全 文 ：作物学报 ACTA AGRONOMICA SINICA 2015, 41(7): 11271135 http://zwxb.chinacrops.org/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

本研究由国家高技术研究计划(863计划)项目(2013AA102604), 科技部国际合作项目(2013DFA31600), 广西农科院团队项目(桂农科
2011YT01), 广西八桂学者、特聘专家专项经费, 广西自然科学基金项目(2014GXNSFBA118085)和广西农科院基本科研业务专项(桂农
科 2014YD01)资助。
* 通讯作者(Corresponding authors): 李杨瑞, E-mail: lyr@gxaas.net; 杨丽涛, E-mail: litaoyang61@yahoo.com
第一作者联系方式: E-mail: lcn560@163.com
Received(收稿日期): 2014-12-08; Accepted(接受日期): 2015-05-04; Published online(网络出版日期): 2015-05-15.
URL: http://www.cnki.net/kcms/detail/11.1809.S.20150515.1542.002.html
DOI: 10.3724/SP.J.1006.2015.01127
水分胁迫下甘蔗差异表达基因筛选及激素相关基因分析
李长宁 谢金兰 王维赞 梁 强 李毅杰 董文斌 刘晓燕
杨丽涛* 李杨瑞*
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
摘 要: 甘蔗是经济和环境上日益重要的 C4作物, 干旱在全球范围内严重限制甘蔗产量。了解甘蔗对水分胁迫反应的
分子机制将有助于甘蔗抗旱性的分子遗传改良。利用基因芯片技术分析水分胁迫下甘蔗叶片的 15 593个基因的表达谱,
结果表明, 中、重度胁迫下的差异表达基因数量分别为 300个和 853个, 中度胁迫中差异基因以上调表达为主, 重度胁
迫中下调表达占多数。功能注释分析显示, 差异表达基因分子功能主要为结合、载体和催化活性, 主要参与代谢、细胞
和生物调控等生物过程。此外, 功能未明确的假定蛋白和无匹配信息的基因序列仍占据注释结果的相当一部分, 表明还
有大量的基因尚待发掘。在水分胁迫下, 甘蔗内源 ABA和 IAA含量显著上升而 GA含量显著受到抑制。以参与生物进
程分类, 对植物激素相关基因进行筛选并分析, 发现激素响应表达基因代谢途径具有多样性, 显示了激素代谢网络的
交叉性与复杂性。挑选 9个差异表达程度不同的基因进行实时荧光定量 PCR检测, 表明芯片数据具有良好的重复性。
关键词: 甘蔗; 水分胁迫; 基因芯片; 植物激素; 基因表达谱
Screening of Differentially Expressed Genes and Analysis of Plant Hormones
Related Genes under Water Stress in Sugarcane
LI Chang-Ning, XIE Jin-Lan, WANG Wei-Zan, LIANG Qiang, LI Yi-Jie, DONG Wen-Bin, LIU Xiao-Yan,
YANG Li-Tao*, and LI Yang-Rui*
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
Abstract: Sugarcane is an increasingly economically and environmentally important C4 crop. Water stress limits enormously sug-
arcane productivity worldwide, and understanding the molecular mechanisms for sugarcane stress responses will be useful for
sugarcane improvement by genetic manipulation. To investigate the transcriptome changes in response to water stress, we used
microarrays to profile expressions of 15 593 genes in sugarcane exposed to drought. The results indicated that 300 and 853 dif-
ferentially expressed genes were detected under moderate and severe water stresses, respectively. The expression of differentially
expressed genes treated with moderate water stress was mainly up-regulated, however that treated with severe water stress was
mainly down-regulated. To further characterize these genes, we used Gene Ontology (GO) for their annotation, the results showed
that differentially expressed genes possessed the functions of binding, transporter, molecular transducer and catalytic activities and
were involved in metabolic, biological regulation and cellular processes. Besides, hypothetical protein and no match annotated
results were found to fill a large part of those genes, indicating that effective approach should be adopted to discover novel genes
in sugarcane genomics. Water stress resulted in an increase in ABA and IAA contents but a depression in GA content. Classified
by biological process, 46 plant hormone related genes were selected, further annotation analysis showed that the metabolic path-
ways of some plant hormone responsive genes were diverse or had crosstalk with each other, indicating the intersectionality and
complexity of plant hormone signaling pathway. Additionally, the relative expressions of nine selected genes were validated by
1128 作 物 学 报 第 41卷


quantitative Real-time PCR (qRT-PCR), further confirming the reliability of microarray results.
Keywords: Sugarcane; Water stress; Microarray; Plant hormone; Gene expression profiles
甘蔗为重要的糖料和能源作物 , 在维持社会可持续
发展中扮演重要角色。干旱致使甘蔗减产和品质降低[1]。
作物能感受刺激和传递信号 , 启动各种生理生化反应以
响应和适应水分胁迫[2]。植物内源激素作为化学信号参与
调节众多植物生长发育代谢进程 , 并在适应环境胁迫中
发挥重要作用 [3-4]。在水分胁迫下, 脱落酸(ABA)可诱导
气孔关闭 , 减少蒸腾量及水分散失 [3]; 细胞分裂素参与
植物对水分胁迫的响应并通过与 ABA协作而实现。在渗
透胁迫下, 随着 ABA 含量增加, 与 ABA 具有拮抗作用
的细胞分裂素含量下降 , 气孔得以顺利关闭 , 水分蒸腾
量随之减少 [5]; 赤霉素对逆境胁迫的反应与生长素类似,
就是含量降低, 植物正常生长发育受阻[6]; 水分胁迫下植
物体内乙烯浓度急剧增加[7], 呼吸作用增强、衰老加速、
成熟加快[8], ROS 导致的膜脂过氧化作用与乙烯浓度密切
相关[9], 逆境下乙烯合成量与植物耐胁迫能力呈负相关[7]。
单一植物激素生理功能的实现离不开其他类激素的协同
作用 , 进而通过影响激素合成和信号转导关键基因的表
达及分子转导, 调节各激素的含量水平[10-11], 从而实现激
素的相互调节, 这已经成为植物生长发育中一种重要的调
控手段[12-13]。
基因差异表达谱为植物在特定环境及生理状态下的
一个基因表达汇总。应用基因芯片筛选逆境胁迫下差异表
达基因, 分析基因的功能, 不仅能使我们更好地理解作物
响应及耐受胁迫的分子机制 , 也为利用这些基因对作物
进行定向遗传改良以提高作物的抗胁迫能力奠定一定的
基础[14-15]。通过该技术, 前人已从各处理条件下的多种作
物的转录水平上挖掘出许多的差异表达基因并应用于作
物分子改良[16-20]。本研究目的在于利用基因芯片技术分析
水分胁迫下甘蔗叶片中基因表达谱的差异 , 并对激素相
关基因作进一步分析 , 探讨甘蔗水分胁迫响应的激素调
节分子基础。
1 材料与方法
1.1 试验材料与处理
试验材料为耐旱品种桂糖 21 号(GT21)。蔗种以单芽方
式种植于泥沙混合培养基质上, 50 d后, 选取长势一致的
甘蔗苗移栽至桶中, 每桶 2~3株。桶规格约 30 cm×35 cm
(直径×高), 每桶装混合土 18 kg (土∶有机肥∶沙=70∶
20∶10, w/w), 桶底钻孔以增强透气性。把桶移至智能温
室大棚, 日常管理, 使其正常生长 5个月处于伸长盛期时,
把材料分为 2 组。第一组正常淋水, 为对照, 第二组停止
淋水, 分别于断水 5 d和 7 d后取样, 清晨采集+1叶样品,
速冻于液氮中, 带回实验室–80℃冰箱保存待分析。处理
期间 , 对照及处理土壤绝对含水量分别为 20%±2%和
9%±2%。
1.2 内源激素含量测定
称取新鲜甘蔗叶片 0.5 g, 用 80%预冷甲醇置弱光下
冰浴研磨至匀浆, 4℃避光放置 24 h, 中间振荡混匀 4~5次,
10 000g 冷冻离心 15 min, 取上清液过 C-18 柱, 真空干
燥后, 以样品稀释液(含 0.1% Tween-20 和 0.1%明胶的磷
酸盐缓冲液, pH 7.5)溶解即得样品激素提取液[21]。采用酶
联免疫吸附法(ELISA)按说明书测定 ABA、IAA和 GA的
含量, 酶联免疫试剂盒购于中国农业大学, 使用 ANTHOS-
2010酶标仪测定吸光值, 每样品重复测定 3次, 取平均值。
1.3 芯片规格、杂交和扫描
选用 Agilent 4×44K 规格芯片。甘蔗基因数据来自
NCBI 的 Unigene 库 , 共包含有代表不同基因序列的
15 593 条探针。依照 Agilent基因表达谱芯片(单标)操作
指南, 使用 TRIzol 法提取叶片的总 RNA 并纯化, 以 T7
Promotor primer为引物配制 cDNA合成体系, 应用一步
法合成 cDNA 的第 1和第 2链。以上述 cDNA为模板并
添加 aa-UTP, 在 T7 RNA 聚合酶作用下合成 cRNA, 经
纯化及浓度测定后的 cRNA 加到荧光染料(Cy3)中进行
标记 , 纯化标记好的 cRNA, 加到片段化溶液中进行样
品片段化。
取 100 µL片段化样品上芯片, 65℃滚动杂交 17 h (10
转 min–1), 杂交完毕 , 取出芯片于洗涤液中清洗干净待
扫描。采用Agilent扫描仪来获取图像, 分辨率为 5 μm, 扫
描仪自动以 100%和 10% PMT各扫描一次, 以GeneSpring
软件读取原始数据, Agilent软件可自动合并 2次结果。
1.4 数据分析
采用 Aligent Scanner获取图像, 通过 Imagene转化为
数值后应用Genespring软件将数值标准化, 取得Ratio值。
之后对芯片的杂交数据进行比较 , 差异表达基因筛选标
准为表达倍数 Fold change≥2或≤0.5 (表达上调或下调),
FDR (False discover rate)< 0.05, 即 q < 0.05, 且方差分析
的 P≤0.01, 以上 3 个条件必须同时满足。使用 AmiGO
的 Blast Query 在线软件对筛选出的差异基因进行 Gene
Ontology 分析, 取舍阈值为比对 E-value≤1E–20。通过
BlastX 将参与植物激素生物过程的基因序列比对到蛋白
数据库(E-value≤1E–20), 得到该基因功能注释信息。
1.5 实时定量 RT-PCR检测
随机挑选 9个筛选出来的差异表达基因, 设计特异引
物, 提取同期样品 RNA, 逆转录为 cDNA, 进行实时荧光
定量 PCR分析(表 1), 选用 GAPDH基因为内参[22]。反应
体系含 10 μL 2×All-in-One qPCR Mix (Gene Copoeia)、2
μL cDNA、4 μmol L–1的正反向引物各 1 μL、6 μL双蒸水。
程序为 95℃预变性 10 min; 95℃变性 10 s, 60℃退火 20 s,
72℃延伸 20 s, 40个循环。反应完成后进行熔解曲线检验,
采用 2−ΔΔCt法进行相对表达量计算[23]。
第 7期 李长宁等: 水分胁迫下甘蔗差异表达基因筛选及激素相关基因分析 1129


表 1 实时定量 PCR选用基因及引物
Table 1 Genes and primers list for qRT-PCR
序号
No.
登录号
Accession No.
基因功能
Putative function
正向引物
Forward primer (5–3)
反向引物
Reverse primer (5–3)
0 EF189713 GAPDH TGGTGCTGACTATGTCGTGGA CATGGGTGCATCTTTGCTTG
1 CA191067 RR4 Corn type A response regulator ATGACGGTGGTGGATGCC TCACTTGGTAGGACGAGTTCTTG
2 DQ494704 Phytochrome B ATGGCAGTCATCATTAGCAGTG GAAACTCGCAAGCATACCTCA
3 CA280103 Serine/threonine-protein kinase ACAGCAGGGCGATTCAGTG TTCACGCGAGGTGTTGGA
4 BU103703 Δ-1-pyrroline-5-carboxylate synthetase CCTAAAGCCAGGAAAGATAACA AATAATGAGCAGAACACCCAAT
5 CA289286 Protein WAX2 GACCTTGTTCACCTCACGCA ACGGTCTGGACGCTATGGA
6 CA110802 Mannitol dehydrogenase AAGCACATTGGCGTAGTTGG GACGGTCACCCTCATCCC
7 CA134623 Asparagine synthetase CATCATCCCATCGTTTCCCTA GTCTGTCATCAGCCGTTTTACC
8 CA093454 Protein phosphatase 2C CTGTCCGACCAGATCAGAAAC CCAGTTCACATTCGCTTCG
9 CA277793 bZIP transcription factor protein AGTCGGCTCGGAGGTCTA GGTTCTGGCTGGTTATGC

2 结果与分析
2.1 内源激素含量变化
水分胁迫处理提高了甘蔗叶片(图 1) ABA 和 IAA 含
量, 在处理第 5、第 7 天, 两者分别比对照增加 32.3%、
9.1%和 71.8%、19.7%, 增量显著; 相反, GA 含量显著受
到水分胁迫的抑制, 处理第 5、第 7 天, 其含量分别比对
照低 15.2%和 26.5%。

图 1 水分胁迫对甘蔗品种 GT21叶片内源 ABA、IAA和 GA含量的影响
Fig. 1 Effect of water stress on ABA, IAA and GA contents in sugarcane cultivar GT21
同一处理时间内, 标有不同字母的处理间在 0.05水平差异显著。
Bars superscripted by a different letter are significantly different at the 0.05 probability level at the same treatment.

2.2 差异表达基因筛选
Agilent 单通道表达谱芯片用重复探针点(10 次重复)
信号的变异系数(CV 值)来计算芯片的稳定性, 其质控标
准是 CV值小于 15%。本次 12张基因芯片(每处理 3个重
复 )的 CV 值为 5.23%~8.15%, 芯片检出率在 93.22%~
94.26%之间, 表明结果合格可用。经筛选, 水分胁迫处理
第 5天的差异表达基因数量为 300个, 上、下调表达基因
数量分别为 175个和 125个; 从第 7天的样品中筛选到差
异表达基因 853个, 上、下调表达基因数量分别为 418个
和 435 个; 第 5、第 7 天共有的差异表达基因数目为 183
个(图 2-A)。
2.3 差异表达基因功能注释
分别对筛选出来的差异表达基因进行功能注释和 GO
分析, 其中第 5、第 7天 183个共有差异表达基因中, 获得
功能注释的基因数为 103 个, 编码假定蛋白的基因数为 58
个, 22个基因未找到匹配信息, 3 种注释情况占比分别为
56.3%、31.7%、12.0%, 除此以外, 第 5、第 7 天特有差异
表达基因 3 种注释情况占比分别为 55.6%、31.6%、12.8%
和 63.6%、20.7%、15.7% (图 2-B)。第 5、第 7天 183个共
有差异表达基因中, 获得有效GO注释基因 118个, 占比为
64.5%, 其余两组基因的注释数量分别为 67个和 420个, 占
比为 57.3%和 62.7% (图 2-C)。118个第 5、第 7天共有差
异表达基因共获得 369 条 GO 注释信息, 生物进程、细胞
组分和分子功能分别占 61.5%、10.6%和 27.9%, 处理第 5、
7天特有的 67和 420个基因分别获得 263和 930条 GO注
释信息, 两组基因的生物进程、细胞组分和分子功能分别
占 70.7%、9.1%、20.2%和 64.7%、10.5%、24.7% (图 2-D)。
2.4 差异表达基因 GO分类
以生物进程分类 , 筛选出的差异表达基因参与的生
物进程主要有细胞过程 (cellular process), 代谢过程
1130 作 物 学 报 第 41卷


(metabolic process)、应激反应(response to stimulus)、生物
调节(biological regulation)、生物过程调节(regulation of
biological process)、发育过程(developmental process)、定
位(localization)、多器官进程 (multi-organism process)等 ;
以细胞组分分类, 差异基因被归于细胞组件(cell part)、细
胞器(organelle)、大分子复合体(macromolecular complex)
和膜关闭内腔(membrane-enclosed lumen); 以分子功能分
类, 差异基因被归于催化活性(catalytic activity)、载体活
性(transporter activity)、结合活性(binding)、分子转导活
性(molecular transducer activity)(图 3)。

图 2 水分胁迫下甘蔗品种 GT21叶片中差异表达基因筛选和功能注释
Fig. 2 Screening and annotation of differential expression genes in sugarcane cultivar GT21 under water stress

图 3 水分胁迫下甘蔗品种 GT21叶片中差异表达基因 GO分类
Fig. 3 GO classification of differential expression genes in sugarcane cultivar GT21 under water stress
横坐标下的数字代表 GO分类信息, 详细见英文注释。
The numeric values under x-axis represent the GO classified information as follows: 1: cellular process; 2: metabolic process; 3: response to
stimulus; 4: biological regulation; 5: regulation of biological process; 6: establishment of localization; 7: localization; 8: multi-organism
process; 9: developmental process; 10: multicellular organismal process; 11: immune system process; 12: cellular component organization;
13: reproductive process; 14: growth; 15: negative regulation of biological process; 16: cellular component biogenesis; 17: positive regulation
of biological process; 18: cell part; 19: organelle; 20: macromolecular complex; 21: membrane-enclosed lumen; 22: catalytic activity;
23: transporter activity; 24: binding; 25: molecular transducer activity.
第 7期 李长宁等: 水分胁迫下甘蔗差异表达基因筛选及激素相关基因分析 1131


2.5 植物激素相关基因分析
以参与生物进程分类, 对植物激素相关基因进行筛选。
第 5、第 7天共有差异表达基因 9个(表 2), 其中 6个上调、
3个下调表达, 第 5、第 7天特有差异表达基因分别为 6 (表
3)和 31个(表 4), 上调表达数量分别为 4个和 14个, 下调表
达数量为 2 个和 17 个, 所编码的基因产物包括蛋白磷酸酶
(CA093454, CA078060, CA282798)、蛋白激酶(CA160805,
CA285332, CA280103, CA135993)、受体蛋白(CA252520,
CA076654, CA138168), 转运蛋白 (CA151192, CA279772,
CA080185, CA106306, CA186908)等, 这些基因参与了包括
赤霉素、生长素、细胞分裂素、脱落酸、乙烯的生物合成、
代谢、转运、响应或信号转导通路等生物进程。

表 2 水分胁迫第 5、第 7天共有的激素相关差异表达基因功能注释和 GO分析
Table 2 Annotation and GO terms of genes related to phytohormone pathways in both 5th and 7th day under water stress
表达倍数
Fold change NCBI登录号
NCBI No.
注释描述
Annotation description
E-value
Day 5 Day 7
GO层次
GO term
GO层次描述
GO term description
GO:0009737 Response to abscisic acid EF517495 Ornithine-oxo-acid aminotransferase 0.0E+00 0.08 0.10
GO:0009733 Response to auxin
CA273285 Gibberellin-regulated protein 2 precursor 5.0E–38 0.39 0.42 GO:0009739 Response to gibberellin
CA266878 Protein HVA22-like 3.0E–73 0.46 0.47 GO:0009737 Response to abscisic acid
CA167529 Cold acclimation WCOR413-like protein 3.0E–76 2.84 2.88 GO:0009737 Response to abscisic acid
CA138168 ADIPOR-like receptor 4.0E–82 3.89 3.17 GO:0009725 Response to hormone
GO:0009737 Response to abscisic acid
GO:0009733 Response to auxin
GO:0009738 Abscisic acid-activated signaling pathway
GO:0009723 Response to ethylene
CA093454 Protein phosphatase 2C 1.0E–46 10.26 9.54
GO:0009788 Negative regulation of abscisic acid-activated signaling pathway
CA093200 Homeobox-leucine zipper protein
HOX1-like
3.0E–31 10.86 11.69 GO:0009725 Response to hormone
GO:0009737 Response to abscisic acid
GO:0009738 Abscisic acid-activated signaling pathway
CA078060 Protein phosphatase 2C 5.0E–94 16.30 14.23
GO:0009788 Negative regulation of abscisic acid-activated signaling pathway
GO:0009737 Response to abscisic acid
GO:0009733 Response to auxin
GO:0009738 Abscisic acid-activated signaling pathway
GO:0009723 Response to ethylene
BU103681 G-box binding factor 0.0E+00 53.45 27.16
GO:0009873 Ethylene-activated signaling pathway

表 3 水分胁迫 5 d特有的激素相关差异表达基因功能注释和 GO分析
Table 3 Annotation and GO terms of genes related to phytohormone pathway in 5th day under water stress
NCBI登录号
NCBI No.
注释描述
Annotation description
E-value 表达倍数
Fold change
GO层次
GO term
GO层次描述
GO term description
GO:0009851 Auxin biosynthetic process CA224212 4-coumarate-CoA ligase-like
7-like isoform X1
6.0E–125 0.14
GO:0009850 Auxin metabolic process
CA252520 Glutamate receptor 9.0E–84 0.44 GO:0071215 Cellular response to abscisic acid stimulus
CA076654 Glutamate receptor 5.0E–84 2.09 GO:0071215 Cellular response to abscisic acid stimulus
CA085551 Inositol-3-phosphate synthase 2.0E–86 2.25 GO:0009733 Response to auxin
GO:0009733 Response to auxin
GO:0009851 Auxin biosynthetic process
GO:0009737 Response to abscisic acid
GO:0009723 Response to ethylene
CA123082 Syntaxin 121 5.0E–65 2.75
GO:0009738 Abscisic acid-activated signaling pathway
AY596597 Programmed cell death protein 4 0.0E+00 3.57 GO:0009734 Auxin-activated signaling pathway

1132 作 物 学 报 第 41卷


表 4 水分胁迫 7 d特有的激素相关差异表达基因功能注释和 GO分析
Table 4 Annotation and GO terms of genes related to phytohormone pathway in the 7th day under water stress
NCBI登录号
NCBI No.
注释描述
Annotation description
E-value
表达倍数
Fold
change
GO层次
GO term
GO层次描述
GO term description
CA223665 Ent-copalyl diphosphate synthase 1.0E–129 0.06 GO:0009685 Gibberellin metabolic process
CA191067 RR4-Corn type-A response regulator 1.0E–92 0.13 GO:0009735 Response to cytokinin
CA252857 Auxin-responsive Aux/IAA family member 2.0E–17 0.21 GO:0009733 Response to auxin
CA216109 Phytochrome B 2.0E–137 0.23 GO:0009687 Abscisic acid metabolic process
CA103845 Cellulose synthase10 5.0E–128 0.29 GO:0009873 Ethylene-activated signaling pathway
CA128805 Auxin response factor 2.0E–101 0.34 GO:0009733 Response to auxin
DQ494704 Phytochrome B 1.0E–132 0.34 GO:0009687 Abscisic acid metabolic process
GO:0009739 Response to gibberellin CA135716 Xyloglucan endotransglucosylase hydrolase
protein
6.0E–86 0.35
GO:0009740 Gibberellic acid mediated signaling pathway
GO:0009737 Response to abscisic acid
GO:0009733 Response to auxin
GO:0009723 Response to ethylene
GO:0009873 Ethylene-activated signaling pathway
GO:0009738 Abscisic acid-activated signaling pathway
CA160805 Leucine-rich repeat receptor-like protein kinase
family protein
6.0E–87 0.35
GO:0009755 Hormone-mediated signaling pathway
CA252377 26S proteasome non-ATPase regulatory subunit 8 4.0E–147 0.37 GO:0009733 Response to auxin
GO:0010540 Basipetal auxin transport
GO:0010541 Acropetal auxin transport
CA151192 ABC transporter B family member 5.0E–67 0.39
GO:0010315 Auxin efflux
GO:0009738 Abscisic acid-activated signaling pathway CA088031 RNA binding protein 3.0E–84 0.40
GO:0009693 Ethylene biosynthetic process
CA143147 RNA-binding protein-like protein 1.0E–46 0.43 GO:0009737 Response to abscisic acid
GO:0009737 Response to abscisic acid
GO:0009733 Response to auxin
GO:0009723 Response to ethylene
CA254394 MYB DNA-binding domain superfamily protein 9.0E–86 0.43
GO:0009739 Response to gibberellin
CA279772 ABC transporter 2.0E–135 0.46 GO:0009737 Response to abscisic acid
GO:0009737 Response to abscisic acid
GO:0009733 Response to auxin
GO:0009723 Response to ethylene
GO:0009873 Ethylene-activated signaling pathway
GO:0009738 Abscisic acid-activated signaling pathway
GO:0010540 Basipetal auxin transport
GO:0009926 Auxin polar transport
GO:0010315 Auxin efflux
CA080185 ABC transporter B family member 4.0E–51 0.46
GO:0060919 Auxin influx
CA106306 Copper transporter 2.0E–53 0.48 GO:0009737 Response to abscisic acid
CA186391 Ent-kaurene oxidase KO4 4.0E–32 2.04 GO:0009686 Gibberellin biosynthetic process
CA162286 Indole-3-acetic acid-amido synthetase 1.0E–135 2.10 GO:0009733 Response to auxin
CA285332 Serine/threonine-protein kinase 3.0E–164 2.17 GO:0009737 Response to abscisic acid
GO:0009723 Response to ethylene CA069791 Protein REVERSION TO ETHYLENE
SENSITIVITY1
2.0E–69 2.31
GO:0010105 Negative regulation of ethylene-activated signaling pathway
BU103689 Cold acclimation protein COR413-PM1 8.0E–116 2.63 GO:0009737 Response to abscisic acid
第 7期 李长宁等: 水分胁迫下甘蔗差异表达基因筛选及激素相关基因分析 1133


(续表 4)
NCBI登录号
NCBI No.
注释描述
Annotation description
E-value
表达倍数
Fold
change
GO层次
GO term
GO层次描述
GO term description
GO:0009737 Response to abscisic acid CA282798 Type I inositol 1,4,5-trisphosphate 5-phosphatase 2.0E–99 3.71
GO:0009850 Auxin metabolic process
GO:0009737 Response to abscisic acid CA280103 Serine/threonine-protein kinase 0.0E+00 4.09
GO:0009738 Abscisic acid-activated signaling pathway
CA186908 Auxin Efflux Carrier family protein 4.0E–46 4.29 GO:0009926 Auxin polar transport
GO:0009737 Response to abscisic acid
GO:0009723 Response to ethylene
CA093392 MYB-related transcription factor 1.0E–79 4.55
GO:0009739 Response to gibberellin
CA122105 Cytochrome P450 superfamily protein 4.0E–111 5.09 GO:0009687 Abscisic acid metabolic process
GO:0009737 Response to abscisic acid
GO:0009733 Response to auxin
CA264504 Aconitate hydratase 6.0E–151 6.91
GO:0009873 Ethylene-activated signaling pathway
CA135993 CBL-interacting protein kinase 2.0E–109 7.31 GO:0010540 Basipetal auxin transport
GO:0009733 Response to auxin
GO:0009926 Auxin polar transport
CA174832 Carotenoid cleavage dioxygenase 6.0E–47 9.83
GO:0009850 Auxin metabolic process
BU103703 Delta 1-pyrroline-5-carboxylate synthetase1 0.0E+00 28.97 GO:0009737 Aesponse to abscisic acid

2.6 芯片实时荧光定量 PCR验证
为了验证芯片数据的可靠性, 挑选 9个差异表达程度
不同的基因(表 1), 实时荧光定量 PCR检测表明(图 4), 叶
片的 qRT-PCR 结果虽然在比值上存在差异, 但是其上调
或下调的趋势却是一致的 , 说明本研究利用芯片杂交分
析获得的基因差异表达信息具有较高可靠性和可重复性。

图 4 差异表达基因的实时荧光定量 PCR验证
Fig. 4 Differentially expressed genes validated by quantitative real-time PCR
横坐标下的数字 1~9代表所选基因的序号, 各序号对应基因的登录号和基因功能详见表 1。
Number 1–9 under x-axis represent the sequence numbers of genes chosen for quantitative Real-time PCR, the accession number and gene
function of the chosen genes can be found in Table 1.

3 讨论
作物的抗旱性是由多基因控制的复杂数量性状 , 涉
及信号转导、编码保护、防御和胁迫耐受蛋白、能量代谢、
膜运输和转录翻译等方面[24-25]。干旱条件的复杂性和作物
基因型的多样性决定了作物对干旱的适应性存在多条途
径, 不同强度或类型的水分胁迫很可能导致不同程度或
方式的适应性[26-27]。本研究采用基因芯片技术构建甘蔗品
种 GT21 在中度和重度水分胁迫下叶片基因表达谱数据
库。结果显示, 在重度水分胁迫下差异表达基因数量远高
于中度胁迫, 中度胁迫差异基因以上调表达为主, 重度胁
迫差异基因下调表达占多数 , 这也许与上述胁迫适应调
节机制密切相关, 但仍需进一步研究揭示。
作物在形态结构和生理生化水平上的抗旱性 , 都是
1134 作 物 学 报 第 41卷


内在相关基因表达调控的结果[24-25]。水分胁迫下, 作物细
胞迅速感知外界信号, 通过信号转导、基因转录、转录后
3 个水平调节, 激活胁迫应答基因的表达, 大量特异蛋白
得以生成 , 协同作用调节植物体内的生理生化和代谢水
平, 提高作物抗旱性[28-29]。本研究对筛选出来的差异基因
进行功能注释和 GO分类, 结果显示这些基因参与的代谢
进程有脂类代谢、信号转导作用、蛋白质代谢、ABA 代
谢、光合作用、碳水化合物代谢、细胞代谢、呼吸作用、
能量代谢等, 或者是编码蛋白激酶、渗透调节物质、转录
因子、碳水化合物和次生代谢相关酶类等, 表明甘蔗对水
分胁迫适应代谢途径的多样性和复杂性。由于甘蔗的遗传
背景和基因组的复杂性 , 迄今为止还尚未见有关其全基
因组测序的报道 , 功能未明确的假定蛋白和无匹配信息
的基因序列仍占据注释结果的相当一部分 , 表明进一步
揭示甘蔗响应水分胁迫的详细机理仍需大量的研究工作。
水分胁迫可以引起作物一系列的生理生化反应 , 其
中植物内源激素含量的变化及相互间的平衡调节是植物
响应水分胁迫的重要生理活动[10-13,30]。大量研究表明, 脱
落酸(ABA)、吲哚乙酸(IAA)、赤霉素(GA)等内源激素在
植株受水分胁迫期间起着信号传导并主动适应的作用 ,
直接影响作物对逆境的适应性 , 反映植株对干旱胁迫的
响应程度[31]。水分胁迫会促使植物的内源 ABA含量增加,
作为信号分子 ABA可激活多种离子通道以及相关的生理
生化反应酶类, 促进气孔关闭, 降低蒸腾速率, 减少水分
散失, 进而减少干旱对植物的伤害[32]。胁迫初期植物内源
IAA 合成会减少以减缓生长, 随着胁迫程度的增强, 由于
侧根和不定根的生长需要, 内源 IAA合成量可能增多, 但内
源 IAA含量变化因作物种类而定, 具体机制较为复杂[33-34]。
IAA、GA这类与作物生长发育相关植物激素含量降低, 使
植物生长速率减慢, 从形态、生理等各方面发生相应变化,
以提高自身的抗旱力, 减轻逆境造成的伤害。本研究表明,
水分胁迫下 , 随着 ABA 含量显著上升 , 甘蔗叶片中的
IAA 含量也随着上升, 而 GA 含量显著受到抑制, 显示了
ABA、IAA和 GA在甘蔗响应逆境胁迫时的重要联系。
植物对逆境胁迫的响应主要有 ABA 依赖和非 ABA
依赖两条代谢途径[35]。本研究对筛选出来的激素相关的
46个基因作进一步分析发现, 与 ABA响应相关的有 26个基
因, 其中与 ABA 信号转导有关的 SnRK2 基因(CA285332,
CA280103), PP2Cs基因(CA093454, CA078060)。在正常条件
下, PP2Cs可与 SnRK2s结合, 使 SnRK2s发生去磷酸化作
用而失活; 在水分胁迫下, 植物内源 ABA 的含量急剧增
加, ABA与受体蛋白结合后, 再与 PP2C相互作用, PP2C
构象改变, 其酶活性受到抑制, 受 PP2C 负调控的 SnRK2
酶活性得以释放[36-37], SnRK2 的作用使下游成员, 包括
AREB 等转录因子磷酸化[38-39], 最终激活响应 ABA 信号
靶基因的表达, 实现植物对水分胁迫响应的调节[40-41]。此
外, ABA响应表达基因代谢途径具有多样性[42-43]。本研究
注释结果显示, 上述 PP2Cs基因不仅与 ABA信号转导有
关 , 还参与生长素和乙烯的响应进程 ; G-box 结合因子
(BU103681) 、 syntaxin 121 (CA123082) 、 蛋 白 激 酶
(CA160805)、ABC转运蛋白(CA151192、CA080185)等基
因也参与众多激素响应代谢进程 , 表明激素代谢网络的
交叉性与复杂性。
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