秀珍菇S-腺苷甲硫氨酸合成酶基因(PpSAMS)的克隆与表达分析

doi:10.3969/j.issn.1004-1524.2021.01.08

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (1): 62-68.DOI: 10.3969/j.issn.1004-1524.2021.01.08

秀珍菇S-腺苷甲硫氨酸合成酶基因(PpSAMS)的克隆与表达分析

王伟科(), 陆娜, 闫静, 宋吉玲, 袁卫东, 周祖法

杭州市农业科学研究院,浙江杭州 310024

收稿日期:2020-04-17 出版日期:2021-01-25 发布日期:2021-01-25
作者简介:王伟科(1981—),男,浙江宁海人,学士,高级农艺师,主要从事食用菌育种与栽培技术研究。E-mail:akeok@126.com
基金资助:
浙江省农业(食用菌)新品种选育重大科技专项(2016C02057)

Cloning and expression analysis of PpSAMS gene of Pleurotus pulmonarius

WANG Weike(), LU Na, YAN Jing, SONG Jiling, YUAN Weidong, ZHOU Zufa

Hangzhou Academy of Agricultural Sciences, Hangzhou 310024,China

Received:2020-04-17 Online:2021-01-25 Published:2021-01-25

摘要/Abstract

摘要：

在对不同生长阶段的秀珍菇样品进行高通量转录组测序的基础上,经分析筛选获得S-腺苷甲硫氨酸合成酶全长基因(PpSAMS)。为了探究PpSAMS在低温刺激后秀珍菇原基形成过程中的功能与表达差异,从秀珍菇中克隆得到了PpSAMS基因,对其进行了系统分析,并利用实时荧光定量PCR的方法,分析该基因在低温刺激后原基形成过程中不同生长发育阶段的表达情况。生物信息学分析表明,PpSAMS基因全长由1 152个核苷酸组成,编码384个氨基酸,具有蛋氨酸腺苷转移酶活性,并具有结合ATP、金属离子的功能。氨基酸序列系统进化树分析结果显示,PpSAMS基因编码蛋白与糙皮侧耳亲缘关系最近。经实时荧光定量PCR检测,该基因在菌丝体经低温诱导后恢复至室温时期(原基形成前期)表达量最高,推测其有可能在诱导真菌DNA甲基化并促使原基形成过程中起到重要作用。

关键词: 秀珍菇, 克隆, 实时荧光定量PCR, S-腺苷甲硫氨酸合成酶基因, 表达分析

Abstract:

Based on the high-throughput transcriptome sequencing of different growth stages of Pleurotus pulmonarius samples, the full-length gene of S-adenosylmethionine synthetase was obtained by analysis and screening. In order to explore the function and expression difference of PpSAMS in the formation of primordium of Pleurotus pulmonarius after low temperature stimulation, the gene of PpSAMS was cloned and analyzed systematically in order to explore the function and expression difference of PpSAMS in the primordium formation of Pleurotus pulmonarius after low temperature stimulation. The expression of the gene in different growth stages was analyzed by real time fluorescence quantitative PCR. Bioinformatics analysis showed that PpSAMS gene was composed of 1 152 nucleotides and encoded 384 amino acids. It had the function of binding ATP and metal ions, and had the activity of S-adenosylmethionine synthetase. The phylogenetic tree analysis of amino acid sequence showed that the protein encoded by PpSAMS gene was closely related to Pleurotus ostreatus. Real time fluorescence quantitative PCR indicated that the expression of PpSAMS was the highest when the mycelium recovered to room temperature after low temperature induction (prophase of primordium formation). It might play an important role in the process of DNA methylation and primordium formation, and participate in the development of fruiting body.

Key words: Pleurotus pulmonarius, clone, real time fluorescence quantitative PCR, PpSAMS, expression analysis

中图分类号:

S646

王伟科, 陆娜, 闫静, 宋吉玲, 袁卫东, 周祖法. 秀珍菇S-腺苷甲硫氨酸合成酶基因(PpSAMS)的克隆与表达分析[J]. 浙江农业学报, 2021, 33(1): 62-68.

WANG Weike, LU Na, YAN Jing, SONG Jiling, YUAN Weidong, ZHOU Zufa. Cloning and expression analysis of PpSAMS gene of Pleurotus pulmonarius[J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 62-68.

图/表 5

表1 实时荧光定量PCR反应引物序列

Table 1 Primers for real time fluorescence quantitative PCR

引物名称 Primer name	引物序列 Primer sequence(5'-3')
PpSAMS-F	GCAACAATCTCCTGATATTGC
PpSAMS-R	GACGAAGCGGCCAGAGGGTTG
Actin-F	CGTTCATTGCCGACGGTGATG
Actin-R	CTCTGAACCCCAAGGCAAACC

图1 PpSAMS基因的ORF读码框与翻译结果

Fig.1 ORF and transcription of PpSAMS

图2 PpSAMS基因氨基酸序列系统进化树

Fig.2 Phylogenetic tree of amino acid sequence of PpSAMS between Pleurotus pulmonarius and other fungi

图3 PpSAMS基因KEGG代谢通路分析

Fig.3 KEGG pathway analysis of PpSAMS

图4 PpSAMS在秀珍菇不同生长阶段的表达情况

Fig.4 Expression profile of PpSAMS in Pleurotus pulmonarius at different growth stages

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秀珍菇S-腺苷甲硫氨酸合成酶基因(PpSAMS)的克隆与表达分析

Cloning and expression analysis of PpSAMS gene of Pleurotus pulmonarius

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