华山松alpha-pinene synthase基因和(-)-limonene synthase基因的克隆及表达

doi:10.11707/j.1001-7488.20210620

摘要/Abstract

摘要：

目的: 研究华山松alpha-pinene synthase基因和（-）-limonene synthase基因及其表达特性，为揭示其在华山松抵御华山松大小蠹和蓝变真菌秦岭细粘束孢的入侵机制提供理论依据。方法: 运用PCR和RACE技术克隆华山松alpha-pinene synthase基因和（-）-limonene synthase基因cDNA序列；利用生物信息学方法分析基因的核苷酸序列和编码蛋白的氨基酸序列，并构建系统发育树；通过实时荧光定量PCR技术分析华山松alpha-pinene synthase基因和（-）-limonene synthase基因在MeJA处理和秦岭细粘束孢侵染后的表达差异。结果: 华山松alpha-pinene synthase基因编码区全长为1 887 bp，编码的蛋白由628个氨基酸组成，分子质量为71.59 kDa，理论等电点为5.86。华山松（-）-limonene synthase基因编码区全长为1 905 bp，编码的蛋白由634个氨基酸组成，分子质量为73.01 kDa，理论等电点为5.97。经实时荧光定量PCR分析，MeJA处理后alpha-pinene synthase基因和（-）-limonene synthase基因在不同时间均被诱导表达，其中alpha-pinene synthase基因表达量在第2天达到最高峰，（-）-limonene synthase基因表达量在第4天达到最高峰；秦岭细粘束孢侵染后alpha-pinene synthase基因和（-）-limonene synthase基因均被诱导表达，但表达量在处理后不同时间有明显差异。结果表明华山松alpha-pinene synthase基因和（-）-limonene synthase基因是响应MeJA处理（模拟华山松大小蠹入侵危害）和秦岭细粘束孢侵染重要的基因。结论: 华山松alpha-pinene synthase基因和（-）-limonene synthase基因能够响应MeJA处理和秦岭细粘束孢侵染，这对进一步揭示寄主华山松抵御华山松大小蠹和蓝变真菌入侵机制提供了理论依据。

关键词: 华山松, 萜类合酶, 基因表达, 茉莉酸甲酯, 秦岭细粘束孢

Abstract:

Objective: This paper aims to study the alpha-pinene synthase gene and (-)-limonene synthase gene of Pinus armandi and their expression characteristics, and so as to provide theoretical support for revealing the terpene synthase gene in P. armandi to defense against Dendroctonus armandi and its blue-stain fungus Leptographium qinlingensis. Method: The cDNA sequences of alpha-pinene synthase gene and (-)-limonene synthase gene of P. armandi were cloned by PCR and race techniques, and the nucleotide sequence and amino acid sequence of the encoded protein were analyzed by bioinformatics method, and the phylogenetic tree was constructed. The expression differences of alpha-pinene synthase gene and (-)-limonene synthase gene were analyzed by real-time fluorescent quantitative PCR after being treated with MeJA and L. qinlingensis. Result: The full length of the coding region of alpha-pinene synthase gene was 1 887 bp. The protein was composed of 628 amino acids with a molecular weight of 71.59 kDa and the theoretical isoelectric point of 5.86. The full-length coding region of (-)-limonene synthase gene was 1 905 bp. The encoded protein was composed of 634 amino acids with a molecular weight of 73.01 kDa and the theoretical isoelectric point of 5.97. Real-time fluorescence quantitative PCR analysis showed that the expression of alpha-pinene synthase gene and (-)-limonene synthase gene was up-regulated at different time after MeJA treatment. The expression level of alpha-pinene synthase gene reached the peak on the second day, and that of (-)-limonene synthase gene reached the peak on the fourth day. Alpha-pinene synthase and (-)-limonene synthase gene was induced to express by L.qinlingensis, but the expression levels were significantly different at different time points after treatment with L. qinlingensis. The result showed that alpha-pinene synthase gene and (-)-limonene synthase gene were crucial genes in response to L. qinlingensis and MeJA treatments. Conclusion: The alpha-pinene synthase gene and (-)-limonene synthase gene of P. armandi are able to respond to the treatment of L. qinlingensis and MeJA, and play an important regulatory role in the defense of D. armandi and blue-stain fungus. This study provides a theoretical basis for further control of D. armandi.

Key words: Pinus armandi, terpene synthases, gene expression, MeJA, Leptographium qinlingensis

中图分类号:

S718.7

康晓彤,陈辉. 华山松alpha-pinene synthase基因和(-)-limonene synthase基因的克隆及表达[J]. 林业科学, 2021, 57(6): 180-188.

Xiaotong Kang,Hui Chen. Cloning and Expression of alpha-pinene synthase and (-)-limonene synthase Genes in Pinus armandi[J]. Scientia Silvae Sinicae, 2021, 57(6): 180-188.

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用途 Application	引物名称 Primer name	正向和反向引物 Forward and reverse primer (5′-3′)
cDNA克隆 cDNA cloning	alpha-pinene-F alpha-pinene-R (-)-limonene-F (-)-limonene-R	AGCCTCAACATTCTGGATT TTCCAGGATTGTCTTTCAT GTTATGGAAGAGGCAGAA CGAATCTTTCCACCATCT
5′RACE PCR	alpha-pinene Outer Primer alpha-pinene Inner Primer (-)-limonene Outer Primer (-)-limonene Inner Primer	GCCTCCGCAGACATTTCACT TGTGGAAGGCAATCTGTGGC TGTGGCTTATTCTTGTTGAG CTTGTGAAAGACTGGAGACC
3′RACE PCR	alpha-pinene Outer Primer alpha-pinene Inner Primer (-)-limonene Outer Primer (-)-limonene Inner Primer	CACAGATTGCCTTCCACA CAGGCTTGGGAGGATTAT CTTTCACAAGAGATAGAGTA CTCAACAAGAATAAGCCACA
qRT-PCR	alpha-pinene-F alpha-pinene-R (-)-limonene-F (-)-limonene-R 18S-F 18S-R	GCATAGGTGGTTTCCATTCC TGTTTCGCCATCTTCGTTTG GGCTCAACAAGAATAAACCA CAAATCCGAATCTTTCCACC AAGACGGACCACTGCGAAAGC TCGGCATCGTTTATGGTTGAGAC

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