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作物学报 ›› 2026, Vol. 52 ›› Issue (2): 421-432.doi: 10.3724/SP.J.1006.2026.51068

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大麦亮氨酸富集重复型类受体激酶基因HvLRR-RLK-510的克隆和表达分析

余开航(), 周洪斌(), 罗亮扎, 王玫郦, 姜瑞梅, 董陈文华, 李仕金, 毛孝强, 陈升位()   

  1. 云南农业大学农学与生物技术学院, 云南昆明 650201
  • 收稿日期:2025-07-21 接受日期:2025-10-30 出版日期:2026-02-12 网络出版日期:2025-11-13
  • 通讯作者: *陈升位 E-mail:ynkmcsw@ynau.edu.cn
  • 作者简介:余开航, E-mail: ynau_ykh@163.com;周洪斌, E-mail: 2021210133@stu.ynau.edu.cn

    **同等贡献

  • 基金资助:
    国家自然科学基金项目(32360469)

Cloning and expression analysis of the HvLRR-RLK-510 gene encoding a leucine-rich repeat receptor-like kinase in barley

Yu Kai-Hang(), Zhou Hong-Bin(), Luo Liang-Zha, Wang Mei-Li, Jiang Rui-Mei, Dong-Chen Wen-Hua, Li Shi-Jin, Mao Xiao-Qiang, Chen Sheng-Wei()   

  1. College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China
  • Received:2025-07-21 Accepted:2025-10-30 Published:2026-02-12 Published online:2025-11-13
  • Contact: *Chen Sheng-Wei E-mail:ynkmcsw@ynau.edu.cn
  • About author:

    **Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(32360469)

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摘要：

解析亮氨酸富集重复型类受体激酶(leucine rich repeat receptor-like kinase, LRR-RLK)基因的功能和作用机制有利于揭示大麦发育和胁迫响应的分子机制。本研究以北青7号、Ynbs突变体和Morex等8个大麦品种(或品系)为材料, 克隆了HvLRR-RLK-510基因的2904 bp CDS序列。8个材料HvLRR-RLK-510基因的CDS序列和编码蛋白氨基酸序列的相似性均高于99.00%。该蛋白具有亮氨酸富集重复结构域、跨膜结构域和激酶结构域等LRR-RLK的关键结构域, 与小麦、水稻和拟南芥等作物LRR-RLK聚为1个亚类。RT-PCR检测和荧光定量检测结果表明, 该基因在8个材料拔节期幼穗、叶鞘、叶、根和茎中差异表达, 具有器官特异性和基因型依赖性, 在幼穗中的表达更高效、基因型依赖性更低; 亚细胞定位结果表明, 编码蛋白位于本氏烟草叶细胞的细胞膜和液泡膜上。本研究为全面解析大麦HvLRR-RLK-510基因的功能和作用机制提供了基因资源和理论支持。

关键词: 大麦, 亮氨酸富集重复型类受体激酶, 基因克隆, 亚细胞定位, 表达特性

Abstract:

Elucidating the function and regulatory mechanisms of LRR-RLK (leucine-rich repeat receptor-like kinase) genes is essential for understanding the developmental processes and stress responses in barley. In this study, eight barley varieties or lines, including Beiqing 7, the Ynbs mutant, and Morex, were used as experimental materials. A 2904 bp coding sequence (CDS) of the HvLRR-RLK-510 gene was successfully cloned. The CDS and corresponding amino acid sequences of HvLRR-RLK-510 all showed over 99.00% similarity, across the eight genotypes. The encoded protein contains key conserved domains typical of LRR-RLKs, including leucine-rich repeat domains, transmembrane regions, and a kinase domain. Phylogenetic analysis grouped this protein into a subclass shared with LRR-RLKs from wheat, rice, and Arabidopsis. RT-PCR and quantitative real-time PCR analyses revealed that HvLRR-RLK-510 was differentially expressed in young spikes, leaf sheaths, leaves, roots, and stems of the above-mentioned eight materials at the jointing stage, exhibiting both organ specificity and genotype dependence. Notably, expression in young spikes was consistently higher and less affected by genotype. Subcellular localization experiments showed that the protein is localized to the plasma membrane and vacuolar membrane in Nicotiana benthamiana leaf cells. This study provides a valuable gene resource and theoretical foundation for further investigation into the functional roles and molecular mechanisms of HvLRR-RLK-510 in barley.

Key words: barley, LRR-RLK, gene clone, subcellular localization, expression characteristics

表1

所使用引物的编号、序列和用途"

引物编号
Primer number		 引物序列
Primer sequence (5′-3′)		 引物用途
Primer usage
HvLRR-RLK510-F ATGACTCCCCTGCTCCCCCTCC 基因克隆Gene clone
HvLRR-RLK510-R TCACGCACCAAGAGGTTCCTTCTC
qHvLRR-RLK510-F GCGTCACTCGCTGATTTGTC RT-PCR和qRT-PCR检测
RT-PCR and qRT-PCR detection
qHvLRR-RLK510-R AAGGTCGAGCTGTGTAAGCC
HvActin-F GGTCCATCCTAGCCTCACTC RT-PCR和qRT-PCR检测
RT-PCR and qRT-PCR detection
HvActin-R GATAACAGCAGTGGAGCGCT
Hg-F GCTCCACCATGTTGGCAAGC 农杆菌阳性克隆鉴定
Identification of agrobacterium positive clone
HgHvLRR-RLK510-R GCCTTTCCGAAACAGAAAAATTTGCTT
M13-F GAGGAAACAGCTATGAC 大肠杆菌阳性克隆鉴定
Identification of agrobacterium positive clone
M13-R ACTGGCCGTCGTTTTAC
BS-F TTCATTTGGAGAGAACACGGGGGAC 亚细胞定位 Subcellular localization
BS-HvLRR-RLK510-R CGGTATCTGGCCGACGAG

图1

8个大麦材料HvLRR-RLK-510基因CDS序列的碱基突变"

图2

8个大麦材料HvLRR-RLK-510基因编码蛋白的氨基酸序列突变"

图3

HvLRR-RLK-510基因编码蛋白的保守结构域(A)和三级结构(B)"

图4

38个亮氨酸富集重复类受体蛋白激酶的系统发生关系和保守结构域"

图5

4个大麦材料不同器官中HvLRR-RLK-510基因的差异表达 A: HvLRR-RLK-510基因; B: 内参基因HvActin。M: marker; 1: 叶; 2: 茎; 3: 穗; 4: 根; 5: 叶鞘。I: 北青7号; II: Golden promise; III: Morex; IV: Ynbs。"

图6

4个大麦材料不同器官中HvLRR-RLK-510基因的表达差异 小写字母表示5%的显著性, 大写字母表示1%的显著性。"

图7

4个大麦材料相同器官中HvLRR-RLK-510基因的表达差异 小写字母表示5%的显著性, 大写字母表示1%的显著性。"

图8

HvLRR-RLK-510基因编码蛋白的亚细胞共定位 A: 转化空白载体pBWA(V)BS-GFP的对照细胞; B~D: 转化pBWA(V)BS-HvLRRPK-GFP载体的细胞, 即3个独立的生物学重复视图。标尺为20 μm。"

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