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作物学报 ›› 2020, Vol. 46 ›› Issue (5): 680-689.doi: 10.3724/SP.J.1006.2020.94096

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

对马铃薯类受体激酶CRK基因家族的鉴定及响应病原真菌信号的表达分析

张卫娜,范艳玲,康益晨,杨昕宇,石铭福,要凯,赵章平,张俊莲,秦舒浩()   

  1. 甘肃农业大学园艺学院, 甘肃兰州 730070
  • 收稿日期:2019-06-28 接受日期:2019-12-26 出版日期:2020-05-12 网络出版日期:2019-10-11
  • 通讯作者: 秦舒浩
  • 作者简介:E-mail:844741204@qq.com
  • 基金资助:
    本研究由甘肃农业大学园艺学科建设基金项目(GAU-XKJS-2018-225);国家自然科学基金项目(31260311);甘肃省自然科学基金项目(1606RJZA034);中国博士后科学基金项目(2012M512042);中国博士后科学基金项目(2014T70942);国家现代农业产业技术体系(马铃薯)建设专项资助(CARS-09-P14)

Genome wide identification and expression analysis of CRK gene family in response to fungal pathogen signals in potato

Wei-Na ZHANG,Yan-Ling FAN,Yi-Chen KANG,Xin-Yu YANG,Ming-Fu SHI,Kai YAO,Zhang-Ping ZHAO,Jun-Lian ZHANG,Shu-Hao QIN()   

  1. College of Horticulture, Gansu Agricultural University, Lanzhou 730070, Gansu, China
  • Received:2019-06-28 Accepted:2019-12-26 Published:2020-05-12 Published online:2019-10-11
  • Contact: Shu-Hao QIN
  • Supported by:
    This study was supported by the Discipline Construction Fund Project of Gansu Agricultural University(GAU-XKJS-2018-225);the National Natural Science Foundation of China(31260311);the Natural Science Foundation of Gansu Province(1606RJZA034);the China Postdoctoral Science Foundation(2012M512042);the China Postdoctoral Science Foundation(2014T70942);the China Agriculture Research System (Potato)(CARS-09-P14)

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摘要：

富含半胱氨酸的类受体激酶(cysteine-rich receptor-like kinase, CRK)在植物生长发育和环境适应过程中发挥重要的作用。本研究鉴定了马铃薯CRK (StCRK)家族成员, 并对其理化性状、进化特征、亚细胞定位、染色体位置和表达模式进行分析。鉴定获得8个StCRKs, 其氨基酸序列大小为459~686 aa, 分子量介于50.75~77.50 kD, 等电点介于5.84~8.75, 主要位于质膜。进化分析将来自马铃薯、拟南芥、香蕉、苹果、水稻、番茄和棉花的CRKs分为9个亚组, 2号、3号和5号染色体上的StCRKs分布于亚组I (6个成员)和VI (2个成员); 存在2个串联重复基因簇, 包含4个成员。StCRKs启动子区域存在多种顺式调控元件, 主要响应激素、低温、防卫和逆境等信号。接种晚疫病菌(Phytophthora infestans, Pi)和干腐病菌(Fusarium sulphureum, Fs)后, 分别发现8个和6个StCRKs为差异表达。其中, StCRK4和StCRK8响应Pi和Fs信号, 在接种以上2种病原菌后, 表达量上调8倍以上, 推测其响应多个真菌信号, 可能在马铃薯对真菌病害的广谱抗性中起重要作用, 可作为进一步抗病研究和功能分析的候选基因。

关键词: CRK, 顺式表达元件, 生物信息学分析, 硫色镰刀菌, 马铃薯晚疫病菌

Abstract:

Cysteine-rich receptor-like kinase (CRK) plays an important role in plant growth and environmental adaptation. In this study, potato CRK (StCRK) family members were identified, and their physical and chemical characteristics, evolutionary characteristics, subcellular location, chromosome location and expression patterns were analyzed. Eight StCRK members were identified, with amino acid size from 459 to 686 aa, molecular weight of 50.75-77.50 kD, and isoelectric point of 5.84-8.75. StCRKs were mainly located on plasma membrane. CRKs from potato (Solanum tuberosum), apple (Malus pumila Mill.), Thale Cress (Arabidopsis thaliana), rice (Oryza sativa), cotton (G. hirsutum), banana (Musa acuminata), and tomato (Solanum lycopersicum) could be divided into nine subgroups, and StCRKs were belonged to subgroups I (6 members) and VI (2 members). Moreover, StCRKs distributed on chromosomes 2, 3, and 5, contained two tandem repeat gene clusters, including four members. There were many cis-regulated elements in the StCRKs promoter region, which mainly respond to hormones, low temperature, defense and stress signals. After inoculating Phytophthora infestans (Pi) and Fusarium sulphureum (Fs), eight and six StCRKs gene differentially expressed, among them, StCRK4 and StCRK8 had the expression levels by more than eight times. It is speculated that they may respond to multiple fungal signals, and play an important role in potato's broad-spectrum resistance to fungal diseases, and can be used as candidate genes for further needed on disease resistance and functional analysis.

Key words: CRK, cis-elements, bioinformatic analysis, Fusarium sulphureum (Fs), Phytophthora infestans (Pi)

表1

马铃薯CRKs引物"

基因登录号
Gene accession number		 上游引物
Forward primer (5′-3′)		 下游引物
Reverse prime (5′-3′)
AB061263 ATTGGAAACGGATATGCTCCA TCCTTACCTGAACGCCTGTCA
PGSC0003DMG402000515 ACTCTGGCTCTCTACTACAAACAGT CTTCCACACCTGAAACCAAAATGAC
PGSC0003DMG400021394 AAGAGTCCCCTTGTATACAGAACCA ATCGAATTCATACAAGAGGTCCAGC
PGSC0003DMG400013525 CAACACAACCACCATTCCTCAATTC AAGATTGTGTAGAGGAGCTTGGTTG
PGSC0003DMG400018101 CTCCTCCAGATACAAGCAGTTCATC TCACCTGATTGAGAGCTAGAAATGC
PGSC0003DMG400006912 TCGTGTTCAAGAGTTATGTCACCAG AATTCCAAGCAAGTCGAAGTCAGAT
PGSC0003DMG400013524 CATATGTAGTACGCAACCTGAGCAT CTTCAGCATAGCATAGGACACAGTC
PGSC0003DMG400015170 TCATGATCAGACTGTTGACTTCGTC AAGCAGTGAATGGAGCAACAAAATC
PGSC0003DMG400015171 TACAAAAGCCAGTGAAGGAGAAGAC TATAATTGCCTGTTTCTTGAGCGGA

表2

马铃薯CRKs"

基因登录号Gene accession number 基因编号
Genetic code		 氨基酸数
AA size		 分子量
Molecular weight (kD)		 等电点
Isoelectric point		 亚细胞定位
Subcellular localization
Gene ID# STWG 1.0b
PGSC0003DMG402000515 PGSC0003DMP400001023/
PGSC0003DMP400001024		 StCRK1 616 67.40 8.53 PMa, Chlob
PGSC0003DMG400021394 PGSC0003DMP400037082 StCRK2 608 67.23 8.07 PMa, Chlob
PGSC0003DMG400013525 PGSC0003DMP400023929 StCRK3 656 72.73 8.75 PMa, Chlob
PGSC0003DMG400018101 PGSC0003DMP400031539 StCRK4 681 75.52 6.37 PMa, Chlob
PGSC0003DMG400006912 PGSC0003DMP400012233/
PGSC0003DMP400012234/
PGSC0003DMP400012235		 StCRK5 668 75.21 5.84 PMa, Vacub
PGSC0003DMG400013524 PGSC0003DMP400023928/
PGSC0003DMP400023926		 StCRK6 656 73.21 6.42 PMa,b
PGSC0003DMG400015170 PGSC0003DMP400026615/
PGSC0003DMP400026616/
PGSC0003DMP400026617		 StCRK7 459 50.75 7.27 PMa, Cytob
PGSC0003DMG400015171 PGSC0003DMP400026620 StCRK8 686 77.50 7.23 PMa, b

图1

马铃薯、拟南芥、香蕉、苹果、水稻、番茄和棉花CRK家族进化树分析"

图2

马铃薯CRKs染色体定位 *代表串联重复基因簇。"

图3

马铃薯CRKs的基因结构和蛋白功能结构域分析 A: 马铃薯CRKs进化树; B: 马铃薯CRKs基因结构; C: 马铃薯CRK蛋白的功能结构域。"

图4

预测马铃薯CRKs启动子中顺式调控元件 A和B: 脱落酸响应元件; C: GARE-motif; D和E: 赤霉素响应元件; F和G: 生长素响应元件; H: 茉莉酸甲酯响应元件; I: 水杨酸响应元件; J: 玉米醇溶蛋白代谢调控元件; K: 厌氧诱导响应元件; L: 缺氧响应元件; M: 防御和压力响应元件; N: 黄酮类代谢响应元件; O: 低温响应元件。"

图5

SA和JA处理后6个马铃薯CRKs的表达情况"

图6

马铃薯CRKs响应晚疫病菌侵染的表达分析"

图7

马铃薯CRKs响应干腐病菌侵染的表达分析"

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