大麦HvERECTA基因的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1077

摘要/Abstract

摘要：

目的跨膜蛋白ERECTA是富含亮氨酸受体样丝氨酸/苏氨酸的蛋白激酶（LRR-RLK），该基因控制细胞增殖生长、调节气孔结构并对各种胁迫作出反应。解析大麦HvERECTA的结构以及功能表达，为其生物学功能鉴定及作物分子育种奠定基础。方法以大麦Morex为材料，克隆大麦HvERECTA，并对其进行生物信息学和基因功能分析。通过RT-qPCR分析大麦HvERECTA在不同器官中和不同逆境下的功能特性，研究模拟干旱胁迫下大麦HvERECTA与大麦气孔发育相关的典型bHLH转录因子表达基因HvSPCH、HvMUTE和HvFAMA的表达模式。结果大麦HvERECTA开放阅读框长2 934 bp，编码977个氨基酸，具有典型的LRR-RLK结构，预测定位在质膜上且含信号肽。系统进化树表明，其属于禾本科的ERECTA亚家族，蛋白互作预测其属于调控气孔发育的关键基因，预测启动子区域内含多个与生长发育及激素调节相关的顺式调控元件。RT-qPCR结果显示，在不同器官中，大麦HvERECTA在时空上呈现出特异性表达特征，其在早期叶片中表达量最高而在根部最低；在ABA、高温、低温、模拟干旱（20% PEG6000）非生物胁迫下，HvERECTA的表达量差异显著；模拟干旱胁迫下，HvERECTA与HvSPCH的表达量表现出相似趋势，与HvMUTE表达量则呈相反趋势。结论克隆获得大麦HvERECTA，它是调控气孔发育的关键基因，在大麦早期叶片中特异性表达，参与ABA信号转导途径，响应高温、低温和干旱等非生物胁迫。

关键词: 大麦, 气孔发育, ERECTA基因, 非生物胁迫, 基因表达分析

Abstract:

Objective Transmembrane protein ERECTA, a protein kinase (LRR-RLK) rich in leucine receptor -like serine/threonine, controls cell proliferation, regulates stomatal structure and responds to various stresses. The aim of this study is to elucidate the structure and functional expression of the HvERECTA gene in barley and lay a foundation for its biological function identification and crop molecular breeding. Method Using barley Morex as material, the HvERECTA gene of barley was cloned and its biological information and gene function were analyzed. RT-qPCR was used to analyze the functional characteristics of barley HvERECTA gene in different organs and under different stresses, and to study the expression patterns of HvERECTA gene in barley and typical bHLH-transcription factors HvSPCH, HvMUTE and HvFAMA related to barley stomatal development. Result The length of open reading frame of barley HvERECTA was 2 934 bp, encoding 977 amino acids, having typical LRR-RLK structure, localization predicted in the plasma membrane and containing signal peptide. The phylogenetic tree showed that it belonged to the ERECTA subfamily of Gramineae, and protein interaction predicted that it was a key gene regulating stomatal development. The results of RT-qPCR showed that the expression of HvERCTA was the highest in early leaves and lowest in the roots, and significantly different in abiotic stress of ABA, high temperature, low temperature and simulated drought (20% PEG6000). Under simulated drought stress, the gene expression of HvERECTA and HvSPCH showed similar trend, but opposite trend with the expression of HvMUTE gene. Conclusion HvERECTA gene is cloned from barley. It is a key gene to regulate the stomatal development. It is expressed in early leaves of barley, participates in ABA signal transduction pathway, and responds the abiotic stresses such as high- or low- temperature and drought.

Key words: Hordeum vulgare, stomatal development, ERECTA gene, abiotic stress, gene expression analysis

韩燚, 侯昌林, 唐露, 孙璐, 谢晓东, 梁晨, 陈小强. 大麦HvERECTA基因的克隆及功能分析[J]. 生物技术通报, 2025, 41(7): 106-116.

HAN Yi, HOU Chang-lin, TANG Lu, SUN Lu, XIE Xiao-dong, LIANG Chen, CHEN Xiao-qiang. Cloning and Preliminary Functional Analysis of HvERECTA Gene in Hordeum vulgare[J]. Biotechnology Bulletin, 2025, 41(7): 106-116.

图/表 11

表1 本研究所用引物以及用途

Table 1 Primers used in this study and their uses

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	Tm （℃）	用途 Purpose
HvERECTA-F	ACGGTGACTCTCCGCAGC	58.8	基因扩增 Gene amplification
HvERECTA-R	CGACTACCCGAATCATCCTCC	60.9	基因扩增 Gene amplification
qHvERECTA-F	TGGCATTGCTAAGAGTTTGTGT	57.9	RT-qPCR
qHvERECTA-R	GATTGCACTCGTTGTCCACG	59.4
qHvSPCH-F	CCGAAGACTTATTCAGCATCC	56.7
qHvSPCH-R	CTCATGCGATATTCCGTCAC	54.7
qHvMUTE-F	TCCTTCGTCCTCAAGATTGG	57.4
qHvMUTE-R	ATATCGCCATGGATGAGTAGTC	56.1
qFAMA-F	TGGCGGAGAACAAGTCGAG	59.2
qFAMA-R	GTGTGGAGGATGGACATGTG	55.8
Actin-F	TGGATCGGAGGGTCCATCCT	63.5

图1 大麦HvERECTA的扩增M： 2K Plus Ⅱ marker； 1‒3： HvERECTA

Fig. 1 Amplification of HvERECTA in barley

图2 序列比对

Fig. 2 Sequence alignment

图3 HvERECTA保守结构域分析A：多重序列比对（黑色虚线为STK_BAK1_like，红色框为ATP结合位点；Hv：大麦；Ta：小麦；Bd：二穗短柄草；Sb：高粱；Zm：玉米；Qs：亚洲稻；Gm：大豆；At：拟南芥）；B：保守结构域分析；C：保守基序预测

Fig. 3 Conservative structure analysis of HvERECTAA: Multiple sequence alignment (STK_BAK1_like by black dashed line, ATP binding site by red box. Hv: Hordeum vulgare; Ta: Triticum aestivum; Bd: Brachypodium distachyon; Sb: Sorghum bicolo; Zm: Zea mays; Qs: Oryza sativa Japonica Group; Gm: Glycine max; At: Arabidopsis thaliana). B: Analysis of conservative domain. C: Prediction of conservative base sequence

图4 HvERECTA蛋白质的二级（A）、三级（B）结构

Fig. 4 Secondary structure (A) and tertiary structure (B) of HvERECTA protein

图5 HvERECTA蛋白互作预测线条颜色表示交互证据的类型，线条的数量表示数据支持的强度

Fig. 5 Prediction of protein interactions of HvERECTALine colors indicate the type of interactive evidence and the number of lines indicates the strength of data support

图6 HvERECTA及其他同源家族基因ERfs的系统发育树蓝色扇形为ERECTA，红色扇形为ERL，红色分支为禾本科植物，蓝色分支为双子叶植物

Fig. 6 Phylogenetic tree of HvERECTA and other homologous gene ERfsThe blue sector is ERECTA, the red sector is ERL, the red branch is grasses, and the blue branch is dicotyledonous

图7 大麦HvERECTA启动子顺式作用元件预测

Fig. 7 Prediction of cis-acting elements of HvERECTA promoter in barley

图8 大麦不同器官HvERECTA的相对表达量C：胚芽鞘；2L：2 d龄叶；4L：4 d龄叶；6L：6 d龄叶；8L：8 d龄叶；S：茎；R：根。小写字母表示显著差异（P<0.05）

Fig. 8 Relative expressions of HvERECTA in different organs of barleyC: Coleoptile; 2L: 2 d leaf age; 4L: 4 d leaf age; 6L: 6 d leaf age; 8L: 8 d leaf age; S: stem; R: root. Lowercase letters for significant differences (P<0.05)

图9 ABA处理和高温、低温胁迫下大麦HvERECTA的表达量A：高温处理；B：低温处理；C：ABA处理。*表示处理组和对照之间基因表达量差异达显著水平（0.01≤P<0.05），**表示处理组和对照之间基因表达量差异达较显著水平（0.001≤P<0.01），***表示处理组和对照之间基因表达量差异达极显著水平（P<0.001）。ns表示处理组和对照之间基因表达量差异无显著水平（P>0.05）。下同

Fig. 9 Expressions of HvERECTA gene in barley treated with ABA and under high temperature, low temperatureA: High temperature treatment. B: Low temperature treatment. C: ABA treatment.* indicates significant difference in gene expression between treatment group and control group (0.01≤P<0.05), ** indicating that the difference in gene expression between the treatment group and the control group was significant (0.001≤P<0.01), *** indicating that the difference in gene expression between the treatment and control groups was extremely significant (P<0.001).nsindicates no significant difference in gene expression between treatment group and control group (P>0.05). The same below

图10 干旱胁迫（20% PEG6000）下气孔发育相关基因的表达量

Fig. 10 Expressions of genes related to stomatal development under drought stress (20% PEG6000)

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