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作物学报 ›› 2022, Vol. 48 ›› Issue (12): 3057-3070.doi: 10.3724/SP.J.1006.2022.11115

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

栽培黑麦光敏色素PHYA、PHYB和PHYC基因转录丰度对不同光质处理的响应

杨陆浩1(), 王立建1,2(), 孙广华1, 王少瓷1, 崔连花1, 陈昌1, 宋梅芳3, 张艳培1, 姜良良1(), 杨建平1(), 王晨阳1()   

  1. 1河南农业大学农学院 / 省部共建小麦玉米作物学国家重点实验室和作物分子育种国家工程中心, 河南郑州 450046
    2河南警察学院, 河南郑州 450046
    3北京市辐照中心, 北京100875
  • 收稿日期:2021-12-22 接受日期:2022-03-25 出版日期:2022-12-12 网络出版日期:2022-04-20
  • 通讯作者: 姜良良,杨建平,王晨阳
  • 作者简介:杨陆浩, E-mail: yangluhao226@163.com;
    王立建, E-mail: wanglijian2012@163.com第一联系人：

    **同等贡献

  • 基金资助:
    2022年拉萨市区域科技协同创新专项(QYXTZX-LS2022-01);国家自然科学基金项目(31871709);北京市自然科学基金(重点)项目(6151002);河南农业大学青年英才项目(30501038);河南农业大学青年英才项目(30500823)

Transcription abundances of PHYA, PHYB, and PHYC genes in response to different light treatments in Secale cereale

YANG Lu-Hao1(), WANG Li-Jian1,2(), SUN Guang-Hua1, WANG Shao-Ci1, CUI Lian-Hua1, CHEN Chang1, SONG Mei-Fang3, ZHANG Yan-Pei1, JIANG Liang-Liang1(), YANG Jian-Ping1(), WANG Chen-Yang1()   

  1. 1College of Agronomy / State Key Laboratory of Wheat and Maize Crop Science, and Center for Crop Genome Engineering, Henan Agricultural University, Zhengzhou 450046, Henan, China
    2Henan Police College, Zhengzhou 450046, Henan, China
    3Beijing Radiation Center, Beijing Academy of Science and Technology, Beijing 100875, China
  • Received:2021-12-22 Accepted:2022-03-25 Published:2022-12-12 Published online:2022-04-20
  • Contact: JIANG Liang-Liang,YANG Jian-Ping,WANG Chen-Yang
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    Lhasa Regional Science and Technology Collaborative Innovation Project in 2022(QYXTZX-LS2022-01);National Natural Science Foundation of China(31871709);Key Project of Beijing Natural Science Foundation(6151002);Young Talents Project of Henan Agricultural University(30501038);Young Talents Project of Henan Agricultural University(30500823)

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摘要：

栽培黑麦(Secale cereal L.)属于禾本科黑麦属, 是一种有价值的粮食和饲料作物, 对生物和非生物胁迫具有很高的抗性。黑麦1RS染色体臂携带的抗病基因通过染色体易位转入小麦基因组, 1BL/1RS易位系在世界范围内提高了小麦对白粉病和条锈病抗性。光敏色素是一类红光/远红光受体, 调控植物的种子萌发、株高、避荫性反应和开花等适应性反应。本试验克隆了黑麦光敏色素基因(phytochrome) ScPHYA、ScPHYB和ScPHYC, 利用荧光定量PCR (qRT-PCR)方法分析ScPHYs在不同组织中的转录丰度, 及其在幼苗期对不同光质和光周期处理的响应。研究发现, 黑麦与拟南芥、水稻及普通小麦等植物的phys蛋白具有相似的结构域, 且与小麦、水稻等禾本科植物的phys蛋白有较高的氨基酸序列一致性, 暗示它们可能具有相似的功能。ScPHYs在根中的表达水平最高, 并在黑暗和远红光条件下的转录丰度较高; 由黑暗转红光、远红光、蓝光和白光条件下三者的表达模式略有不同。在光周期反应中, 长日照条件下三者的表达模式相似, 短日照条件下三者的表达模式各不相同。以上结果表明, ScPHYs可能在黑麦光形态建成中发挥重要功能。本文通过对3个黑麦光敏色素基因的转录分析, 为研究黑麦的光信号系统提供参考, 同时也为进一步探究光敏色素在黑麦遗传改良及分子育种中的应用提供依据。

关键词: 黑麦, 光敏色素, 基因克隆, 光处理, 转录丰度

Abstract:

Cultivated rye (Secale cereal L.), belonging to the rye genus of the poaceae family, is a valuable food and feed crop that is highly resistant to biotic and abiotic stresses. The 1BL/1RS translocation line has improved wheat resistance to powdery mildew and stripe rust worldwide. Phytochromes are an red/far-red receptors that regulate seed germination, plant height, shade avoidance response, flowering, and other adaptive responses in plants. In this study, three phytochrome genes, ScPHYA, ScPHYB, and ScPHYC were cloned. The transcriptional abundance of ScPHYs in different tissues and light treatments was analyzed by qRT-PCR. The results showed that phys of rye had similar domains with those of Arabidopsis, rice, and wheat, and had higher amino acid sequence consistency with those of wheat and rice, suggesting that they might have similar function. The transcriptional abundances of ScPHYs were the highest in roots, and higher under dark and far-red light than other continuous light conditions, while the relative expression patterns of the three genes were slightly different in the transformation from dark to red light, far-red light, blue light, and white light. In photoperiodic response, the relative expression patterns of the three genes were similar under long-day conditions, but different under short-day conditions. These results indicated that ScPHYs may play an important role in the photomorphogenesis of rye. The transcriptional analysis of three rye phytochrome genes provides a reference for the study of rye optical signal system and a basis for exploring the application of phytochrome in rye genetic improvement and molecular breeding.

Key words: Secale cereale, phytochromes, gene cloning, light treatment, transcription abundance

表1

基因克隆所用引物"

基因名称
Gene name		 基因编号
Accession number		 正向引物序列
Forward sequence (5'-3')		 反向引物序列
Reverse sequence (5'-3')
ScPHYA ScWN4R01G009900 CGGGGGACTCTTGACCATGGCA ATGTCTTCCTCAATGCCTGCTT AGTTCTTCTCCTTTACTAGT GTGCCCCATTGCCGTT
ScPHYB ScWN7R01G236700 CGGGGGACTCTTGACCATGGCA ATGGCCTCGGGAAGCCG AGTTCTTCTCCTTTACTAGT GCTCCGATCCCTACTTTCT
ScPHYC ScWN4R01G173600 CGGGGGACTCTTGACCATGGCA
ATGTCGTCGTCGCGGTCC		 AGTTCTTCTCCTTTACTAGT
GAAGTTGCTCTTGCTCGTC

表2

qRT-PCR所用引物"

基因名称
Gene name		 基因编号
Accession number		 正向引物序列
Forward sequence (5'-3')		 反向引物序列
Reverse sequence (5'-3')
ScPHYA ScWN4R01G009900 CAGTGAAGTTCTCTCCTGTTG TCCCTGGTGCTTGATCC
ScPHYB ScWN7R01G236700 GCTATGGGCAAATCATTAG TGGTCCTTTAGACTGCTCTGAC
ScPHYC ScWN4R01G173600 GCAAGGAACCGAAGAGCAA CCTGTCAAATCTTGTGCTACG
ScActin ScWN1R01G374800 CGTGTTGGATTCTGGTGATG AGCCACATATGCGAGCTTCT

图1

光敏色素蛋白的系统发育分析 在Uniprot网站和WheatOmics网站获得全长氨基酸序列, 利用 MEGA Version 10对其进行系统发育分析。AtphyA: 拟南芥, P14712; AtphyB: 拟南芥, P14713; AtphyC: 拟南芥, P14714; OsphyA: 水稻, A2XLG5; OsphyB: 水稻, A2XFW2; OsphyC: 水稻, A2XM23; ZmphyA1: 玉米, P19862; ZmphyA2: 玉米, Q53ZT7; ZmphyB1: 玉米, Q6XFQ3; ZmphyB2: 玉米, Q6XFQ2; ZmphyC1: 玉米, Q6XFQ1; ZmphyC2: 玉米, Q6XFQ0; SbphyA: 高粱, Q53YS9; SbphyB: 高粱, Q6S525; SbphyC: 高粱, P93528; TaphyA: 小麦, Q5K5K6; TaphyB: 小麦, A9JR06; TaphyC: 小麦, Q8VWN1; ScphyA: 黑麦, ScWN4R01G009900; ScphyB: 黑麦, ScWN7R01G236700; ScphyC: 黑麦, ScWN4R01G173600; HvphyA: 大麦, Q2I7M3; HvphyB: 大麦, Q2I7M0; HvphyC: 大麦, Q2I714; AetphyA: 粗山羊草, A0A453H8S4; AetphyB: 粗山羊草, A0A453I867; AetphyC: 粗山羊草, A0A453LU83; TuphyA: 乌拉尔图小麦, M8B484; TuphyB: 乌拉尔图小麦, TuG1812G0400002191; TuphyC: 乌拉尔图小麦, M8A187。"

图2

黑麦、拟南芥、水稻和小麦phyA蛋白的氨基酸序列比对和结构域分析 使用DNAMAN version 9、Pfam网站和WheatOmics网站进行氨基酸序列多重比对及蛋白结构域分析。AtphyA: 拟南芥, P14712; OsphyA: 水稻, A2XLG5; TaphyA: 小麦, Q5K5K6; ScphyA: 黑麦, ScWN4R01G009900。"

图3

黑麦、拟南芥、水稻和小麦phyB蛋白的氨基酸序列比对和结构域分析 使用DNAMAN version 9、Pfam网站和WheatOmics网站进行氨基酸序列多重比对及蛋白结构域分析。AtphyB: 拟南芥, P14713; OsphyB: 水稻, A2XFW2; TaphyB: 小麦, A9JR06; ScphyB: 黑麦, ScWN7R01G236700。"

图4

黑麦、拟南芥、水稻和小麦phyC蛋白的氨基酸序列比对和结构域分析 使用DNAMAN version 9、Pfam网站和WheatOmics网站进行氨基酸序列多重比对及蛋白结构域分析。AtphyC: 拟南芥, P14714; OsphyC: 水稻, A2XM23; TaphyC: 小麦, Q8VWN1; ScphyC: 黑麦, ScWN4R01G173600。"

图5

ScPHYA、ScPHYB及ScPHYC基因在不同部位中的转录丰度分析 以威宁黑麦不同组织为材料, qRT-PCR分析ScPHYA、ScPHYB及ScPHYC的转录丰度。以叶片中ScPHYB转录丰度作为对照(ScPHYB/ScActin设为1), 柱状图表示进行3次独立重复试验后的平均值, 其中, 误差线代表标准差。*: P < 0.05; **: P < 0.01; ***: P < 0.001。"

图6

ScPHYA、ScPHYB及ScPHYC在不同持续光下的转录丰度 威宁黑麦在黑暗(Dk)、红光(R, 22.3 μmol m-2 s-1)、远红光(FR, 1.9 μmol m-2 s-1)、蓝光(B, 13.0 μmol m-2 s-1)和白光(W, 17.0 μmol m-2 s-1)培养箱中生7 d。以黑暗中ScPHYB转录丰度为对照(ScPHYB/ScActin设为1)柱状图表示进行3次独立重复试验后的平均值, 其中, 误差线代表标准差。** P < 0.01; *** P < 0.001。"

图7

ScPHYA、ScPHYB及ScPHYC基因在黑暗到不同光质转换的转录丰度 威宁黑麦幼苗在黑暗(Dk)条件生长7 d, 分别转入红光(R, 22.3 μmol m-2 s-1)、远红光(FR, 1.9 μmol m-2 s-1)、蓝光(B, 13.0 μmol m-2 s-1)和白光(W, 17.0 μmol m-2 s-1) 0、0.25、0.5、1、2、4、8、16和24 h的表达模式, 以黑暗条件下ScPHYB转录丰度为对照(ScPHYB/ScActin设为1), 折线图代表3次实验的平均值, 误差线代表标准差。"

图8

光周期处理下ScPHYA、ScPHYB及ScPHYC的转录丰度 威宁黑麦幼苗在长日照(LD, 16 h光照/8 h)和短日照(SD, 8 h光照/16 h黑暗)条件生长10 d后, 每隔2 h取样。分别以每个基因在长日照和短日照黑暗结束时转录丰度作为对照(设为1), 折线图代表3次实验的平均值, 误差线代表标准差。"

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