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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (6): 1480-1495.doi: 10.3724/SP.J.1006.2023.24113

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

谷子MAPK家族成员的鉴定及其对生物胁迫的响应分析

刘佳1,**(), 邹晓悦1,2,**(), 马继芳1, 王永芳1, 董志平1, 李志勇1,*(), 白辉1,*()   

  1. 1河北省农林科学院谷子研究所/农业农村部特色杂粮遗传改良与利用重点实验室(省部共建)/河北省杂粮研究实验室, 河北石家庄 050035
    2河北师范大学生命科学学院, 河北石家庄 050024
  • 收稿日期:2022-05-05 接受日期:2022-07-21 出版日期:2023-06-12 网络出版日期:2022-08-09
  • 通讯作者: *李志勇, E-mail: lizhiyongds@126.com;白辉, E-mail: baihui_mbb@126.com
  • 作者简介:刘佳, E-mail: 15031210252@126.com;
    邹晓悦, E-mail: z_xiaoyue@126.com第一联系人：**同等贡献
  • 基金资助:
    国家重点研发计划项目(2018YFD1000703);国家重点研发计划项目(2018YFD1000700);国家自然科学基金项目(31872880);河北省农林科学院基本科研业务费包干制项目(HBNKY-BGZ-02);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-06-14.5-A25);河北省重点研发计划项目(21326338D)

Genome-wide identification and characterization of MAPK genes and their response to biotic stresses in foxtail millet

LIU Jia1,**(), ZOU Xiao-Yue1,2,**(), MA Ji-Fang1, WANG Yong-Fang1, DONG Zhi-Ping1, LI Zhi-Yong1,*(), BAI Hui1,*()   

  1. 1Institute of Millet Crops, Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Genetic Improvement and Utilization for Featured Coarse Cereals (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs/Key Laboratory of Minor Cereal Crops of Hebei Province, Shijiazhuang 050035, Hebei, China
    2College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, Hebei, China
  • Received:2022-05-05 Accepted:2022-07-21 Published:2023-06-12 Published online:2022-08-09
  • Contact: *E-mail: lizhiyongds@126.com;E-mail: baihui_mbb@126.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Key Research & Development Program of China(2018YFD1000703);National Key Research & Development Program of China(2018YFD1000700);Natural Science Foundation of China(31872880);HAAFS Basic Science and Technology Contract Project(HBNKY-BGZ-02);China Agriculture Research System of MOF and MARA(CARS-06-14.5-A25);Hebei Key Research & Development Program(21326338D)

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摘要：

MAPK在植物的生长发育调节、生物和非生物胁迫反应、激素信号转导中具有重要作用。系统分析谷子SiMPKs基因家族成员全基因组分布、结构、进化及其响应不同胁迫的表达特性, 对于阐明其生物学功能具有重要意义。本研究利用谷子和水稻MAPK蛋白保守结构域及特异TXY基序的氨基酸序列在全基因组水平鉴定了谷子SiMPKs家族成员, 分析其蛋白质理化性质、系统进化、染色体定位、基因结构、蛋白质保守基序、启动子顺式作用元件及共线性等。利用荧光定量PCR技术, 分析了谷子SiMPKs在不同组织部位和谷锈菌、玉米螟病虫害生物胁迫以及不同激素处理下的表达模式。结果显示, 共鉴定到15个谷子SiMPK成员, 其编码的蛋白质含有220~611个氨基酸, 相对分子量范围25.77~69.63 kD, 等电点范围5.46~9.34。系统进化分析表明, SiMPK基因分为4组, A、B、C组包含TEY基序, D组包含TDY基序。SiMPK基因分布在1号、3号、4号、5号、8号和9号染色体上, 含有3~11个外显子, 所有SiMPK蛋白均含有motif 1与motif 2。上游2000 bp启动子区域预测到多个与胁迫、激素和植物生长发育等相关的顺式作用元件。qRT-PCR结果表明, 大部分基因具有明显的组织表达特异性; 除SiMPK21-2和SiMPK6外, 其余成员对谷锈菌侵染、玉米螟取食、SA和MeJA激素处理等1~3种胁迫具有明显响应。以上结果为进一步研究SiMPK基因在谷子应对病虫害生物胁迫中的功能奠定了理论基础。

关键词: 谷子, MAPK家族, 谷锈菌, 玉米螟, 表达分析

Abstract:

MAPK plays an important role in plant growth and development regulation, biotic and abiotic stress responses, and hormone signal transduction. In order to elucidate the biological function of the SiMPK genes in foxtail millet, we identified the SiMPK family members in the genome and analyzed the distribution, structure, evolution, and its expression characteristics in response to different stresses. In this study, the SiMPK gene family members were identified in the genome-wide level using the amino acid sequences of conserved domains and specific TXY motifs of MAPK proteins between foxtail millet and rice. The protein physicochemical property, phylogenetic evolution, chromosome distribution, gene structure, protein conserved motif, promoter cis-acting regulatory elements, and collinearity were analyzed. The relative expression patterns of SiMPK genes in the different tissue, under the biotic stresses of Uromyces setariae-italicae Yoshino and Ostrinia furnacalis and with different hormone treatments were analyzed by qRT-PCR. The results showed that a total of 15 SiMPK genes were identified, and the encoded proteins contained 220-611 amino acids, the relative molecular weight ranged from 25.77 kD to 69.63 kD, and the isoelectric point ranged from 5.46 to 9.34. Phylogenetic analysis showed that SiMPK genes were divided into four groups. Group A, B, and C contained TEY motifs, and group D contained TDY motifs. SiMPK genes were distributed on chromosomes 1, 3, 4, 5, 8, and 9, and contained 3-11 exons. All SiMPK proteins contained motif 1 and motif 2. A number of cis-acting elements related to stress, hormones and plant growth and development were predicted in the promoter regions of the SiMPK genes. Most genes had obvious tissue expression specificity. Except for SiMPK21-2 and SiMPK6, the other members had obvious responses to 1 to 3 kinds of stresses, such as Uromyces setariae-italicae Yoshino infection, Ostrinia furnacalis feeding, and SA and MeJA treatments. The results laid a theoretical foundation for further research on the function of SiMPK genes in the biotic stresses of disease and pest in foxtail millet.

Key words: foxtail millet (Setaria italica), MAPK gene family, Uromyces setariae-italicae Yoshino, Ostrinia furnacalis, relative expression analysis

表1

引物序列表"

引物
Primer		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
SiActin_F CGCATATGTGGCTCTTGACT GGGCACCTAAATCTCTCTGC
SiMPK3 AGCGACATGATGACGGAGTA TAGTCGGTGGAGTTGAGCAG
SiMPK4-1 GTGCAGTCGATTTGTTGGAG ATGCAGAGCCTCATCAACTG
SiMPK4-2 CAGCACGACCAGAAGAAGAA TATGGGTGTCCAGAGCAGAG
SiMPK4-3 AATCAGAGATGATGCACGGA TAACGCTCTGCAGAACCAAC
SiMPK6 ATGAGCCTGTCTGCTCAATG TTGTTCTTCGGACAGTGCAT
SiMPK7 TGTACCCACAAGCACATCCT CAGAGCCTCGGTGACACTAA
SiMPK14 TGTGCCTATCAAGCCCATAG CTTAGTGCATCCACACGGTT
SiMPK17-1 ACGGATTATGTGGCAACAAG TCAGCAAATATGCACCCAAT
SiMPK17-2 CTTCCAGATGCGCTATCAAA CACCCTGGGTACCTTGTCTAA
SiMPK20-1 TTGCAGGCTGAAAGGTACAC TCATGTCCAGAGCAACATCA
SiMPK20-2 TTCGGTGGGAGATCTGGTG TCTGTCGAGTGTGCAGATCA
SiMPK20-4 CCAAGTCATCAAGGCAAATG TTCTTTGGCTTCAAATCACG
SiMPK20-5 GTCGCCGATCAACACATTAC CCGGTCTTTCTCAAGCTCTC
SiMPK21-1 GAAAGCATCAGTGCTTCCAA AGCGATCTTCTTCGACACCT
SiMPK21-2 GACCCAATGGCTCTTCATTT GTGTTATAGGCTCGCGTTCA

表2

谷子SiMPK家族成员特征"

基因名称
Gene name		 基因编号
Gene ID		 氨基酸数目
Number of amino acids (aa)		 分子量
Molecular weight (kD)		 等电点
Isoelectric point (pI)		 亚细胞定位
Subcellular
location		 信号肽
Signal
peptide
SiMPK3 Seita.9G444100 375 42.43 5.46 C 无 No
SiMPK4-1 Seita.9G344000 372 42.29 5.96 C 无 No
SiMPK4-2 Seita.3G058000 557 63.46 8.83 N 无 No
SiMPK4-3 Seita.8G104500 535 61.17 8.79 C 无 No
SiMPK6 Seita.4G069900 347 39.13 5.71 C 无 No
SiMPK7 Seita.4G243400 369 42.34 6.63 N 无 No
SiMPK14 Seita.1G089400 370 42.41 6.53 N 无 No
SiMPK17-1 Seita.4G273900 574 65.25 6.64 N 无 No
SiMPK17-2 Seita.1G095300 506 57.85 7.67 N 无 No
SiMPK20-1 Seita.5G235700 611 69.63 9.14 N 无 No
SiMPK20-2 Seita.3G131800 220 25.77 9.17 C 无 No
SiMPK20-4 Seita.5G261300 589 67.37 9.34 N 无 No
SiMPK20-5 Seita.3G145200 591 67.26 9.20 N 无 No
SiMPK21-1 Seita.3G136100 590 66.86 6.74 C 无 No
SiMPK21-2 Seita.5G241700 508 57.77 7.64 C 无 No

图1

谷子、水稻和拟南芥中MAPK基因家族系统发育进化树 Si: 谷子; Os: 水稻; At: 拟南芥。"

图2

SiMPK基因在谷子染色体上的分布"

图3

谷子SiMPK家族成员保守基序和基因结构 Motif: 保守基序; UTR: 非翻译区; CDS: 编码区序列。"

图4

谷子MAPK保守基序的序列Logo"

图5

谷子15个SiMPK基因启动子中顺式作用元件"

图6

谷子MAPK基因家族共线性分析"

图7

谷子与水稻MAPK基因的共线性分析"

图8

SiMPK家族基因在谷子不同组织部位中的表达分析 SD: 苗期; BT: 孕穗期; shoot: 苗期地上部; root: 苗期地下部或孕穗期根。不同字母表示差异水平显著(P < 0.05)。"

图9

谷子SiMPK基因在谷锈菌侵染的谷子叶片中的表达分析 * 代表基因在同一时间点的抗病反应与感病反应中的相对表达量在0.05概率水平差异显著。"

图10

谷子SiMPK基因在玉米螟取食的谷子叶片中的表达分析 * 代表基因在玉米螟取食后不同时间点的叶片中的相对表达量与0 h相比在0.05概率水平差异显著。** 代表在0.01概率水平差异显著, *** 代表在0.001概率水平差异显著。"

图11

SiMPK基因在激素SA和MeJA处理的谷子叶片中的表达分析 * 代表基因在SA/JA处理谷子叶片后不同时间点的相对表达量与0 h相比在0.05概率水平差异显著, ** 代表在0.01概率水平差异显著, *** 代表在0.001概率水平差异显著。"

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