铁皮石斛<i>DoLIS</i>基因克隆与茉莉酸甲酯诱导表达分析

林江波; 邹晖; 王伟英; 戴艺民

doi:10.19303/j.issn.1008-0384.2020.10.004

铁皮石斛DoLIS基因克隆与茉莉酸甲酯诱导表达分析

doi: 10.19303/j.issn.1008-0384.2020.10.004

福建省农业科学院亚热带农业研究所，福建　漳州　363005

基金项目: 福建省科技计划公益类专项(2018R1024-3)

详细信息

作者简介:
林江波（1976-），男，硕士，副研究员，研究方向：农业生物技术（E-mail：345953257@qq.com）

通讯作者:
戴艺民(1969-)，男，博士，研究员，研究方向：农业生物技术（E-mail：dymttcn@163.com）

中图分类号: S 567
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出版历程
- 收稿日期: 2020-08-15
- 修回日期: 2020-09-11
- 刊出日期: 2020-10-28

Cloning and Expressions of LIS in Dendrobium officinale

Subtropical Agriculture Research Institute, Fujian Academy of Agricultural Sciences, Zhangzhou, Fujian　363005, China

摘要

摘要: 目的克隆铁皮石斛（Dendrobium officinale）芳樟醇合酶（linalool synthase, LIS）基因，分析该基因在铁皮石斛开花期花、叶、茎和根中的表达及茉莉酸甲酯（Methyl Jasmonate, MeJA）诱导表达模式，以期为进一步鉴定其功能及分析铁皮石斛单萜类代谢机制奠定基础。方法利用RACE-PCR和RT-PCR技术克隆DoLIS基因全长cDNA序列和开放阅读框（open reading frame, ORF），利用ProtParam和BLAST P在线软件进行理化性质分析和氨基酸同源性比对，采用MEGA 6.0构建系统进化树。利用qPCR方法分析DoLIS基因在开花期铁皮石斛花、叶、茎和根中的表达及MeJA处理后叶片中的表达模式。结果 DoLIS基因cDNA序列全长2 844 bp，含有1个2 538 bp的ORF，编码845个氨基酸。分子量为98.298 kD，理论等电点为7.04，不稳定系数是46.29，属不稳定蛋白。DoLIS蛋白具有Terpene_cyclase_plant_C1保守结构域。系统进化分析表明，DoLIS与其他物种的（S）-（+）-LIS聚在同一支，与姬蝴蝶兰（XP_020576697）的LIS亲缘关系最近。qPCR分析结果显示，DoLIS基因在铁皮石斛开花期叶片中的相对表达量最高，在MeJA处理后相对表达量呈先上升后下降的趋势，处理后5 h相对表达量达到最高，是诱导前的3.88倍。结论本研究克隆了铁皮石斛DoLIS基因cDNA全长，该基因在叶片中的相对表达量极显著高于花、茎和根中的表达量。MeJA处理能显著诱导DoLIS基因的表达。
- 铁皮石斛 /
- 芳樟醇合酶 /
- 基因克隆 /
- 表达分析 /
- 茉莉酸甲酯
Abstract: Objective Linalool synthase gene, LIS, of Dendrobium officinale was cloned. Expression patterns of the gene in flower, leaf, stem, and root at flowering stage as well as those in leaf induced by methyl jasmonate (MeJA) were determined to help decipher the monoterpene metabolism mechanism involved. Method Full-length cDNA of D. officinale LIS (DoLIS) was cloned using RACE-PCR and RT-PCR. Physiochemical properties and amino acid homology were analyzed by ProtParam and BLAST P, and phylogenetic tree constructed by MEGA 6.0. Expressions of DoLIS in the flowers, leaves, stems, and roots of D. officinale at flowering stage, as well as those in the MeJA-treated leaves were determined by quantitative real time PCR. Result The full-length of DoLIS was 2 844 bp with a 2 538 bp ORF encoding 845 amino acids. The protein had a molecular weight of 98.298 KD, a theoretical isoelectric point of 7.04, and an instability coefficient of 46.29. An unstable protein, DoLIS contained a conservative domain of Terpene_cyclase_plant_C1. The phylogenetic analysis showed that DoLIS was closely related to Phalaenopsis equestris (XP_02057697) and clustered in the same branch with the (s)-(+)-LIS of other species. The qPCR results on relative expression of DoLIS indicated that the highest level at flowering stage was found in the leaves. The MeJA induction produced the peak DoLIS expression, which was 3.88-fold of the original, in 5h after the treatment. Conclusion This study cloned the full-length cDNA of DoLIS and discovered the relative expression of the gene to be significantly higher in the leaves than the flowers, stems or roots of a D. officinale plant at the flowering stage. In addition, the expression could be upregulated by MeJA induction.
- Dendrobium officinale /
- linalool synthase /
- gene cloning /
- expression analysis /
- methyl jasmonate

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图 1 PCR电泳结果

注：M：分子量标准；A：3’RACE；B：开放阅读框。

Figure 1. PCR products by electrophoresis

Note: M: DNA marker; A: 3’RACE; B: ORF.

下载: 全尺寸图片幻灯片

图 2 铁皮石斛DoLIS基因cDNA序列及其推导的氨基酸序列

Figure 2. cDNA and deduced amino acid sequence of DoLIS from D. officinale

下载: 全尺寸图片幻灯片

图 3 不同物种LIS的系统进化树

Figure 3. Phylogenetic trees of LlS from different species

下载: 全尺寸图片幻灯片

图 4 DoLIS基因在不同器官中的相对表达量

注：柱上不同字母表示差异极显著（P＜0.01）；图5同。

Figure 4. Relative expression of DoLIS in different organs

Note: Different letters on columns indicate significant difference at 0.01 level.The same as figure 5.

下载: 全尺寸图片幻灯片

图 5 MeJA对铁皮石斛DoLIS基因表达的影响

Figure 5. Relative expression of DoLIS in response to MeJA treatment

下载: 全尺寸图片幻灯片

表 1 PCR引物及其序列

Table 1. PCR primers and sequences

引物名称 Primer name	引物序列（5′-3′） Primer sequence（5′-3′）
3LIS-F1	CTCAACACAGAGTCAAGAAAGG
3LIS-F2	TCATTTGACTCGCCAACCGCAC
dT-adapt	CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTTTT
adapt	CTGATCTAGAGGTACCGGATCC
DoLIS-F	CCGGAATTCATGGAGCAGTCATTGTGGTT
DoLIS-R	CCGCTCGAGCTTTCTCCCTTCATTTGCTC
LIS-F	TGGATCATTTGGAGCACAG
LIS-R	CATTTGGCTGCTTCCTTTC
DoACT-F	AGGAAGGCGGCTTTGAATC
DoACT-R	CCATGCCAACCATGACACC
注：下划线碱基为酶切位点。 Note: The underlined bases were the enzyme site.

下载: 导出CSV

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