Cloning and Preliminary Functional Analysis of HvERECTA Gene in Hordeum vulgare

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

Abstract

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

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.

Figures/Tables 11

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

Fig. 1 Amplification of HvERECTA in barley

Fig. 2 Sequence alignment

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

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

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

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

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

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)

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

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

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