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作物杂志,2021, 第6期: 78–87 doi: 10.16035/j.issn.1001-7283.2021.06.013

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

薰衣草芳樟醇合酶基因的克隆、表达及酶活性检测

龚林涛(), 苏秀娟(), 廖燕, 克热木汗·吾斯曼, 周迪, 尹松松   

  1. 新疆农业大学农学院/新疆农业大学农业生物技术重点实验室,830052,新疆乌鲁木齐
  • 收稿日期:2020-10-16 修回日期:2021-08-13 出版日期:2021-12-15 发布日期:2021-12-16
  • 通讯作者: 苏秀娟
  • 作者简介:龚林涛,研究方向为作物遗传育种,E-mail： gonglintao@yeah.net
  • 基金资助:
    国家自然科学基金地区科学基金(31760429);国家自然科学基金地区科学基金(31960452);新疆维吾尔自治区研究生科研创新项目(XJ2019G155)

Cloning, Expression and Enzyme Activity Detection of Linalool Synthase Gene in Lavender

Gong Lintao(), Su Xiujuan(), Liao Yan, Keremuhan·Wusiman , Zhou Di, Yin Songsong   

  1. College of Agriculture, Xinjiang Agricultural University/Key Laboratory of Agricultural Biotechnology, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
  • Received:2020-10-16 Revised:2021-08-13 Online:2021-12-15 Published:2021-12-16
  • Contact: Su Xiujuan

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摘要：

从高油薰衣草品种“杂花”中克隆得到芳樟醇合酶基因LIS1和LIS2,并对其进行了生物信息学和表达模式分析、原核表达和酶活性检测,以低油品种“法国蓝”为对照。结果表明,LIS1基因编码602个氨基酸,LIS2基因编码564个氨基酸。LIS1蛋白与阔叶薰衣草、一串红的芳樟醇合酶亲缘关系相近,LIS2蛋白与狭叶薰衣草、杂薰衣草的芳樟醇合酶亲缘关系相近。LIS1基因在“杂花”花器官半开期、盛开期和衰败期的表达量均高于“法国蓝”;LIS1基因在“杂花”花萼中的表达量最高,且仅在“法国蓝”雄蕊中少量表达。LIS2基因在“杂花”花器官不同组织和不同发育时期表达量均低于其在“法国蓝”中表达量,该基因在“杂花”花器官衰败期和“法国蓝”花器官半开期表达量最高,在2个品种花萼中高表达。LIS2基因在2个品种不同发育时期及组织中的表达量均明显高于LIS1基因。LIS1和LIS2重组蛋白具有单萜合酶活性,且均参与合成芳樟醇。

关键词: 薰衣草, 芳樟醇合酶, 克隆, 基因表达, 原核表达

Abstract:

In this study, the LIS1 and LIS2 genes of lavender were cloned from the oil-rich lavender variety ‘Zahua’, and bioinformatics analysis, expression pattern analysis and prokaryotic expression and enzyme activity detection with the control of ‘French bule’ were carried out. The results showed that LIS1 gene could encode 602 amino acids and LIS2 gene could encode 564 amino acids. LIS1 protein was closely related to the linalool synthase in broadleaf Lavandula latifolia and Salvia splendens. The LIS2 protein was closely related to the linalool synthase in Lavandula angustifolia and Lavandula x intermedia. The expression level of the LIS1 gene in half-opening stage, full blooming stage and senescence stage of flower organs of ‘Zahua’ were higher than that of ‘French blue’ at the same stages. The expression of the LIS1 gene was the highest in calyx of ‘Zahua’, and only slight expression in the stamen of ‘French blue’. The expression level of the LIS2 gene in different tissues and different development stages of ‘Zahua’ flower organs were lower than that of ‘French blue’ in the same tissues and stages. The LIS2 gene had the highest expression in ‘Zahua’ flower organs at senescence stage and in ‘French blue’ flower organs at the half-opening stage, and was strongly expressed in the calyx of the two varieties. The LIS2 gene expression was significantly higher than that of the LIS1 gene in different development stages and tissues of the two varieties flower organs. The recombinant proteins LIS1 and LIS2 had monoterpene synthase activity and were involved in the synthesis of linalool.

Key words: Lavender, Linalool synthase, Cloning, Gene expression, Prokaryotic expression

表1

试验所用引物序列及用途

引物
Primer		 序列 (5'-3')
Sequence(5'-3')		 用途
Application
F1 ATGTCTATCATTAGCATGCATGTGGG 基因克隆
R1 TTAGGGATATGGCTCGAACATCAGGGT 基因克隆
F2 ATGTCGATCAATATCAACATGCCTGC 基因克隆
R2 TCATGCGTACGGCTCGAACA 基因克隆
F3 AATAGGCCTGCAGCTTATAACCAT 荧光定量
R3 TGGCGCGGCTTTGAAG 荧光定量
F4 AATCGGCCGACTTCCTGAAT 荧光定量
R4 TCGGAAACAAAGTTGTGGATTG 荧光定量
β-Actin-F TGTGGATTGCCAAGGCAGAGT 荧光定量内参
β-Actin-R AATGAGCAGGCAGCAACAGCA 荧光定量内参
F5
CGGAATTCATGTCTATCATTAGCATGCA
TGTGGG		 载体构建
R5
CCAAGCTTGGGATATGGCTCGAACATCA
GGGT		 载体构建
F6
GGAATTCATATGATGTCGATCAATATCA
ACATGCCTGC		 载体构建
R6 CGGAATTCTGCGTACGGCTCGAACA 载体构建

表2

生物信息学分析工具

网站
Website		 网址
URL		 预测内容
Predict the content
ProtParam http://web.expasy.org/protparam/ 蛋白理化参数
InterProScan
& Pfam		 http://www.ebi.ac.uk/Tools/pfa/iprscan/
http://pfam.xfam.org/search		 蛋白功能结构域
DNAMAN – 氨基酸序列比对
PSIPRED http://bioinf.cs.ucl.ac.uk/psipred/ 蛋白二级结构
SWISS-Model http://swissmodel.expasy.org/ 蛋白三级结构
MEGA 5.0 – 同源树构建

图1

薰衣草花器官不同组织和不同发育时期示意图

图2

LIS基因扩增产物琼脂糖凝胶电泳

图3

LIS1和LIS2蛋白的功能结构域

图4

LIS1和LIS2蛋白二级结构预测

图5

LIS1和LIS2蛋白的三级结构预测

图6

LIS1和LIS2蛋白与其他物种芳樟醇合酶蛋白的多序列比对 第1个红色框线为RRX8W功能基团,第2个红色框线为DDXXD功能基团,第3个红色框线为DDXXTXXXE功能基团

图7

LIS1和LIS2蛋白的系统进化树分析 分支点的数值表示bootstrap验证中基于1000次重复的置信度

图8

LIS1基因表达量及显著性分析 不同字母表示在0.05水平上差异显著,下同

图9

LIS2基因表达量及显著性分析

图10

重组质粒pET-28a(+)-LIS1和pET-28a(+)-LIS2的双酶切鉴定 M：Marker 15 000;1：pET-28a(+)-LIS1质粒经EcoRⅠ、Hind Ⅲ双酶切;2：pET-28a(+)-LIS1线性质粒;3：pET-28a(+)-LIS2质粒经NdeⅠ、EcoRⅠ双酶切;4：pET-28a(+)-LIS2线性质粒

图11

LIS1和LIS2重组蛋白原核表达分析 M：蛋白ProteinRuler®Ⅰ,下同;1：pET-28a(+)未诱导;2：pET-28a(+)诱导后;3：pET-28a(+)-LIS2蛋白诱导前;4：pET-28a(+)-LIS2蛋白未诱导;5：pET-28a(+)-LIS2蛋白诱导后;6：pET-28a(+)未诱导;7：pET-28a(+)诱导后;8：pET-28a(+)-LIS1蛋白诱导前;9：pET-28a(+)-LIS1蛋白未诱导;10：pET-28a(+)-LIS1蛋白诱导后

图12

LIS1和LIS2重组蛋白可溶性鉴定 1：诱导前蛋白样品;2：未诱导蛋白样品;3：诱导后蛋白样品;4：诱导后可溶样品;5：诱导后不可溶样品

图13

LIS1、LIS2重组蛋白回收鉴定 1：包涵体溶解液;2：溶解蛋白流穿液;3：溶解蛋白洗脱液1;4：溶解蛋白洗脱液2

图14

LIS1重组蛋白添加底物输出产物GC-MS检测

图15

LIS2重组蛋白添加底物输出产物GC-MS检测 芳樟醇,单萜,1,6-octadien-3-ol, 3,7-dimethyl-,分子式：C10H18O

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