Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (11): 318-327.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0551
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YOU Chui-huai1(), XIE Jin-jin1, ZHANG Ting1, CUI Tian-zhen2, SUN Xin-lu2, ZANG Shou-jian2, WU Yi-ning1, SUN Meng-yao2, QUE You-xiong2, SU Ya-chun2()
Received:
2023-06-12
Online:
2023-11-26
Published:
2023-12-20
Contact:
SU Ya-chun
E-mail:you123chui@163.com;syc2009mail@163.com
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.
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 | |
GeLOX1-G-F | GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGGACTCCTGGGGCCTT | Gateway入门载体构建 Construction of gateway entry vector |
GeLOX1-G-R | GGGGACCACTTTGTACAAGAAAGCTGGGTCGATGGAGACACTGTTAGGAA | |
GeLOX1-Q1F | AAATCTACGCCAGCCGAACTAT | RT-qPCR |
GeLOX1-Q1R | GCCAAACTGAGCCTTCAATACCA | |
GeCUL-Q1F | CAAATTGGGCAGAGGCCACC | |
GeCUL-Q1R | TCTGGGGCTGGCTGTAGAAT |
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 | |
GeLOX1-G-F | GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGGACTCCTGGGGCCTT | Gateway入门载体构建 Construction of gateway entry vector |
GeLOX1-G-R | GGGGACCACTTTGTACAAGAAAGCTGGGTCGATGGAGACACTGTTAGGAA | |
GeLOX1-Q1F | AAATCTACGCCAGCCGAACTAT | RT-qPCR |
GeLOX1-Q1R | GCCAAACTGAGCCTTCAATACCA | |
GeCUL-Q1F | CAAATTGGGCAGAGGCCACC | |
GeCUL-Q1R | TCTGGGGCTGGCTGTAGAAT |
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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