Identification of the Lipoxygenase Gene GeLOX1 and Expression Analysis Under Low Temperature Stress in Gelsmium elegans

doi:10.13560/j.cnki.biotech.bull.1985.2023-0551

Abstract

Abstract:

In recent years, the medicinal and feeding value of Gelsemium elegans has become increasingly prominent. However, G. elegans is not tolerant to low temperatures during its growth process. Exploring its low temperature response genes may lay the foundation for the research of cold resistance breeding of G. elegans. Lipoxygenase（LOX）has a significant impact on the physiological and biochemical processes of plants in seed aging, stress resistance, and other aspects. The LOX genes of G. elegans in response to low temperature stress were mined from the transcriptome database of G. elegans constructed by our team. A full-length cDNA sequence of the GeLOX1 gene was cloned from the leaves of G. elegans using RT-PCR technology, and its bioinformatics, subcellular localization, gene expression, prokaryotic expression, and plate stress were analyzed. The results showed that the amino acid length of the protein encoded by the GeLOX1 was 761 aa, and the relative molecular weight of the protein was 87.00 kD. It was predicted to be an unstable hydrophilic protein, containing 28 serine phosphorylation sites, 22 threonine phosphorylation sites, and 9 tyrosine phosphorylation sites. The analysis results of evolutionary tree indicated that GeLOX1 belonged to the 9-LOX family. The subcellular localization test results showed that the GeLOX1 was located in the cytoplasm. Real time fluorescence quantitative PCR analysis revealed that the GeLOX1 was highly expressed in the roots of the G. elegans, and its expressions showed a downward trend under 4℃ low temperature stress. After prokaryotic expression induction, the recombinant protein of the GeLOX1 showed a target band at approximately 111 kD, and the accumulation of the recombinant protein reached its peak at 8 h of induction. In addition, the plate stress experiment showed that the prokaryotic expression strain of the GeLOX1 was more sensitive to low temperature stress compared to the control group. The research results indicate that GeLOX1 of G. elegans can respond to low temperature stress.

Key words: Gelsemium elegans, lipoxygenase（LOX）, sequence analysis, low temperature stress, expression analysis

YOU Chui-huai, XIE Jin-jin, ZHANG Ting, CUI Tian-zhen, SUN Xin-lu, ZANG Shou-jian, WU Yi-ning, SUN Meng-yao, QUE You-xiong, SU Ya-chun. Identification of the Lipoxygenase Gene GeLOX1 and Expression Analysis Under Low Temperature Stress in Gelsmium elegans[J]. Biotechnology Bulletin, 2023, 39(11): 318-327.

Figures/Tables 7

Table 1 Primer sequence and usage

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Usage
GeLOX1-F	ACCAAGAGGGAAACTAGGAAA	基因克隆Gene cloning
GeLOX1-R	CAAACATCTGAATAAACGAGTGA	基因克隆Gene cloning
GeLOX1-G-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGGACTCCTGGGGCCTT	Gateway入门载体构建 Construction of gateway entry vector
GeLOX1-G-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCGATGGAGACACTGTTAGGAA	Gateway入门载体构建 Construction of gateway entry vector
GeLOX1-Q1F	AAATCTACGCCAGCCGAACTAT	RT-qPCR
GeLOX1-Q1R	GCCAAACTGAGCCTTCAATACCA
GeCUL-Q1F	CAAATTGGGCAGAGGCCACC
GeCUL-Q1R	TCTGGGGCTGGCTGTAGAAT

Fig. 1 PCR amplification of GeLOX1 gene M: DNA marker; 1: PCR amplification products

Fig. 2 Phylogenetic tree analysis of GeLOX1 protein in G. elegans and LOX proteins in other plants OsLOX and r9-LOX: Oryza sativa LOX; ZmLOX: Zea mays LOX; AtLOX: Arabidopsis thaliana LOX; NtLOX: Nicotiana tabacum LOX; CaLOX: Capsicum annuum LOX; TomLOX: Lycopersicon esculentum LOX; ScLOX: Saccharum spp. LOX; StLOX: Solanum tuberosum LOX

Fig. 3 RT-qPCR analysis of GeLOX1 gene in different tissues of G. elegans（A）and under 4℃ low-temperature treatment（B） Different lowercase letters indicate significant differences at P<0.05

Fig. 4 Observations of Agrobacterium-transformed pFAST-R05-GFP empty vector（35S::GFP）and recombinant plasmid pFAST-GeLOX1-GFP（35S::GeLOX1::GFP）at 3 d after the injection into the leaves of Nicotiana benthamiana The epidermal cells of N. benthamiana leaf are used for capturing images of epidermal cells in bright-field, green fluorescence, and merged light. Cholorophy II indicates chlorophyll autofluorescence. Scale bar is 50 μm. 35S::GFP: Agrobacterium strains carrying empty vector pFAST-R05-GFP; 35S::GeLOX1::GFP: Agrobacterium strains carrying recombinant vector pFAST-GeLOX1-GFP

Fig. 5 Prokaryotic expressions of GeLOX1 recombinant protein in G. elegans in Escherichia coli BL21（DE3） M: Protein marker. 1: Empty bacteria E. coli BL21（DE3）without induction. 2: Empty bacteria induced for 22 h. 3: Empty vector without induction. 4: Empty vector induction for 22 h. 5: Recombinant bacteria without induction. 6-9: Recombinant bacteria induced for 2, 8, 10 and 22 h. Black arrow: The induced target protein GeLOX1

Fig. 6 Growth status of prokaryotic expression recombinant strain BL21/pEZYHb-GeLOX1 on LB plate under low temperature stress The growth performance of BL21/pEZYHb and BL21/pEZYHb-GeLOX1 cells on LB plates was used as a control. After spotting the plates on LB agar plates, they were placed in dark culture at 4℃ for 3 d, 7 d and 10 d, and then cultured overnight at 37℃ for photography

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