ScSAM, 基因登录号为KC172558。序列分析发现其包含一个375 bp的3'端非编码区, 一个1191 bp的完整开放阅读框(图2-B), 编码396个氨基酸(图3)。

	Figure Option ScSAM gene '>View Download New Window
ScSAM gene '>	图2 ScSAM基因3'RACE (A)和编码框(B)的扩增产物M: DL2000 marker; 1: 目的片段。 M: DL2000 marker; 1: target fragment.Fig. 2 Amplified products of 3'RACE (A) and ORF (B) of ScSAM gene

	Figure Option View Download New Window
	图3 ScSAM编码区核苷酸序列及推导出的氨基酸序列起始密码子ATG和终止密码子TAA用下画线表示, S-腺苷甲硫氨酸合成酶特征序列为灰色。Fig. 3 Nucleotide and predicted amino acid sequences of ScSAMThe start codon (ATG) and stop codon (TAA) are underlined. The characteristic sequence of SAM is shaded by grey.

2.3 ScSAM基因生物信息学分析

在线软件预测 ScSAM基因所编码的氨基酸序列

的等电点为5.40, 蛋白质分子量为43.07 kD; 使用频率较高的氨基酸有Gly、Val、Asp、Ala, 分别占总氨基酸的10.1%、8.6%、7.8%和7.6%, 频率较低的氨基酸有Sec、Trp、Met、Cys, 仅占0、1.0%、1.8%和1.8%; 以SOSUI Signal软件预测, SAM蛋白为可溶性蛋白, 不含信号肽; 二级结构预测结果显示α-螺旋占36.11%, 无规卷曲占40.66%, 延伸链占15.40%, β-转角占7.83%。疏水性最大值为1.73, 最小值为-2.10; 含有1个跨膜区域, 序列为VHT; 蛋白质功能结构域预测结果显示ScSAM包含3个SAMS的特征序列, 分别为GHPDK (17~21)、GAGDQGHMFGY (122~132)和GGGAFSGKD (269~ 277)(图3), 还含有1个酰胺化位点, 1个糖基化位点为, 6个肌酸激酶2磷酸化位点, 6个肉豆蔻基位点和6个蛋白激酶C磷酸化位点。

甘蔗ScSAM的氨基酸序列与其他作物SAM氨基酸序列多重比对分析(图4)发现, 它们之间有较高的同源性, 不同植物SAM存在高度保守序列, 但也有较明显组成变异。可见 S-腺苷甲硫氨酸合成酶在进化的过程中保持了足够的遗传稳定性和演化趋同性。

Figure Option
View Download New Window

图4 不同作物SAM氨基酸序列多重比对gi:242086805: 高粱; gi:195645456: 玉米; gi:288300148: 无芒隐子草; gi:17529621: 水稻; gi:474019723: 小麦; gi:304273278: 剑兰; gi:444436384: 桉树; gi:224124942: 杨树; gi:166872: 拟南芥。Fig. 4 Multiple sequence alignment of amino acids of SAM proteins isolated from different speciesgi:242086805: Sorghum bicolor; gi:195645456: Zea mays; gi:288300148: Cleistogenes songorica; gi:17529621: Oryza sativa; gi:474019723: Triticum urartu; gi:304273278: Gladiolus grandiflorus; gi:444436384: Eucalyptus cladocalyx; gi:224124942: Populus trichocarpa; gi:166872: Arabidopsis thaliana.

系统进化树分析(图5)显示甘蔗 S-腺苷甲硫氨酸合成酶与高粱的SAM聚在一起, 表明它们之间亲缘关系最近。另外杨树和桉树聚在一起, 草莓和拟南芥聚在一起。但不同植物间SAM也有很高的同源性。

	Figure Option View Download New Window
	图5 ScSAM与其他植物SAM氨基酸序列的系统进化树各节点处数值表示BootStrap值(重复1000次)。Fig. 5 Phylogentic relationship of amino acid sequences between ScSAM and other SAM proteinsThe numbers next to the nodes give BootStrap values of 1000 replicates.

2.4 ScSAM基因在不同组织中的表达量分析

利用qRT-PCR技术分析 ScSAM在甘蔗幼苗中的表达情况表明, ScSAM在根、茎、叶中均有表达, 但表达量存在明显差异, 在根中表达量最高, 是叶片中相对表达量的3.6倍(图6)。

	Figure Option View Download New Window
	图6 甘蔗 ScSAM的组织特异性表达Fig. 6 Tissue-specific expression of ScSAM gene in root, stalk, and leaf of sugarcane

2.5 ScSAM基因在黑穗病病原菌胁迫条件下的表达量分析

人工接种黑穗病病原菌后 ScSAM的表达总体表现为先下降后上升再下降的变化趋势(图7), 接种1 d后 ScSAM的相对表达量下降, 接种2 d后表达量迅速回升, 在接种3 d后基因的表达量达最高, 为对照的1.89倍, 但4 d后下降到正常水平。

	Figure Option View Download New Window
	图7 ScSAM在黑穗病病原菌胁迫条件下的表达特性Fig. 7 Expression profiles of ScSAM gene in sugarcane incubated with smut pathogen

2.6 ScSAM基因在4种非生物胁迫条件下的表达量分析

在4种非生物胁迫条件下 ScSAM均被诱导表达, 但响应模式不尽相同(图8)。在4℃低温胁迫下 ScSAM的相对表达量随着胁迫时间的延长整体表现缓慢升高的模式, 48 h后表达量最高, 为对照的2.18倍。在PEG模拟的干旱胁迫下 ScSAM的表达变化不明显, 24 h后表达量最高, 为对照的1.23倍。在NaCl胁迫下 ScSAM表达量表现出先升高后下降再升高的变化特点, 2次高峰分别出现在胁迫12 h和72 h。在H₂O₂胁迫下 ScSAM的表达整体表现为下降, 12 h后表达量接近对照, 24 h时表达量最低, 仅为对照的0.2倍。

	Figure Option View Download New Window
	图8 ScSAM在4种非生物胁迫条件下的表达特性Fig. 8 Expression profiles of ScSAM gene under four abiotic stress conditions

3 讨论

S-腺苷甲硫氨酸由Cantoni^{[ 17]}于1952年首次发现, 是 S-腺苷甲硫氨酸代谢途径关键的中间产物。它参与40多种不同的代谢过程, 其中主要有合成多胺(精胺、亚精胺和腐胺等), 合成乙烯及合成谷胱甘肽^{[ 18]}。这些物质在植物抵御逆境胁迫时起着重要的作用^{[ 19, 20]}。 S-腺苷甲硫氨酸合成酶催化ATP与甲硫氨酸反应, 是目前可知的生成 S-腺苷甲硫氨酸的唯一途径^{[ 21]}, 因此研究 S-腺苷甲硫氨酸合成酶对作物抗逆尤为重要。

生物信息学分析发现ScSAM蛋白含有典型的SAM特征序列如ATP结合域、磷酸盐结合区和GHPDK甲硫氨酸结合域, 及一个控制基因催化效率的活性中心等等, 这些结构特点与已报道的无芒隐子草和向日葵的SAM相似^{[ 22, 23]}。由此可见甘蔗ScSAM是典型的 S-腺苷甲硫氨酸合成酶, 是SAM蛋白质家族的一员。同源性分析发现甘蔗 ScSAM编码的氨基酸序列与其他物种 SAM基因编码的氨基酸序列同源性很高, 进化树分析结果表明 ScSAM与高粱 SAM亲缘关系最近, 但不同科属间的进化差异也不是很大, 推测SAM作为 S-腺苷甲硫氨酸代谢途径的重要中间产物 S-腺苷甲硫氨酸合成途径的关键酶, 在进化过程中保持了足够的演化趋同性和遗传稳定性。

组织特异性表达分析发现 ScSAM为组成型表达, 其相对表达量为根>茎>叶, 这与朱晶莹等^{[ 12]}发现的玉米 S-腺苷甲硫氨酸合成酶基因的表达模式相同。前人研究还发现 SAM基因的组织特异性表达受发育时期^{[ 24]}、环境因子^{[ 25]}和植物激素^{[ 26]}等的调节。

在甘蔗黑穗病病原菌胁迫下 Sc SAM的相对表达量整体呈上调表达, 在病菌侵染3 d后其相对表达量最高, 这与蛋白质双向电泳分析的结果吻合, 可见 ScSAM积极参与了甘蔗对黑穗病的响应过程。关于其在病原菌胁迫时的响应特性和机制的研究有待进一步探索, 但已有研究证实该基因受到真菌和细菌的激发子的诱导表达^{[ 7]}。推测 ScSAM表达量的提高会促使其下游代谢产物多胺的积累, 多胺作为一类植物体内重要的次生代谢物质, 在提高植物抗逆性方面具有重要作用^{[ 5]}。Hazarika等^{[ 27]}也发现在转基因番茄中多胺的积累会提高其对尖孢镰刀菌和番茄早疫病的抗性。所以可以推测 ScSAM在甘蔗幼苗抵御黑穗病菌入侵的过程中发挥重要作用。

在4种非生物胁迫条件下, ScSAM的表达模式不尽相同。在4℃低温胁迫下甘蔗 ScSAM相对表达量总体呈升高趋势, 这与甘薯 S-腺苷甲硫氨酸合成酶在低温胁迫的表达特性是一致的^{[ 28]}。在PEG模拟的干旱胁迫条件下甘蔗 ScSAM表达量变化不明显。这与陈锐等^{[ 29]}和林凡云等^{[ 30]}的研究结果不同, 推测是因为 SAM由一个家族基因编码的, 不同的 SAM行使的功能侧重不同。陈锐等^{[ 29]}研究发现小麦 SAMS基因在水分胁迫早期上调表达, 当水分胁迫程度严重时表达受到抑制。林凡云等^{[ 30]}发现糜子 SAMS基因在干旱早期(土壤含水量36%)明显上调表达, 而干旱程度更严重时(土壤含水量为24%)其表达受到严重抑制, 严重干旱后复水2 h, 其表达量增强至干旱早期的表达量, 而复水6 h后表达量降低至对照(干旱处理前)水平。在NaCl模拟的盐胁迫下 ScSAM表现“扬-抑-扬”的变化特点, 推测SAM在植物抵御高盐胁迫过程中发挥了重要作用。周凯等^{[ 31]}研究发现陆地棉 SAMS的表达受盐胁迫的诱导, 不同敏感材料诱导响应的时间不同, 盐敏感材料中诱导延迟。李燕等^{[ 32]}也发现在盐胁迫下, 盐地碱蓬的 S-腺苷甲硫氨酸合成酶基因的cDNA表达上调, 同时 S-腺苷甲硫氨酸合成酶的活性也增加。番茄中 SAM1和 SAM3的mRNA表达水平在盐处理8 h后增加, 并且长时间持续较高水平。较多的 S-腺苷甲硫氨酸可促使植物根部细胞壁的合成及修复, 还可增加细胞间水分的运输。这些改变使得植物根部的选择性更强, 可减少木质部对离子的吸收, 也能够补偿质外体运输过程中水分和有机溶质的流失^{[ 33]}。在H₂O₂模拟的氧化胁迫下甘蔗体内 ScSAM表达量整体表现下降, 具体原因尚不清楚, 需要进一步验证分析。

4 结论

克隆获得甘蔗 ScSAM基因全长, GenBank登录号为KC172558。它包含ATP结合域(GAGDQGHMF GY)、磷酸盐结合区(GGGAFSGKD)和甲硫氨酸结合域(GHPDK) 3个 S-腺苷甲硫氨酸合成酶的特征序列。甘蔗ScSAM与高粱SAM的关系最近; ScSAM在甘蔗根、茎和叶中均有表达, 在根中的表达量最高。其表达受黑穗病病原菌胁迫和低温(4℃)、聚乙二醇(PEG)、NaCl非生物胁迫的诱导但被H₂O₂胁迫所抑制, 推测它参与甘蔗应答黑穗病过程且也在甘蔗抵抗低温、高盐、干旱和氧化胁迫的机制中发挥作用。

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.

参考文献

View Option

[1]	李杨瑞, 杨丽涛. 20世纪90年代以来我国甘蔗产业和科技的新发展. 西南农业学报, 2009, 22: 1469-1476 Li Y R. Yang L T. New developments of sugarcane industry and technology in China since 1990s. Southwest China J Agric Sci, 2009, 22: 1469-1476 (in Chinese with English abstract) [本文引用:1]
[2]	Kumar T, Uzma, Khan M R, Abbas Z, Ali G M. Genetic improvement of sugarcane for drought and salinity stress tolerance using Arabidopsis vacuolar pyrophosphatase (AVP1) gene. Mol Biotechnol, 2014, 56: 199-209 [本文引用:1] [JCR: 2.262]
[3]	Mato J, Alvarez L, Ortiz P, Pajares M A. S-adenosylmethionine synthesis: molecular mechanisms and clinical implications. Pharmacol Therapeutics, 1997, 73: 265-280 [本文引用:1] [JCR: 7.793]
[4]	Fontecave M, Atta M, Mulliez E. S-adenosylmethionine: nothing goes to waste. Trends Biochem Sci, 2004, 29: 243-249 [本文引用:1] [JCR: 13.076]
[5]	Belbahri L, Chevalier L, Bensaddek L, Gillet F, Fliniaux M A, Boerjan W, Inzé D, Thomas D, Thomasset B. Different expression of an S-adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis. Biotechnol Bioeng, 2000, 69: 11-20 [本文引用:2] [JCR: 3.648]
[6]	Grundy F J, Henkin T M. Regulation of gene expression by effectors that bind to RNA. Curr Opin Microbiol, 2004, 7: 126-131 [本文引用:1] [JCR: 8.23]
[7]	Winkler W C, Nahvi A, Sudarsan N, Barrick J E, Breaker R R. An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat Struct Mol Biol, 2003, 10: 701-707 [本文引用:2] [JCR: 11.902]
[8]	Kevbrin V V, Zengler K, Lysenko A M, Wiegel J. Anoxybacillus kamchatkensis sp. nov. , a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka. Extremophiles, 2005, 9: 391-398 [本文引用:1] [JCR: 2.203]
[9]	Foyer C H, Bloom A J, Queval G, Noctor G. Photorespiratory metabolism: genes, mutants, energetics, and redox signaling. Annu Rev Plant Biol, 2009, 60: 455-484 [本文引用:1] [JCR: 25.962]
[10]	Espartero J, Pintor-Toro J A, Pardo J M. Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress. Plant Mol Biol, 1994, 25: 217-227 [本文引用:1] [JCR: 3.518]
[11]	Ma X L, Wang Z L, Qi Y C, Zhao Y X, Zhang H. Isolation of S-adenosylmethionine synthetase gene from Suaeda salsa and its differential expression under NaCl stress. Acta Bot Sin, 2003, 45: 1359-1365 [本文引用:1] [JCR: 0.599]
[12]	朱晶莹, 王寒玉, 张晏萌, 余爱丽. 玉米S-腺苷甲硫氨酸合成酶基因家族成员在盐胁迫条件下的差异表达. 核农学报, 2011, 25: 427-431 Zhu J Y, Wang H Y, Zhang Y M, Yu A L. Differential expression of maize S-adenosylmethionin synthetase gene family members during salt stress. J Nucl Agric Sci, 2011, 25: 427-431 (in Chinese with English abstract) [本文引用:2] [CJCR: 1.237]
[13]	Li X D, Xia B, Wang R, Xu S, Jiang Y M, Yu F B, Peng F. Molecular cloning and characterization of S-adenosylmethionine synthetase gene from Lycoris radiata. Mol Biol Rep, 2013, 40: 1255-1263 [本文引用:1] [JCR: 2.506]
[14]	樊金萍, 柏锡, 李勇, 纪巍, 王希, 才华, 朱延明. 野生大豆S-腺苷甲硫氨酸合成酶基因的克隆及功能分析. 作物学报, 2008, 34: 1581-1587 Fan J P, Bo X, Li Y, Ji W, Wang X, Cai H, Zhu Y M. Cloning and function analysis of gene SAMS from Glycine soja. Acta Agron Sin, 2008, 34: 1581-1587 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.667]
[15]	Santiago R, de Armas R, Fontaniella B, Vicente C, Legaz M E. Changes in soluble and cell wall-bound hydroxycinnamic and hydroxybenzoic acids in sugarcane cultivars inoculated with Sporisorium scitamineum sporidia. Eur J Plant Pathol, 2009, 124: 439-450 [本文引用:1] [JCR: 1.61]
[16]	宋修鹏, 黄杏, 莫凤连, 杨丽涛, 李杨瑞, 陈保善. 甘蔗苯丙氨酸解氨酶基因(PAL)的克隆和表达分析. 中国农业科学, 2013, 46: 2856-2868 Song X P, Hang X, Mo F L, Yang L T, Li Y R, Chen B S. Cloning and expression analysis of sugarcane phenylalanin ammonia- lyase (PAL) gene. Sci Agric Sin, 2013, 46: 2856-2868 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.889]
[17]	Cantoni G. The nature of the active methyl donor formed enzymatically from L-methionine and adenosinetriphosphate. J Am Chem Soc, 1952, 74: 2942-2943 [本文引用:1] [JCR: 10.677]
[18]	柴兴苹, 张玉秀, 谭金娟, 冯珊珊, 柴团耀. Zn胁迫下小麦S-腺苷甲硫氨酸代谢途径关键基因表达模式分析. 植物生理学报, 2013, 49: 375-384 Chai X P, Zhang Y X, Tan J J, Feng S S, Chai T Y. Analysis of expression patterns of genes participated in S-adenosylme-thionine (SAM) metabolic pathway in wheat under Zn stress. Plant Physiol J, 2013, 49: 375-384 (in Chinese with English abstract) [本文引用:1] [JCR: 2.699]
[19]	Kasukabe Y, He L, Nada K, Misawa S, Ihara I, Tachibana S. Overexpression of spermidine synthase enhances tolerance to multiple environmental stresses and up-regulates the expression of various stresS-regulated genes in transgenic Arabidopsis thaliana. Plant Cell Physiol, 2004, 45: 712-722 [本文引用:1] [JCR: 4.134]
[20]	陈坤明, 张承烈. 干旱期间春小麦叶片多胺含量与作物抗旱性的关系. 植物生理学报, 2000, 26: 381-386 Chen K M, Zhang C L. Polyamine contents in the spring wheat leaves and their relations to drought-resistance. Plant Physiol J, 2000, 26: 381-386 (in Chinese with English abstract) [本文引用:1] [JCR: 2.699]
[21]	鞠妍, 侯和胜. SAM合成酶基因的研究进展与应用现状. 天津农业科学, 2012, 18: 27-29 Ju Y, Hou H S. Research and application status of SAM synthase gene. Tianjin Agric Sci, 2012, 18: 27-29 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.5293]
[22]	周向红, 王萍. 向日葵S-腺苷甲硫氨酸合成酶基因克隆与分析. 作物杂志, 2011, (6): 10-13 Zhou X H, Wang P. Molecular cloning and bioinformatic analysis of S-adenosylmethionine synthetase gene from Helianthus annuus. Crops, 2011, (6): 10-13 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.6276]
[23]	孔令芳, 张吉宇, 刘志鹏, 王彦荣. 无芒隐子草SAMS1基因的克隆及干旱胁迫下的表达分析. 草业学报, 2013, 22: 268-275 Kong L F, Zhang J Y, Liu Z P, Wang Y R. Cloning of a S-adenosyl methionine synthetase gene from Cleistogenes songorica and its expression under droght stress. Acta Pratac Sin, 2013, 22: 268-275 (in Chinese with English abstract) [本文引用:1]
[24]	Jennings J C, Apel-Birkhold P C, Mock N M, Baker C J, Anderson J D, Bailey B A. Induction of defense responses in tobacco by the protein Nep1 from Fusarium oxysporum. Plant Sci, 2001, 161: 891-899 [本文引用:1] [JCR: 2.922]
[25]	Kodaki T, Tsuji S, Otani N, Yamamoto D, Rao K S, Watanabe S, Tsukatsune M, Makino K. Differential transcriptional regulation of two distinct S-adenosylmethionine synthetase genes (SAM1 and SAM2) of Saccharomyces cerevisiae. Nucl Acids Res, 2003, 3(suppl): 303-304 [本文引用:1]
[26]	Mathur M, Sharma N, Sachar R. Differential regulation of S-adenosylmethionine synthetase isozymes by gibberellic acid in dwarf pea epicotyls. Biochim Biophys Acta (BBA)-Protein Struct Mol Enzymol, 1993, 1162: 283-290 [本文引用:1]
[27]	Hazarika P, Rajam M V. Biotic and abiotic stress tolerance in transgenic tomatoes by constitutive expression of S-adenosylmethionine decarboxylase gene. Physiol Mol Biol Plants, 2011, 17: 115-128 [本文引用:1] [JCR: 25.962]
[28]	Yue C W, Xiao J, Ling X, Zeng N. Effect of low temperature stress on sweet potato S-adenosyl methionine synthetase gene expression. Agric Sci Technol, 2008, 9: 11-14 [本文引用:1] [CJCR: 2.0208]
[29]	陈锐, 陈亮, 王士强, 胡银岗. 水分胁迫下小麦S-腺苷甲硫氨酸合成酶基因的半定量表达模式分析. 麦类作物学报, 2009, 29: 954-958 Chen R, Chen L, Wang S Q, Hu Y G. Expression analysis of wheat S-adenosylmethionine synthetase gene during water stress and re-watering by semi-quantitative RT-PCR. J Triticeae Crops, 2009, 29: 954-958 (in Chinese with English abstract) [本文引用:2]
[30]	林凡云, 王士强, 胡银岗, 何蓓如. 糜子SAMS基因的克隆及其在干旱复水中的表达模式分析. 作物学报, 2008, 34: 777-782 Lin F Y, Wang S Q, Hu Y G, He B R. Cloning of a S-adenosylmethionine synthetase gene from broomcorn millet (Panicum miliaceum L. ) and its expression during drought and re-watering. Acta Agron Sin, 2008, 34: 777-782 (in Chinese with English abstract) [本文引用:2] [CJCR: 1.667]
[31]	周凯, 宋丽艳, 叶武威, 王俊娟, 王德龙, 樊保香. 陆地棉耐盐相关基因GhSAMS的克隆及表达. 作物学报, 2011, 37: 1012-1019 Zhou K, Song L Y, Ye W W, Wang J J, Wang D L, Fan B X. Cloning and expression of GhSAMS gene related to salt-tolerance in Gossypium hirsutum L. Acta Agron Sin, 2011, 37: 1012-1019 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.667]
[32]	李燕, 孙伟, 赵彦修, 张慧. 盐地碱蓬甲硫氨酸合成酶基因(SsMS)的克隆与表达分析. 山东师范大学学报(自然科学版), 2003, 18: 76-78 Li Y, Sun W, Zhao Y X, Zhang H. Isolation and characteizing of a methionine synthetase gene in Suaeda salsa under stress. J Shand ong Norm Univ (Nat Sci), 2003, 18: 76-78 (in Chinese with English abstract) [本文引用:1]
[33]	Sánchez-Aguayo I, Rodríguez-Galán J M, García R, Torreblanca J, Pardo J M. Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants. Planta, 2004, 220: 278-285 [本文引用:1] [JCR: 3.347]

2009

0.0

... )是我国最主要的糖料和能源作物^[1] ...

2014

2.262

0.0

... 甘蔗安全生产受到干旱、盐渍、极端温度和病虫害的威胁, 利用基因工程的方法可以有效提高甘蔗的抗逆性^[2], 而挖掘抗逆基因是首要基础 ...

1997

7.793

0.0

... S-腺苷甲硫氨酸合成酶(S-adenosyl-methionine synthetase, SAM)是植物代谢过程中的一个关键酶, 它是生物合成多胺和乙烯等的前体, 参与了植物的转氨丙基、转甲基和转硫等多种重要的生化反应过程, 还能催化ATP与甲硫氨酸反应生成S-腺苷甲硫氨酸^[3,4,5] ...

2004

13.076

0.0

2000

3.648

0.0

... 推测ScSAM表达量的提高会促使其下游代谢产物多胺的积累, 多胺作为一类植物体内重要的次生代谢物质, 在提高植物抗逆性方面具有重要作用^[5] ...

2004

8.23

0.0

. 2004, 7:126-131 DOI:10.1016/j.mib.2004.02.013

Regulation of gene expression by effectors that bind to RNA

Abstract

Recent studies have revealed several genetic systems in bacteria that use complex RNA structural elements to monitor regulatory signals and control expression of downstream genes. These include RNA thermosensors, in which an inhibitory structure melts at high temperature, and systems where binding of small RNAs or cellular metabolites modulates the structure of the RNA. The remarkable feature of these systems is the ability of the regulatory RNA elements to specifically sense the regulatory signal, without accessory components, and convey that information to the gene expression machinery via a structural change in the nascent RNA.

... 此外, SAM还可与RNA结合参与体内基因表达的调控^[6,7] ...

2003

11.902

0.0

... 此外, SAM还可与RNA结合参与体内基因表达的调控^[6,7] ...

... 关于其在病原菌胁迫时的响应特性和机制的研究有待进一步探索, 但已有研究证实该基因受到真菌和细菌的激发子的诱导表达^[7] ...

2005

2.203

0.0

. 2005, 9:391-398 DOI:10.1007/s00792-005-0479-7

Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka

1.Winogradsky Institute of Microbiology RAS 117312 Moscow Russia
2.Diversa Corporation 4955 Directors Place San Diego CA 92121 USA
3.University of Georgia Department of Microbiology Athens GA 30602 USA

A facultative aerobic, moderately thermophilic, spore forming bacterium, strain JW/VK-KG4 was isolated from an enrichment culture obtained from the Geyser valley, a geothermally heated environment located in the Kamchatka peninsula (Far East region of Russia). The cells were rod shaped, motile, peritrichous flagellated stained Gram positive and had a Gram positive type cell wall. Aerobically, the strain utilized a range of carbohydrates including glucose, fructose, trehalose, proteinuous substrates, and pectin as well. Anaerobically, only carbohydrates are utilized. When growing on carbohydrates, the strain required yeast extract and vitamin B₁₂. Anaerobically, glucose was fermented to lactate as main product and acetate, formate, ethanol as minor products. Aerobically, even in well-aerated cultures (agitated at 500 rpm), glucose oxidation was incomplete and lactate and acetate were found in culture supernatants as by-products. Optimal growth of the isolate was observed at pH^{25 C} 6.8–8.5 and 60°C. The doubling times on glucose at optimal growth conditions were 34 min (aerobically) and 40 min (anaerobically). The G+C content was 42.3 mol% as determined by T_m assay. Sequence analysis of the 16S rRNA gene indicated an affiliation of strain JW/VK-KG4 with Anoxybacillus species. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA homology with validly published species of Anoxybacillus, it is proposed that strain JW/VK-KG4 represents a new species in the genus Anoxybacillus as A. kamchatkensis sp. nov. The type strain for the novel species is JW/VK-KG4^T (=DSM 14988, =ATCC BAA-549). The GenBank accession number for the 16S rDNA sequence is AF510985.

... 现已证实多胺和乙烯均积极地参与了植物的抗逆反应, 因而推测SAM在植物抵御逆境的过程中也发挥重要的作用^[8,9] ...

2009

25.962

0.0

... 现已证实多胺和乙烯均积极地参与了植物的抗逆反应, 因而推测SAM在植物抵御逆境的过程中也发挥重要的作用^[8,9] ...

1994

3.518

0.0

... 目前已从番茄^[10]、盐地碱蓬^[11]、玉米^[12]、石蒜^[13]和大豆^[14]等植物中克隆获得S-腺苷甲硫氨酸合成酶基因的cDNA序列, 但迄今尚未见有甘蔗SAM基因克隆及其在不同组织和不同胁迫条件下表达特性研究的报道 ...

2003

0.599

0.0

. 2003, 45:1359-1365

Isolation of S-adenosylmethionine synthetase gene from Suaeda salsa and its differential expression under NaCl stress

硫腺苷甲硫氨酸作为甲基供体在转甲基反应中起到重要作用.为了解硫腺苷甲硫氨酸在盐地碱蓬(Suaedasalsa (L.)Pall)耐盐中的作用,我们对可能编码硫腺苷甲硫氨酸合成酶的基因(SsSAMS2)进行了分析.该基因在经400 mmol/L NaCl处理的盐地碱蓬地上部分的λ-Zap cDNA文库中克隆到,其插入片段全长1 531 bp,包含一个395个氨基酸的开放阅读框架,该基因推断的分子量约为43 kD.SsSAMS2与长春花(Catharanthus roseus)的SAMS2在氨基酸水平上的一致性为93%.Southern杂交显示,SsSAMS2在盐地碱蓬基因组中可能是两个拷贝.Northern分析显示硫腺苷甲硫氨酸合成酶基因受NaCl等胁迫的正调控.酶活性检测表明,NaCl胁迫条件下该酶活性增强.

Abstract：

AdoMet plays numerous roles of being the major methyl-group donor in trans-methylation reactions. To gain insight into the possible functions of the AdoMet protein of Suaeda salsa L. in response to salt stress, S-adenosylmethionine synthetase gene (SAMS2) was analyzed. We isolated SAMS2 cDNA clone (AF321001) from a λ -Zap cDNA library constructed from the halophyte S. salsa Pall aerial tissue treated with 400 mmol/L NaCl. SsSAMS2 was found to encode a S-adenolyl-L-methionine synthetase enzyme (AdoMet synthetase). The fragment was 1 531 bp with an open reading frame of 395 amino acids,the calculated molecular weight was about 43 kD. SsSAMS2 showed the highest homology to SAMS2 gene of Catharanthus roseus G. Don., with 93% identity in deduced amino acid sequence. Southern blotting analysis showed that SsSAMS2 might be a two-copy gene in S. salsa genome. Northern blot indicated that the cDNA was up-regulated by salt and other stresses. Enzyme activity assay indicated that the activity of SAMS2 increased under NaCl stress.

2011

0.0

1.237

. 2011, 25:427-431

Differential expression of maize S-adenosylmethionin synthetase gene family members during salt stress

1. College of Life Sciences, Hebei Agricultural University, Baoding,Hebei 071000;
2. Biological Treatment Center, 255 Hospital of PLA, Tangshan, Hebei 063000

S-adenosylmethionine synthetase SAMS is a key enzyme in plant metabolism,catalyzing the biosynthesis of SAM,which is a donor for methyl groups and the precursor of many compounds. To study the response and functions of SAMS under salt stress in maize, the semi-quantitative RT-PCR was used to investigate the expression patterns of SAMS gene, using maize glyceraldehyde-3-phosphate dehydrogenase gene as a control. The results showed that all of the four maize SAMS genes family members were expressed both under normal and salt conditions, In the control seedlings, the mRNA levels of the four genes were higher in roots and stems than those in leaves. Under the salt stress the expression of SAMS1 was down-regulated in stems, up-regulated in leaves, but no change in roots. The transcript levels of SAMS2 and SAMS4 also increased significantly under salt stress. However, SAMS3 had no response to NaCl in any organs. In maize, the four genes of SAMS family were differentially expressed under salt stress condition, suggesting the possibility of functional differences between their proteins.

S-腺苷甲硫氨酸合成酶(SAMS)是植物代谢过程中的一个关键酶,催化甲基供体和化合物前体S-腺苷甲硫氨酸的合成。为研究S-腺苷甲硫氨酸合成酶基因在盐胁迫条件下的响应和功能,以玉米甘油醛-3-磷酸脱氢酶(GAPDH)基因为内参基因,通过半定量RT-PCR法分析玉米SAMS基因在盐胁迫条件下的表达模式。结果表明,玉米SAMS基因家族4个成员(SAMS1、SAMS2、SAMS3和SAMS4)在正常生长(对照)和盐胁迫条件下都表达,对照根茎中的表达量大于叶中。SAMS1受盐胁迫后,茎中表达量降低,而叶中表达量略有增加,根中无明显变化。SAMS2和SAMS4受盐胁迫明显诱导,而SAMS3似乎不受盐胁迫诱导,处理植株与对照株相比均无差异。说明玉米SAMS基因家族的4个基因在表达模式上存在差异,推测在功能上存在分工的不同。

... 组织特异性表达分析发现ScSAM为组成型表达, 其相对表达量为根>茎>叶, 这与朱晶莹等^[12]发现的玉米S-腺苷甲硫氨酸合成酶基因的表达模式相同 ...

2013

2.506

0.0

. 2013, 40:1255-1263 DOI:10.1007/s11033-012-2168-9

Molecular cloning and characterization of S-adenosylmethionine synthetase gene from Lycoris radiata

1.Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Qian Hu Hou Cun 1#, Nanjing, 210014, Jiangsu Province, China

2.Departments of Environmental Sciences, School of Environment and Resource Sciences, Zhejiang Agricultural and Forestry University, Linan, 311300, Zhejiang Province, China

Abstract
S-adenosylmethionine (SAM) synthetase catalyzes the synthesis of SAM, a molecule important for all cellular organisms. It is also considered to play an important role in salt tolerance of plants. Here, we cloned a Lycoris radiata (L. radiata) SAM synthetase gene LrSAMS to determine its biological function. The gene encodes a protein of 401 amino acids with a calculated molecular weight of 43.9 kDa. Amino acid sequence analysis of the deduced protein LrSAMS reveals high sequence identity to SAM synthetases from other organisms, such as Arabidopsis thaliana and Oryzasativa. The deduced LrSAMS protein contains conserved amino acids residues and sequences motifs that closely related to the function of SAM synthetase. Otherwise, the transcript levels of LrSAMS were significantly induced by NaCl treatment in L. radiata leaves, which implied that LrSAMS might play an important role in tolerance to salt stress in L.radiata. Complete ORF of LrSAMS was inserted into expression vector pET-29a(+) and transformed into Escherichia coli BL21 (DE3). The difference between the growth curve of the transgenic strain and control strain with blank vector showed that over-expressing LrSAMS could provide growth advantage to the engineered strain in high salt concentration.

2008

0.0

1.667

. 2008, 34:1581-1587 DOI:10.3724/SP.J.1006.2008.01581

Cloning and function analysis of gene SAMS from Glycine soja

1 College of Horticulture, Northeast Agricultural University; 2 College of Life Sciences, Northeast Agricultural University, Harbin 150030, Heilong-jiang, China

Drought, salinity, and low temperature are the most severe natural disaster of many abiological adversities. They can make crop yield reduction, even to death. At present, biotechnology is an important way for crops to improve plant resistance. Glycine soja carrying tolerant genes in northeast of China was used as a material for cloning. Total RNA was extracted from the leaf, and the first strand of cDNA was synthesized with reverse transcription under low temperature (4℃), drought (30%PEG), and salinity (200 mmol L^-1 NaCl). S-adenosylmethionine synthetase gene (SAMS gene) was amplified by PCR with the first strand cDNA as template and a pair of primer based on constructed the cDNA library and ESTs sequence under salinity condition. Full-length SAMS gene sequence was obtained by Blast comparison. A expression vector based on pBI121 with CaMV35S promoter was constructed, and 180 transgenic tobacco plants transformed by Agrobacterium tumefaciens were obtained. Transgenic plants resistances to drought, salinity and low temperature were investigated. The gene function was analyzed via measuring the physiological stress indicators under different osmotic pressures. The results showed SAMS gene can improve tobacco resistance to lower temperature, drought, and salinity.

干旱、盐碱和低温等非生物逆境是危害作物诸多因素中最为严重的自然灾害, 它可以使作物减产, 甚至死亡。目前通过生物技术手段获得可利用的抗性基因是提高植物抗性的重要途径。东北野生大豆具有丰富的基因资源, 是基因克隆的理想材料。从低温(4℃)、干旱(30%PEG)及盐胁迫(200 mmol L^-1NaCl)处理的东北野生大豆幼苗叶片中提取总RNA, 经反转录合成cDNA第一链, 并以此链为模板, 从已构建的盐胁迫全长cDNA文库和东北野生大豆盐胁迫EST数据库中筛选出与渗透胁迫直接相关的EST序列, 根据该序列设计一对PCR引物扩增S-腺苷甲硫氨酸(SAM)合成酶基因的全长序列, 经Blast比较分析, 确定所获得的SAMS基因全长序列。构建以pBI121为基础的CaMV35S启动子调控的植物表达载体; 利用农杆菌介导法将其导入烟草, 获得了180株转基因烟草, 并进行了低温、干旱和盐胁迫处理, 通过测定在不同胁迫处理下的生理指标, 分析了基因的功能。野生大豆的SAMS基因在烟草中的超量表达提高了转基因烟草的抗低温、干旱和耐盐能力。

2009

1.61

0.0

. 2009, 124:439-450 DOI:10.1007/s10658-009-9431-5

Changes in soluble and cell wall-bound hydroxycinnamic and hydroxybenzoic acids in sugarcane cultivars inoculated with Sporisorium scitamineum sporidia

1.Havana University Department of Plant Biology, Faculty of Biology Havana Cuba
2.Complutense University Department of Plant Physiology, Faculty of Biology 2 José Antonio Novais Av. Madrid 28040 Spain

The accumulation of soluble and cell wall-bound phenolics in the sugarcane stems of young plants from highly resistant cv. My 5514 and susceptible cv. B 42231, inoculated or not inoculated with smut sporidia, was studied. The ratio of inoculated to uninoculated plants of some cell wall-bound phenolics, such as ferulic, caffeic, and syringic acids increased for the resistant cv. My 5514, whereas it was maintained more or less constantly for the susceptible cv. B 42231. The highest increase of this ratio in the resistant cv. My 5514 corresponded to both caffeic and syringic acids. This could result in a better capacity to cv. My 5514 for an increase in the frequency of bridges between lignin fragments through ester-ether linkages for reinforcing the cell wall and major resistance to the disease. This reinforcement of the cell wall could provide an effective barrier to pathogen entry and spread. Soluble sub-fractions of all phenolics detected showed non-stable patterns. Caffeic acid, that regulates phenylalanine ammonia-lyase activity in sugarcane, showed a significant decrease in its titre at 24 h in the resistant cultivar, principally in the free soluble fraction, whilst the susceptible cultivar enhanced it. We hypothesise that the pathway of hydroxybenzoic acids is only activated once the level of p-coumaric acid justifies the accumulation of hydroxycinnamic acids required for reinforcing the cell wall after inoculation.

... 一组针刺接种黑穗病菌^[15], 将采集的萌发率在95%以上的黑穗病病原菌孢子用灭菌的ddH₂O稀释成5#cod#x000d7 ...

2013

0.0

1.889

. 2013, 46:2856-2868 DOI:10.3864/j.issn.0578-1752.2013.14.002

Cloning and expression analysis of sugarcane phenylalanin ammonia- lyase (PAL) gene

1.College of Agriculture, Guangxi University/State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Nanning 530004
2.Sugarcane Research Center, Chinese Academy of Agricultural Sciences/Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture/Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences / Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007
3.Biotechnology Research Institute, Guangxi Academy of Agriculture Sciences, Nanning 530007

【Objective】The aim of this study was to clone full-length cDNA of sugarcane phenylalanin ammonia-lyase (PAL) gene (ScPAL), a key enzyme gene related to phenylpropanoid metabolism in sugarcane, investigate its sequence characteristics, analyze its expressions in different organs and under five different stress conditions, thus providing a theoretical support for using this gene in sugarcane stress tolerance breeding. 【Method】 The differentially expressed protein was identified by MALDI TOF/TOF and analyzed using Applied Biosystems GPS Explorer software with Mascot analysis against the NCBI and Uniprot database using a combined peptide mass fingerprint and MS/MS search, then the full sequence of ScPAL was cloned from sugarcane variety ROC22 using RT-PCR and RACE techniques. The bioinformatics method was used to analyze the putative amino acid sequence, and real-time PCR method was used to analyze the expression of ScPAL in different tissues and under different stresses. 【Result】 The full-length cDNA of ScPAL (GenBank accession number: KC172559) in sugarcane was cloned. The sequence consists of 2590 bp with an intact open reading frame of 2115 bp, encoding a polypeptide of 704 amino acids. Sequence analysis showed that it contains the typical PAL enzyme active site sequence (GTITASGDLVPLSYIA). Homology analysis showed that the deduced ScPAL protein was highly homologous to other PAL proteins from different species. Phylogenetic tree analysis indicated that ScPAL was very closely related to PAL of sorghum. Real-time PCR results showed that the ScPAL expressed in root, stalk and leaf, respectively, and its expression was different among three organs. The mRNA of ScPAL in root was the highest among three organs and was about sixty-six times higher than that in leaf. Furthermore, ScPAL transcription level was induced under the treatment of low temperature, PEG, NaCl and H2O2 stresses, but the expression patterns were different. 【Conclusion】 The gene ScPAL which was firstly cloned and characterized from sugarcane (ROC22) is a member of PAL family typically. The results of study indicated that it participated in sugarcane resistance to smut, also played a role in the sugarcane resistance to chilling, drought and salt stress processes.

【目的】克隆甘蔗的苯丙氨酸解氨酶基因（PAL），分析其序列特征及其在不同组织和5种胁迫条件下的表达情况，为此基因在甘蔗抗逆育种中的应用提供理论支撑。【方法】利用串联飞行时间质谱仪对差异表达蛋白质进行质谱鉴定分析，通过RT-PCR和RACE技术从甘蔗品种新台糖22号（ROC22）中克隆PAL，以生物信息学方法对其序列进行预测分析，利用real-time PCR分析PAL在不同组织和不同胁迫条件下的表达特性。【结果】克隆获得甘蔗PAL，命名为ScPAL，GenBank登录号为KC172559。该cDNA全长2 590 bp，含有1个2 115 bp的完整开放阅读框（ORF），编码704个氨基酸。序列分析表明，其包含典型的PAL酶活性中心序列（GTITASGDLVPLSYIA），与其它植物的PAL蛋白有很高的相似性。系统进化树分析显示，甘蔗ScPAL与高粱的PAL蛋白亲缘关系较近。real-time PCR分析表明PAL为组成型表达，在根中的表达量最高，是叶中表达量的66倍。其在低温（4℃）、聚乙二醇（PEG）、NaCl和H2O2四种外源胁迫下均诱导表达，但表达模式不同。【结论】从甘蔗品种ROC22中克隆获得苯丙氨酸解氨酶基因（ScPAL），是典型的PAL家族成员，推测其参与了甘蔗抗黑穗病过程，且在甘蔗抗寒、抗旱和抗盐胁迫过程也起到某种作用。

... 以甘蔗GAPDH (NCBI登录号为EF189713)为内参基因, 内参引物为Y-GAPDH-F: 5'-AAGGGTGGTGCC AAGAAGG-3'和Y-GAPDH-R: 5'-CAAGGGGAGCA AGGCAGTT-3' ^[16] ...

1952

10.677

0.0

... 3 讨论S-腺苷甲硫氨酸由Cantoni^[17]于1952年首次发现, 是S-腺苷甲硫氨酸代谢途径关键的中间产物 ...

2013

2.699

0.0

... 它参与40多种不同的代谢过程, 其中主要有合成多胺(精胺、亚精胺和腐胺等), 合成乙烯及合成谷胱甘肽^[18] ...

2004

4.134

0.0

... 这些物质在植物抵御逆境胁迫时起着重要的作用^[19,20] ...

2000

2.699

0.0

. 2000, 26:381-386

Polyamine contents in the spring wheat leaves and their relations to drought-resistance

使用两种抑制剂MCBG(抑制SAMDC活性)和AOA(抑制ACC合成酶活性)研究了干旱期间两个春小麦品种8139(抗旱性较弱)和504(抗旱性较强)叶片多胺(Put、Spd和Spm)含量、RWC水平、SOD和POD活性以及MDA含量的变化，并由此探讨了不同类别多胺与作物抗旱性的关系以及多胺与乙烯在作物对干旱胁迫响应过程中对共同前体SAM的竞争趋向及其生理意义。

Abstract：

The results of applications of MGBG(an inhibitor of SAMDC) and AOA (an inhibitor of ACC synthase) to wheat (Triticum aestivum L.) cv. 8139 and cv. 504 with relatively low and strong resistance drought respectively, showed that the two inhibitors did not affect the trends of the changes in polyamine content of drought-stressed leaves of two wheat cultivars which increased in the earlier period of drought and decreased in the later (Fig. 2), and also did not affect the changes in SOD and POD activities and levels of MDA and RWC in stressed leaves( Figs. 1,3,4). But application of MGBG reduced the leaf Spd and Spm contents, RWC and activities of SOD and POD, increased the levels of MDA. However, the effects of applications of AOA ware the con verse (Figs. 1～4). The drought-resistant strain 504 had higher RWC than drought-sensitive strain 8139, and its Spd and Spm contents and activities of SOD and POD had stronger responses to drought than those of 8139; moreover, it had lower level of MDA( Figs. 1～4).Thus, it can be concluded that Spd and Spm are more important than that of Put in plant response to drought stress, and that Spd and Spm probably play key roles in promoting the plant drought resistance.

... 这些物质在植物抵御逆境胁迫时起着重要的作用^[19,20] ...

2012

0.0

0.5293

... S-腺苷甲硫氨酸合成酶催化ATP与甲硫氨酸反应, 是目前可知的生成S-腺苷甲硫氨酸的唯一途径^[21], 因此研究S-腺苷甲硫氨酸合成酶对作物抗逆尤为重要 ...

2011

0.0

0.6276

... 生物信息学分析发现ScSAM蛋白含有典型的SAM特征序列如ATP结合域、磷酸盐结合区和GHPDK甲硫氨酸结合域, 及一个控制基因催化效率的活性中心等等, 这些结构特点与已报道的无芒隐子草和向日葵的SAM相似^[22,23] ...

2013

0.0

2001

2.922

0.0

... 前人研究还发现SAM基因的组织特异性表达受发育时期^[24]、环境因子^[25]和植物激素^[26]等的调节 ...

2003

0.0

... 前人研究还发现SAM基因的组织特异性表达受发育时期^[24]、环境因子^[25]和植物激素^[26]等的调节 ...

1993

0.0

... 前人研究还发现SAM基因的组织特异性表达受发育时期^[24]、环境因子^[25]和植物激素^[26]等的调节 ...

2011

25.962

0.0

... Hazarika等^[27]也发现在转基因番茄中多胺的积累会提高其对尖孢镰刀菌和番茄早疫病的抗性 ...

2008

0.0

2.0208

... 在4℃低温胁迫下甘蔗ScSAM相对表达量总体呈升高趋势, 这与甘薯S-腺苷甲硫氨酸合成酶在低温胁迫的表达特性是一致的^[28] ...

2009

0.0

... 这与陈锐等^[29]和林凡云等^[30]的研究结果不同, 推测是因为SAM由一个家族基因编码的, 不同的SAM行使的功能侧重不同 ...

... 陈锐等^[29]研究发现小麦SAMS基因在水分胁迫早期上调表达, 当水分胁迫程度严重时表达受到抑制 ...

2008

0.0

1.667

. 2008, 34:777-782 DOI:10.3724/SP.J.1006.2008.00777

Cloning of a S-adenosylmethionine synthetase gene from broomcorn millet (Panicum miliaceum L.) and its expression during drought and re-watering

1 College of Agriculture, Northwest A & F University, Yangling 712100, Shaanxi; 2 Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, Shaanxi, China

To explore more genes for the improvement of drought-tolerance and water use efficiency in plants, broomcorn millet (Panicum miliaceum L.) was used to investigate the differential gene expression during drought stress and re-watering after serious drought stress by constructing SSH-cDNA library in previous study. A full-length cDNA of S-adenosylmethionine synthetase (SAMS) gene was amplified from broomcorn millet using PCR. The PCR primers were designed based on an EST sequence highly similar to SAMS gene in the SSH-cDNA library and the sequences of SAMS genes from rice and barley. The full-length cDNA sequence of SAMS gene amplified from broomcorn millet was 1 293 bp in length and designated as PmSAMS, encoding 396 amino acids with the 3 typical domains of SAMS (N terminal domain, inter domain and C terminal domain). Multiple alignment analysis based on the amino acids encoded by some SAMS genes from broomcorn millet (P. miliaceum L.), potato (S. tuberosum), Arabidopsis (A. thaliana), sugar beet (B. vulgaris), barley (H. vulgare), litchi (L. chinensis), tomato (L. esculentum), and rice (O. sativa) indicated that SAMS was very conserved among different species of plants with 92–97% of sequence similarity of amino acids, and PmSAMS had the highest similarity with that of rice (97%). The expression patterns of PmSAMS during drought and re-watering after serious drought were investigated by means of semi-quantitative RT-PCR. The results showed that the ex-pressions of PmSAMS were great at the beginning of drought treatment (soil relative moisture of 36%), then declined under seri-ous drought (soil relative moisture of 24%) to less than the normal level, and increased at 2 h after re-watering, declined to the normal level at 6 h after re-watering. The expression patterns of PmSAMS during drought and re-watering after serious drought indicated that it was involved in the response of broomcorn millet to drought and re-watering and might be one of the key genes for drought tolerance and water use efficiency of broomcorn millet. The cloning of this gene provides the potential for its utiliza-tion in the improvement of drought-tolerance and water use efficiency in other plants.

以糜子抗旱节水研究中获得的一个与S-腺苷甲硫氨酸合成酶(SAMS)基因同源性较高的EST序列为基础, 采用RT-PCR技术从糜子中分离到一个SAMS基因的全长cDNA序列(命名为PmSAMS), 全长1 293 bp, 编码396个氨基酸, 具有SAMS典型的N端结构域、中间结构域及C端结构域。糜子、马铃薯、拟南芥、甜菜、大麦、荔枝、番茄和水稻8种植物SAMS的氨基酸序列多重比较分析表明, 不同植物的SAMS的氨基酸相似程度非常高(92%~97%), 其中糜子与水稻的相似性最高(97%), 说明SAMS基因在植物进化中非常保守。PmSAMS基因在糜子幼苗干旱及复水过程中的半定量RT-PCR表达模式分析表明, 在干旱早期(土壤含水量36%)诱导该基因大量表达, 而干旱程度更严重时(土壤含水量为24%)其表达受到严重抑制, 表达量比对照还低; 严重干旱后复水2 h, 其表达量增强至干旱早期的表达量, 而复水6 h后表达量降低至对照(干旱处理前)水平。可见, PmSAMS基因的表达涉及糜子响应干旱胁迫及干旱后复水过程, 可能是糜子抗旱节水的关键基因。该基因的克隆为进一步探讨其应用于农作物抗旱节水性的遗传改良奠定了基础。

... 这与陈锐等^[29]和林凡云等^[30]的研究结果不同, 推测是因为SAM由一个家族基因编码的, 不同的SAM行使的功能侧重不同 ...

... 林凡云等^[30]发现糜子SAMS基因在干旱早期(土壤含水量36%)明显上调表达, 而干旱程度更严重时(土壤含水量为24%)其表达受到严重抑制, 严重干旱后复水2 h, 其表达量增强至干旱早期的表达量, 而复水6 h后表达量降低至对照(干旱处理前)水平 ...

2011

0.0

1.667

. 2011, 37:1012-1019 DOI:10.3724/SP.J.1006.2011.01012

Cloning and expression of GhSAMS gene related to salt-tolerance in Gossypium hirsutum L

Cotton Research Institute, Chinese Academy of Agricultural Science, Key Laboratory of Cotton Genetic Improvement of Agriculture Ministry, Anyang 455000, China

As one of main abiotic stresses in nature, salt stress does great harm to plants, and seriously affect plant growth and development. Simultaneously, the crops cultivated in the saline land undergo a wide range of yield decline. To excavate salt-tolerance gene, we cloned the cDNA of S-adenosyl-L-methionine synthetase gene from Gossypium hirsutum by RACE and RT-PCR, which was named GhSAMS, with the cDNA full length of 1 576 bp, ORF of 1 182 bp, and coding 393 amino acid residues. Bioinformatics analysis showed GhSAMS has the similarity of 91%, 93%, and 93% with Arabidopsis thaliana, Suaeda salsa, and Oryza sativa, respectively. Phylogenetic analysis showed GhSAMS was the closest to Suaeda salsa,and Real-time PCR suggested that GhSAMS was induced by salt stress, while the induction was postponed in salt sensitivity material. It showed lower gene expression level on salt sensitive material Zhong S9612 relative to salt resistance material Zhong 9835. At the same time, we established protokaryotic expression vector pET28-GhSAMS and transformed GhSAMS into E. coli after IPTG induction, showing a successful gene expression.

为了挖掘棉花耐盐相关基因，我们根据陆地棉耐盐性抑制消减文库中的一个S-腺苷甲硫氨酸合成酶基因的同源EST设计引物，利用RACE结合RT-PCR技术克隆陆地棉S-腺苷甲硫氨酸合成酶基因的cDNA全长，命名为GhSAMS。该cDNA全长1 576 bp，ORF为1 182 bp，编码393个氨基酸的多肽。生物信息学分析表明GhSAMS蛋白与拟南芥、盐地碱蓬、水稻中该蛋白的相似性分别为91%、93%和93%。系统发育树结果显示GhSAMS与盐地碱蓬中该蛋白的亲缘关系最近。Real-time PCR分析结果表明，GhSAMS的表达受盐胁迫诱导，在盐敏感材料中诱导被推迟，而且，该基因表达水平在耐盐材料中9835中明显高于在盐敏感材料中S9612中。我们构建了原核表达载体pET28-GhSAMS，经IPTG诱导，实现了GhSAMS在大肠杆菌中的表达，为进一步开展GhSAMS的遗传转化工作奠定了有益基础。

... 周凯等^[31]研究发现陆地棉SAMS的表达受盐胁迫的诱导, 不同敏感材料诱导响应的时间不同, 盐敏感材料中诱导延迟 ...

2003

0.0

... 李燕等^[32]也发现在盐胁迫下, 盐地碱蓬的S-腺苷甲硫氨酸合成酶基因的cDNA表达上调, 同时S-腺苷甲硫氨酸合成酶的活性也增加 ...

2004

3.347

0.0

. 2004, 220:278-285 DOI:10.1007/s00425-004-1350-2

Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants

1.Instituto de Recursos Naturales y Agrobiología C.S.I.C. 41012 Sevilla Spain
2.Departamento de Biología Celular, Facultad de Biología Universidad de Sevilla 41012 Sevilla Spain

S-Adenosyl-l-methionine synthase (SAM; ATP:l-methionine adenosyltransferase, EC 2.5.1.6) catalyzes the biosynthesis of S-adenosyl-l-methionine (AdoMet), a universal methyl-group donor. This enzyme is induced by salinity stress in tomato (Lycopersicon esculentum Mill.). To elucidate the role of SAM and AdoMet in the adaptation of plants to a saline environment, the expression pattern and histological distribution of SAM was investigated in control and salt-stressed tomato plants. Immunohistochemical analysis showed that SAM proteins were expressed in all cell types and plant organs, albeit with preferential accumulation in lignified tissues. Lignin deposition was estimated by histochemical tests and the extent of tissue lignification in response to salinity was quantified by image analysis. The average number of lignified cells in vascular bundles was significantly greater in plants under salt stress, with a maximal expansion of the lignified area found in the root vasculature. Accordingly, the greatest abundance of SAM gene transcripts and proteins occurred in roots. These results indicate that increased SAM activity correlated with a greater deposition of lignin in the vascular tissues of plants under salinity stress. A model is proposed in which an increased number of lignified tracheary elements in tomato roots under salt stress may enhance the cell-to-cell pathway for water transport, which would impart greater selectivity and reduced ion uptake, and compensate for diminished bulk flow of water and solutes along the apoplastic pathway.

... 这些改变使得植物根部的选择性更强, 可减少木质部对离子的吸收, 也能够补偿质外体运输过程中水分和有机溶质的流失^[33] ...