Crops ›› 2021, Vol. 37 ›› Issue (6): 78-87.doi: 10.16035/j.issn.1001-7283.2021.06.013

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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 E-mail:gonglintao@yeah.net;smm1980@yeah.net

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

Table 1

Primers sequence and application"

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

Table 2

Bioinformatics analysis tools"

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

Fig.1

Schematic diagram of different tissues of lavender flower organs and different development stages"

Fig.2

Agarose gel electrophoresis of LIS gene amplification products"

Fig.3

Conserved domains in LIS1 and LIS2 proteins"

Fig.4

Predicted secondary structure of LIS1 and LIS2 proteins"

Fig.5

Predicted tertiary structures of LIS1 and LIS2 proteins"

Fig.6

Multiple alignment of amino acid sequences of LIS1 and LIS2 proteins with linalool synthase proteins from other species The first red box line is RRX8W functional group, the second red box line is DDXXD functional group, and the third red box line is DDXXTXXXE functional group"

Fig.7

Phylogenetic tree analysis of LIS1 and LIS2 proteins The numerical values of the branch points represent the confidence of the bootstrap validation based on 1000 repetitions"

Fig.8

LIS1 gene expression level and significance analysis Different letters indicate significant difference at 0.05 level, the same below"

Fig.9

LIS2 gene expression level and significance analysis"

Fig.10

Identification of recombinant plasmid pET-28a(+)-LIS1 and pET-28a(+)-LIS2 by double enzyme digestion M: Marker 15000; 1: pET-28a(+)- LIS1 digested by double enzyme EcoRⅠand Hind Ⅲ; 2: pET-28a(+)-LIS1 linear plasmid; 3: pET-28a(+)- LIS2 digested by double enzyme NdeⅠ and EcoRⅠ; 4: pET-28a(+)-LIS2 linear plasmid"

Fig.11

Prokaryotic expression analysis of LIS1 and LIS2 recombinant proteins M: ProteinRuler®Ⅰ, the same below; 1: transetta with pET-28a(+) uninduced; 2: transetta with pET-28a(+) induced by IPTG; 3: transetta with pET-28a(+)-LIS2 before induction; 4: transetta with pET-28a(+)-LIS2 uninduced; 5: transetta with pET-28a(+)-LIS2 by IPTG; 6: transetta with pET-28a(+) uninduced; 7: transetta with pET-28a(+) induced by IPTG; 8: transetta with pET-28a(+)-LIS1 before induction; 9: transetta with pET-28a(+)-LIS1 uninduced; 10: transetta with pET-28a(+)-LIS1 by IPTG"

Fig.12

Soluble identification of LIS1 and LIS2 recombinant proteins 1: protein samples before induction; 2: non-induced protein sample; 3: protein samples after induction; 4: soluble sample after induction; 5: insoluble sample after induction"

Fig.13

Recovery identification of LIS1 and LIS2 recombinant proteins 1: inclusion body dissolution solution; 2: dissolved protein fluid; 3: dissolved protein eluent 1; 4: dissolved protein eluent 2"

Fig.14

GC-MS detection of output products of LIS1 recombinant protein supplemented with substrate"

Fig.15

GC-MS detection of output products of LIS2 recombinant protein supplemented with substrate Linalool, monoterpene, 1,6-octadien-3-ol, 3,7-dimethyl-, molecular formula: C10H18O"

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