中国农业科学 ›› 2020, Vol. 53 ›› Issue (12): 2460-2476.doi: 10.3864/j.issn.0578-1752.2020.12.013
肖罗丹1,唐磊1,王伟东1,高岳芳1,黄伊凡1,孟阳1,杨亚军1,2(),肖斌1(
)
收稿日期:
2019-12-13
出版日期:
2020-06-16
发布日期:
2020-06-25
通讯作者:
杨亚军,肖斌
作者简介:
肖罗丹,E-mail:1023279673@qq.com。
基金资助:
XIAO LuoDan1,TANG Lei1,WANG WeiDong1,GAO YueFang1,HUANG YiFan1,MENG Yang1,YANG YaJun1,2(),XIAO Bin1(
)
Received:
2019-12-13
Online:
2020-06-16
Published:
2020-06-25
Contact:
YaJun YANG,Bin XIAO
摘要:
【目的】以‘陕茶1号’为材料,克隆CsWRKYIIcs转录因子并分析序列特征,了解其在不同组织和非生物胁迫下的表达模式以及转录活性,为深入研究茶树在逆境胁迫中的功能提供理论依据。【方法】根据茶树基因组数据库注释的WRKY序列设计特异性引物,采用RT-PCR技术从‘陕茶1号’中扩增CsWRKYIIcs的cDNA序列,用生物信息工具分析序列的特点,实时荧光定量PCR(qRT-PCR)研究基因的表达模式,用酵母试验验证其转录活性。【结果】获得9条CsWRKYIIcs的cDNA序列,开放阅读框长度分别为561、960、936、978、897、912、720、1 008和969 bp,分别编码186、319、311、325、298、303、239、335、322个氨基酸。除了CsWRKYIIc7缺少锌指序列外,其他CsWRKYIIcs均含有1个WRKY保守结构域和典型的C2H2型锌指结构。不同物种中的WRKYIIcs具有相似的保守基序,而茶树CsWRKYIIcs和拟南芥、葡萄等双子叶植物的WRKYIIcs氨基酸序列相似性更高。另外,CsWRKYIIcs启动子区域均预测到多个与非生物胁迫相关的顺式作用元件,意味着其可能参与非生物胁迫响应。qRT-PCR结果表明,9个CsWRKYIIcs在根和花中的表达量高于茎和叶,具有一定的特异性。同时,在干旱、ABA、高温和高盐胁迫下被不同程度的诱导表达,其中CsWRKYIIc1和CsWRKYIIc7的表达量变化显著,与推定的顺式作用元件结果相符。酵母试验表明9个茶树CsWRKYIIcs均具有转录激活活性。【结论】克隆获得9个茶树CsWRKYIIcs转录因子,它们参与了茶树对ABA、干旱、高温和高盐胁迫的响应,也可能发挥转录激活因子的调控作用。CsWRKYIIc1和CsWRKYIIc7可作为候选基因深入研究茶树的抗逆功能。
肖罗丹, 唐磊, 王伟东, 高岳芳, 黄伊凡, 孟阳, 杨亚军, 肖斌. 茶树CsWRKYIIcs转录因子的克隆及功能分析[J]. 中国农业科学, 2020, 53(12): 2460-2476.
XIAO LuoDan, TANG Lei, WANG WeiDong, GAO YueFang, HUANG YiFan, MENG Yang, YANG YaJun, XIAO Bin. Cloning and Functional Analysis of CsWRKYIIcs Transcription Factors in Tea Plant[J]. Scientia Agricultura Sinica, 2020, 53(12): 2460-2476.
表1
克隆、荧光定量和构建酵母载体引物"
引物 Primer | 上游序列(5′-3′) Upstream primer | 下游序列(5′-3′) Downstream primer | 作用 Function |
---|---|---|---|
CsWRKYIIc1 | ATC CTT CCA AAC GAT GAC ATT | ACCATTGTAAGTTGTACACATGG | 克隆 Cloning |
CsWRKYIIc2 | TGAAGAAGAATCTGTGCTCCT | TCTAAAGATAACCCAACAAACCTC | |
CsWRKYIIc3 | AGAAACTGCACTTGAAGGAGCT | CTCAACCTCAAGACCAGTCAGT | |
CsWRKYIIc4 | TGATGTAGAAGAAATTGGAGTTGTG | ACATACCAGTAATTGGGTTTCCA | |
CsWRKYIIc5 | TGAGATAACGCGTAGTCCCAATAG | CTGTGGTGAATTAGTTTAGTGCAT | |
CsWRKYIIc6 | CGTGGTGGCTACAGAGACAT | GTACTTACGTGAGACTGGTTAGCC | |
CsWRKYIIc7 | TTCATACTTTGCTCTCTCCCTT | TGAGTTGTTCTTGTACAAAGAGGT | |
CsWRKYIIc8 | AGAGACCTTAGACAAATCTTCCTGT | ATAAAATTAGCAATTGAAAGGGGCT | |
CsWRKYIIc9 | TGACCTTAGAGTCAAACCTATCT | TATGAATTGTCCCATACCTGACT | |
qRT- CsWRKYIIc1 | GAGGAAATATGGACAGAAAGCTG | GCATTCCTTCGTAAGTTGTCAC | 荧光定量qRT-PCR |
qRT- CsWRKYIIc2 | GAATCATGCCAGAAAGTGCC | GGTGTTGGGTTGTGAAAATAGG | |
qRT- CsWRKYIIc3 | TCCACAACCTCAATTCCAACCG | AGCAAAGACGATGACAGTGA | |
qRT- CsWRKYIIc4 | ACCACAATCTCAACCTCAACC | CGACACAATGACAGAGCAAAAG | |
qRT- CsWRKYIIc5 | CAGCATTGTCACCATACAGTTG | AGGCAGCTTCATGATTGACTAG | |
qRT- CsWRKYIIc6 | AAGCCACTTGACTCCTTCAG | TGGGAAGTTGAGGATTTGGC | |
qRT- CsWRKYIIc7 | TTCGGGTTCATGGACTTACTG | AGTCATGGGCTGGTTATTCAAG | |
qRT- CsWRKYIIc8 | AGGTCGTTTGAAGATCCATCAG | CCACATTACCCCTCAGAGTTG | |
qRT- CsWRKYIIc6 | TCGATCTCTTCCTCGTCTACTG | CCTCCATCTTCCACCTCTTTT | |
qRT- CsWRKYIIc7 | GCCATCTTTGATTGGAATGG | GGTGCCACAACCTTGATCTT | |
qRT- CsWRKYIIc8 | AGGTCGTTTGAAGATCCATCAG | CCACATTACCCCTCAGAGTTG | |
qRT- CsWRKYIIc9 | TCGATCTCTTCCTCGTCTACTG | CCTCCATCTTCCACCTCTTTT | |
qRT- Csβ-actin | GCCATCTTTGATTGGAATGG | GGTGCCACAACCTTGATCTT | |
pGBKT7-CsWRKYIIc1 | TCAGAGGAGGACCTGCATATGATGGACAACTACTCATCAAT | CGGCCTCCATGGCCATATGAAATGCCGTGTAAATTTG | 构建酵母载体 Constructing yeast vectors |
pGBKT7-CsWRKYIIc2 | TCAGAGGAGGACCTGCATATGATGGAGAAGAAGAAACAGGA | CGGCCTCCATGGCCATATGCTCTTCTTTTGGCTCCTT | |
pGBKT7-CsWRKYIIc3 | TCAGAGGAGGACCTGCATATGATGCTTGTTGTTGTGAGTGA | CGGCCTCCATGGCCATATGCTCGGGTTTTGCCTCCTT | |
pGBKT7-CsWRKYIIc4 | TCAGAGGAGGACCTGCATATGATGGAGAGTAAAGAAGCTGT | CGGCCTCCATGGCCATATGGTCATCCTTTGTAACAAG | |
pGBKT7-CsWRKYIIc5 | TCAGAGGAGGACCTGCATATGATGGATGAGAACGACAGAGT | CGGCCTCCATGGCCATATGTTGATTGCGCATTCCAGG | |
pGBKT7-CsWRKYIIc6 | TCAGAGGAGGACCTGCATATGATGGATGATGATGATAAGGA | CGGCCTCCATGGCCATATGCTGATTGCGCATTCCAGG | |
pGBKT7-CsWRKYIIc7 | TCAGAGGAGGACCTGCATATGATGGAGAGGAAACAAGCTGT | CGGCCTCCATGGCCATATGTTTGATGAGCCAAATTTC | |
pGBKT7-CsWRKYIIc8 | TCAGAGGAGGACCTGCATATGATGTCTGATGAACACAGAGA | CGGCCTCCATGGCCATATGTGGCTCTTGTTTAAGAAA | |
pGBKT7-CsWRKYIIc9 | TCAGAGGAGGACCTGCATATGATGTCTGATGAACCAGGAGG | CGGCCTCCATGGCCATATGTGGCTCTTGTTTAAAAAA |
表2
CsWRKYIIcs的理化性质分析"
转录因子 Transcription factor | 基因ID Gene ID | 开放阅读框 Open Reading Frame | 氨基酸数量Amino acids number | 相对分子质量 Molecular weight | 理论等电点 Isoelectric point | 平均疏水性Grand average of hydropathy | 核定位预测值 NLSs prediction score | 亚细胞定位预测Subcellular localization prediction |
---|---|---|---|---|---|---|---|---|
CsWRKYIIc1 | TEA006586.1 | 561 | 186 | 21.098 | 9.28 | -0.854 | 5.6 | 细胞核 Nucleus |
CsWRKYIIc2 | TEA028473.1 | 960 | 319 | 35.606 | 6.83 | -0.872 | 4.6 | 细胞核 Nucleus |
CsWRKYIIc3 | TEA008513.1 | 936 | 311 | 34.324 | 6.54 | -0.692 | 7.7 | 细胞核 Nucleus |
CsWRKYIIc4 | TEA017544.1 | 978 | 325 | 36.203 | 8.21 | -0.602 | 4.4 | 细胞核 Nucleus |
CsWRKYIIc5 | TEA001162.1 | 897 | 298 | 32.070 | 5.79 | -0.790 | 4.3 | 细胞核 Nucleus |
CsWRKYIIc6 | TEA022377.1 | 912 | 303 | 33.502 | 5.49 | -0.894 | 4.3 | 细胞核 Nucleus |
CsWRKYIIc7 | TEA007197.1 | 720 | 239 | 27.123 | 8.06 | -0.789 | 4.5 | 细胞核 Nucleus |
CsWRKYIIc8 | TEA023233.1 | 1008 | 335 | 36.952 | 6.00 | -0.840 | 7.9 | 细胞核 Nucleus |
CsWRKYIIc9 | TEA001873.1 | 969 | 322 | 35.717 | 6.31 | -0.762 | 5.5 | 细胞核 Nucleus |
图4
不同物种WRKYIIcs的进化树和保守基序分析 At:拟南芥 Arabidopsis thaliana;Cs:茶树 Camellia sinensis;Zm:玉米 Zea mays;Os:水稻 Oryza sativa;Vv:葡萄 Vitis vinifera 4个物种的WRKYIIcs蛋白ID号ID numbers of WRKYIIcs proteins from four species : AtWRKY8 (AT5G46350), AtWRKY24 (AT5G41570), AtWRKY23 (AT2G47260), AtWRKY28 (AT4G18170), AtWRKY43 (AT2G46130), AtWRKY45 (AT3G01970), AtWRKY48 (AT5G49520), AtWRKY56 (AT1G64000), AtWRKY57 (AT1G69310), AtWRKY68 (AT3G62340), AtWRKY71 (AT1G29860), AtWRKY75 (AT5G13080), ZmWRKY28 (Zm00001d011413), ZmWRKY85 (Zm00001d018656), ZmWRKY95 (Zm00001d043950), VvWRKY1 (CBI27268.3), VvWRKY3 (CBI27681.3), VvWRKY16 (CBI22862.3), VvWRKY21 (CBI36970.3), VvWRKY33 (CBI30827.3), VvWRKY37 (CBI22108.3), VvWRKY44 (CBI21329.3), VvWRKY53 (CBI15677.3), OsWRKY3 (Os03g0758000), OsWRKY8 (Os05g0583000), OsWRKY11 (Os01g0626400), OsWRKY23 (Os01g0734000), OsWRKY29 (Os07g0111400), OsWRKY49 (Os05g0565900), OsWRKY72 (Os11g0490900)"
表3
顺式作用元件预测"
顺式作用元件 Cis-element | 元件数量 Cis-element number | 功能 Function | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
W1 | W2 | W3 | W4 | W5 | W6 | W7 | W8 | W9 | ||
ABRE | 4 | 3 | 3 | 7 | 4 | 10 | 2 | 3 | 2 | 参与脱落酸反应性的顺式作用元件 Cis-acting elements involved in abscisic acid reactivity |
ARE | 5 | 2 | 3 | 1 | 1 | 2 | 3 | 2 | 8 | 厌氧诱导所必需的顺式调节元件 Cis-regulating elements necessary for anaerobic Induction |
Box 4 | 4 | 5 | 2 | 1 | 4 | 7 | 5 | 4 | 光响应DNA模块部分 Optical response DNA module part | |
CGTCA-motif | 1 | 1 | 1 | 3 | 4 | 4 | 5 | 参与MEJA反应的顺式调节元件 Cis-regulating elements involved in MEJA reaction | ||
ERE | 4 | 1 | 3 | 1 | 4 | 2 | 参与乙烯反应的顺式调节元件 Cis-regulating elements involved in ethylene reaction | |||
G-box | 3 | 2 | 1 | 4 | 2 | 4 | 1 | 3 | 光应答元件Cis-regulating elements involved in light reaction | |
MBS | 2 | 1 | 1 | 2 | 1 | 1 | 1 | 干旱诱导MYB结合位点Drought-induced MYB binding site | ||
MYB | 2 | 6 | 3 | 1 | 2 | 1 | 3 | 2 | 4 | 参与干旱、高盐和低温诱导Drought/high salt/low temperature-induced |
MYC | 2 | 7 | 3 | 2 | 5 | 2 | 4 | 2 | 干旱应答元件 Cis-regulating elements involved in drought reaction | |
STRE | 3 | 4 | 4 | 5 | 1 | 2 | 参与热响应 Heat stress responsiveness | |||
TGACG-motif | 1 | 1 | 1 | 3 | 1 | 4 | 4 | 5 | 参与MEJA反应的顺式调节元件 Cis-regulating elements involved in MEJA reaction | |
W box | 4 | 2 | 3 | 防卫和胁迫应答元件 Cis-acting elements involved in defense and stress response |
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