芜菁BrrLOX7基因克隆、表达及生物信息学分析

doi:10.3969/j.issn.1004-1524.2023.04.10

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (4): 831-840.DOI: 10.3969/j.issn.1004-1524.2023.04.10

芜菁BrrLOX7基因克隆、表达及生物信息学分析

燕存尧(), 贾凯, 闫会转(), 高杰

新疆农业大学园艺学院,新疆乌鲁木齐 830052

收稿日期:2021-12-13 出版日期:2023-04-25 发布日期:2023-05-05
作者简介:燕存尧(1997—),男,新疆昌吉人,硕士研究生,主要从事蔬菜栽培与生理研究。E-mail: 1033146390@qq.com
通讯作者: ^*闫会转,E-mail: hzhyan1118@163.com
基金资助:
农业农村部园艺作物生物学与种质创制综合性重点实验室开放课题(IVF201701);新疆维吾尔自治区高校科研计划自然科学项目(XJEDU2017S017);新疆维吾尔自治区园艺学重点学科基金(2016-10758-3)

Cloning, expression and bioinformatics analysis of BrrLOX7 gene in turnip

YAN Cunyao(), JIA Kai, YAN Huizhuan(), GAO Jie

College of Horticulture, Xinjiang Agricultural University, Urumqi 830052, China

Received:2021-12-13 Online:2023-04-25 Published:2023-05-05

摘要/Abstract

摘要：

脂氧合酶(lipoxygenase,EC 1.13.11.12;简称LOX),是一类由非血红素铁组成的脂肪酸双加氧酶,在植物的生长发育和环境胁迫响应中发挥着重要作用,本研究以4片真叶期芜菁幼苗为材料,利用同源克隆技术从芜菁中克隆BrrLOX7基因。利用生物信息学方法分析BrrLOX7基因序列。使用荧光定量PCR技术分析非生物胁迫下BrrLOX7基因表达模式。结果显示,克隆的BrrLOX7基因CDS长度为2 715 bp,编码904个氨基酸,编码的蛋白质分子量为103.10 ku,无跨膜结构和信号肽。亚细胞定位预测显示,该基因编码的蛋白位于叶绿体中,该蛋白与白菜和萝卜的序列同源性和遗传距离最近。不同胁迫下的表达模式显示,BrrLOX7基因参与对4种胁迫(盐胁迫、冷胁迫、热胁迫、干旱胁迫)的响应。研究结果将为BrrLOX7基因的功能研究奠定基础。

关键词: 芜菁, 脂氧合酶, 基因克隆, 生物信息学分析, 表达分析

Abstract:

Lipoxygenase (EC 1.13.11.12; LOX) is a class of fatty acid dioxygenases composed of non-heme iron, which plays an important role in plant growth and development and environmental stress response. In this study, the BrrLOX7 gene was cloned from turnip using homologous cloning techniques. The sequence of BrrLOX7 gene was analyzed by bioinformatics. The expression pattern of BrrLOX7 gene under abiotic stress was analyzed using fluorescence quantitative PCR. The results showed that the CDS length of cloned BrrLOX7 gene was 2 715 bp, it encoded 904 amino acids, protein encoded by BrrLOX7 gene had a relative molecular weight of 103.10 ku and had no transmembrane structure or signal peptide. Subcellular localization prediction showed that the protein encoded by this gene was located in chloroplasts, and the protein had the closest sequence homology and genetic distance to Chinese cabbage and radish. Expression patterns under different stresses showed that the BrrLOX7 gene was involved in response to four stresses (salt stress, cold stress, heat stress and drought stress). The results of this study will provide a basis for functional studies of the BrrLOX7 gene.

Key words: turnip, lipoxygenase, gene cloning, bioinformatics analysis, expression analysis

中图分类号:

S631.3

燕存尧, 贾凯, 闫会转, 高杰. 芜菁BrrLOX7基因克隆、表达及生物信息学分析[J]. 浙江农业学报, 2023, 35(4): 831-840.

YAN Cunyao, JIA Kai, YAN Huizhuan, GAO Jie. Cloning, expression and bioinformatics analysis of BrrLOX7 gene in turnip[J]. Acta Agriculturae Zhejiangensis, 2023, 35(4): 831-840.

图/表 11

图1 BrrLOX7反转录PCR M,DL5000 marker;1和2表示BrrLOX7基因的PCR扩增产物。

Fig.1 RT-PCR of BrrLOX7 M, DL5000 marker; 1 and 2 indicate PCR amplification products of the BrrLOX7 gene.

表1 BrrLOX7基因编码蛋白的氨基酸组成

Table 1 Amino acid composition of the protein encoded by BrrLOX7 gene

氨基酸的种类 Types of amino acid	氨基酸的数目 Number of amino acids	占氨基酸总量的百分比 Percent of total amino acids/%	氨基酸的种类 Types of amino acid	氨基酸的数目 Number of amino acids	占氨基酸总量的百分比 Percent of total amino acids/%
苯丙氨酸Phe	34	3.76	脯氨酸Pro	62	6.86
亮氨酸Leu	89	9.85	苏氨酸Thr	45	4.98
异亮氨酸Ile	47	5.20	丙氨酸Ala	51	5.64
甲硫氨酸Met	16	1.77	酪氨酸Tyr	34	3.76
缬氨酸Val	53	5.86	组氨酸His	22	2.43
半胱氨酸Cys	12	1.33	谷氨酰胺Gln	25	2.77
色氨酸Trp	21	2.32	天冬氨酸Asn	30	3.32
精氨酸Arg	49	5.42	赖氨酸Lys	63	6.97
丝氨酸Ser	60	6.64	天冬氨酸Asp	61	6.75
甘氨酸Gly	55	6.08	谷氨酸Glu	75	8.30

图2 BrrLOX7蛋白编码磷酸化位点预测

Fig.2 Phosphorylation site prediction of BrrLOX7 protein

图3 BrrLOX7基因编码蛋白亲水性

Fig.3 BrrLOX7 gene encodes a protein with hydrophilicity

图4 BrrLOX7蛋白二级结构预测蓝色代表 α-螺旋; 红色代表延伸链; 绿色代表 β-折叠; 紫色代表无规则卷曲。

Fig.4 Secondary structure of BrrLOX7 protein Blue for α-helix; red for extended chain; green for β-turn; purple for irregularly curled.

图5 BrrLOX7蛋白三级结构预测

Fig.5 Tertiary structure of BrrLOX7 protein

图6 BrrLOX7蛋白序列结构域预测

Fig.6 Structural domain prediction of the BrrLOX7 protein sequence

图7 芜菁与其他植物BrrLOX7蛋白的氨基酸序列比对

Fig.7 Comparison of amino acid sequence of BrrLOX7 protein in turnip and other plants

图8 芜菁BrrLOX7蛋白与其他植物相关蛋白序列的系统进化树

Fig.8 Phylogenetic tree of turnip BrrLOX7 protein and other plant-related protein sequences

图9 芜菁BrrLOX7基因启动子区顺式作用元件预测

Fig.9 Prediction of cis-acting elements of the BrrLOX7 gene promoter in turnip

图10 芜菁BrrLOX7基因在非生物胁迫下的表达

Fig.10 Expression of the BrrLOX7 gene in turnip under abiotic stress

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芜菁BrrLOX7基因克隆、表达及生物信息学分析

Cloning, expression and bioinformatics analysis of BrrLOX7 gene in turnip

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