钩吻脂氧合酶基因 GeLOX1 的鉴定及低温胁迫表达分析

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

摘要/Abstract

摘要：

近年来，钩吻（Gelsemium elegans）的药用和饲用价值日益凸显，但钩吻在生长过程中不耐低温，挖掘其低温响应基因，为钩吻的抗寒育种研究奠定基础。植物中，脂氧合酶（lipoxygenase, LOX）在种子老化、抗逆境胁迫等方面的生理生化过程中有重要影响。基于课题组构建的钩吻转录组数据库，挖掘响应低温胁迫的钩吻LOX基因，运用RT-PCR技术，从中克隆到一条GeLOX1的cNDA全长序列，对其进行生物信息学、亚细胞定位、基因表达、原核表达及平板胁迫等分析。结果显示，GeLOX1所编码蛋白的氨基酸长度为761 aa，蛋白相对分子质量为87.00 kD，预测为不稳定的亲水性蛋白，含有28个丝氨酸磷酸化位点，22个苏氨酸磷酸化位点和9个酪氨酸磷酸化位点。进化树分析结果表明，GeLOX1属于9-LOX家族的成员。亚细胞定位检测结果显示，GeLOX1蛋白定位于细胞质中。实时荧光定量PCR分析发现，GeLOX1在钩吻的根中高表达，且其在4℃低温胁迫下的表达量呈现下调的趋势。经原核表达诱导后，GeLOX1的重组蛋白在约111 kD处出现目标条带，且重组蛋白的积累量在诱导8 h时达到峰值。此外，平板胁迫试验表明，GeLOX1的原核表达菌株相较于对照组对低温胁迫更敏感。钩吻GeLOX1能够应答低温胁迫。

关键词: 钩吻, 脂氧合酶（LOX）, 序列分析, 低温胁迫, 表达分析

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

尤垂淮, 谢津津, 张婷, 崔天真, 孙欣路, 臧守建, 武奕凝, 孙梦瑶, 阙友雄, 苏亚春. 钩吻脂氧合酶基因 GeLOX1 的鉴定及低温胁迫表达分析[J]. 生物技术通报, 2023, 39(11): 318-327.

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.

图/表 7

表1 引物序列及用途

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

图1 GeLOX1的PCR扩增 M：DNA marker；1：PCR产物

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

图2 钩吻GeLOX1蛋白与其他植物的LOX蛋白的系统进化树分析 OsLOX、r9-LOX：水稻LOX；ZmLOX：玉米LOX；AtLOX：拟南芥LOX；NtLOX：烟草LOX；CaLOX：辣椒LOX；TomLOX：番茄LOX；ScLOX：甘蔗LOX；StLOX：马铃薯LOX

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

图3 GeLOX1在钩吻不同组织（A）和4℃条件下（B）的RT-qPCR分析不同小写字母表示在P<0.05水平差异显著

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

图4 农杆菌转化的pFAST-R05-GFP空载（35S::GFP）和重组质粒pFAST-GeLOX1-GFP（35S::GeLOX1::GFP）注射本氏烟叶片3 d后的结果本氏烟叶片表皮细胞被用于明场、绿色荧光和合并光捕获表皮细胞的图像；CholorophyII表示叶绿素自发荧光；比例尺为50 μm。35S::GFP：携带空载pFAST-R05-GFP的农杆菌菌株；35S::GeLOX1::GFP：携带重组载体pFAST-GeLOX1-GFP的农杆菌菌株

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

图5 钩吻GeLOX1重组蛋白在大肠杆菌BL21（DE3）中的原核表达分析 M：蛋白marker；1：不经诱导的空菌E. coli BL21（DE3）；2：空菌诱导22 h；3：不经诱导的空载；4：空载诱导22 h；5：不经诱导的重组菌；6-9：重组菌诱导2、8、10和22 h；黑色箭头：被诱导的目标蛋白GeLOX1

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

图6 低温胁迫下原核表达重组菌BL21/pEZYHb-GeLOX1在LB板上的生长状况以BL21/pEZYHb和BL21/pEZYHb-GeLOX1细胞在LB板上的生长性能作为对照，在LB琼脂平板上点板后，置于4℃黑暗培养3、7和10 d，再于37℃过夜培养拍照

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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