糙皮侧耳乳酸脱氢酶鉴定及其菌丝高温胁迫下表达特征分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0631

生物技术通报 ›› 2023, Vol. 39 ›› Issue (11): 350-359.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0631

糙皮侧耳乳酸脱氢酶鉴定及其菌丝高温胁迫下表达特征分析

吴柏增¹^,²(), 何琪²^,³, 姚方杰¹^,³(), 赵梦然²()

1.吉林农业大学园艺学院，长春 130118
2.中国农业科学院农业资源与农业区划研究所农业农村部农业微生物资源收集与保藏重点实验室，北京 100081
3.吉林农业大学食药用菌教育部工程研究中心,长春 130118

收稿日期:2023-07-03 出版日期:2023-11-26 发布日期:2023-12-20
通讯作者: 赵梦然，女，博士，副研究员，研究方向：食用菌遗传育种与栽培创新；E-mail: zhaomengran@caas.cn；
姚方杰，女，博士，教授，研究方向：食用菌遗传育种及高效栽培；E-mail: yaofj@aliyun.com
作者简介:吴柏增，男，硕士研究生，研究方向：食用菌环境响应；E-mail:1772696751@qq.com
基金资助:
现代农业产业技术体系建设专项资金项目(CARS-20)

Identification of Lactate Dehydrogenase in Pleurotus ostreatus and Heat Stress Expression Analysis of Mycelium

WU Bai-zeng¹^,²(), HE Qi²^,³, YAO Fang-jie¹^,³(), ZHAO Meng-ran²()

1. College of Horticulture, Jilin Agriculture University, Changchun 130118
2. Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture and Rural Affairs, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081
3. Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Jilin, Changchun 130118

Received:2023-07-03 Published:2023-11-26 Online:2023-12-20

摘要/Abstract

摘要：

乳酸脱氢酶（lactate dehydrogenase，LDH）可催化丙酮酸与乳酸（lactic acid，LA）之间的可逆转化，为探索乳酸脱氢酶基因在糙皮侧耳（Pleurotus ostreatus）菌丝体高温胁迫响应中的作用，在糙皮侧耳（P. ostreatus）菌株CCMSSC00389基因组中鉴定获得了8个乳酸脱氢酶编码基因。生物信息学分析结果显示，3个基因编码D型乳酸脱氢酶，5个基因编码L型乳酸脱氢酶。氨基酸序列比对、系统发育、三维结构和亚细胞定位分析显示，3个D型乳酸脱氢酶的同源性较高，可能属于细胞色素C依赖的D型乳酸脱氢酶，D-LDH2和D-LDH3位于线粒体中；5个L型乳酸脱氢酶的氨基酸序列同源性较低，并按电子受体和亚细胞定位的不同分为3个类型。通过检测36℃、40℃高温胁迫下乳酸脱氢酶基因的表达量变化，明确D-ldh3、L-ldh5和L-ldh7表达特征相同，L-ldh6和L-ldh8的表达特征相同，D-ldh1、D-ldh2和L-ldh4则表现出不同的基因表达趋势，表明乳酸脱氢酶基因对不同高温胁迫温度的响应方式存在差异。高温胁迫条件下，乳酸脱氢酶总酶活性降低，胞内乳酸含量升高；外源添加乳酸脱氢酶抑制剂和乳酸都能显著抑制菌丝生长，表明乳酸脱氢酶能够调控热胁迫下糙皮侧耳的菌丝生长。推测糙皮侧耳乳酸脱氢酶通过降低基因转录水平和酶活性，影响了胞内乳酸脱氢向丙酮酸的转化，导致胞内乳酸的过量积累造成细胞损伤，最终抑制了菌丝生长速率。

关键词: 糙皮侧耳, 乳酸脱氢酶, 高温响应, 基因表达

Abstract:

Lactate dehydrogenase（LDH）catalyzes the reversible conversion between pyruvate and lactic acid. In order to explore the role of LDH genes in heat stress response of Pleurotus ostreatus, eight coding genes of LDH were identified in the genome of P. ostreatus strain CCMSSC00389. The bioinformatics analyses showed that three genes encoded D-lactate dehydrogenase and five gene encoded L-lactate dehydrogenase. The results from the amino acid sequence alignment, phylogenetic analysis, three-dimensional structure and subcellular localization showed that three D-lactate dehydrogenases sharing a high homolog, which belonged to cytochrome C-dependent D-lactate dehydrogenase, and D-LDH2 and D-LDH3 were located in mitochondria. The five L-lactate dehydrogenases having a low homolog were divided into three different types according to electron acceptor and subcellular localization. The gene transcription changes of lactate dehydrogenase at 36℃ and 40℃ indicated the similar expression characteristics shared by D-ldh3, L-ldh5 and L-ldh7, as well as by L-ldh6 and L-ldh8; while the distinctive expression characteristics within D-ldh1, D-ldh2 and L-ldh4 was observed, which revealed varied response ways of the LDH genes to different stress temperatures. The total lactate dehydrogenase enzyme activity decreased and the intracellular lactic acid content increased under the heat stress. The inhibiting effects of lactate dehydrogenase inhibitor and exogenous lactic acid on the mycelia growth of P. ostreatus suggested that LDH regulated the mycelia growth under heat stress. It is speculated that P. ostreatus lactate dehydrogenase affects the conversion of intracellular LDH to pyruvate by reducing gene transcription level and enzyme activity, resulting in excessive accumulation of intracellular lactic acid and cell damage, and finally inhibits the growth rate of hyphae.

Key words: Pleurotus ostreatus, lactate dehydrogenase, high temperature response, gene express

吴柏增, 何琪, 姚方杰, 赵梦然. 糙皮侧耳乳酸脱氢酶鉴定及其菌丝高温胁迫下表达特征分析[J]. 生物技术通报, 2023, 39(11): 350-359.

WU Bai-zeng, HE Qi, YAO Fang-jie, ZHAO Meng-ran. Identification of Lactate Dehydrogenase in Pleurotus ostreatus and Heat Stress Expression Analysis of Mycelium[J]. Biotechnology Bulletin, 2023, 39(11): 350-359.

图/表 8

表1 荧光定量PCR引物

Table 1 Primers for qPCR

引物Primer	正向序列Forward sequence（5'-3'）	反向序列Reverse sequence（5'-3'）
qPCR-ldh1	GGTCCTGCTTTGTCTCGTAG	GGGAAAGGATGGAGTACAGATG
qPCR-ldh2	AGAGGAATTGGAAAGACATGGG	ATACGGAATGACGGGCATAAG
qPCR-ldh3	CTTGGGAGATGTGATACTGGG	GTTCTCGGTTTGAATTCGCTG
qPCR-ldh4	GCGATTAGTGGCAAGGTTTAC	CATCCAACATGAAATCGGCG
qPCR-ldh5	CACCCGCTCTACCAATTCTG	GCCATTCCTTTACGCTCAATC
qPCR-ldh6	TGCTTCTACCCGTTTCTGTC	ATATGACCCAATACATCCACCG
qPCR-ldh7	TGAACTTGCGTCTACTTGGTG	CAGGCGGAGAGGTTGATATG
qPCR-ldh8	GGCGGAGGTGTCAAGATTC	ATGGCGGAACGAAGGATATG

表2 糙皮侧耳ldh序列特征

Table 2 ldh sequence characteristics of P. ostreatus

名称 Name	互补DNA/基因组DNA cDNA/gDNA/bp	分子量 Molecular weight/kD	等电点 pI
D-ldh1	1473/2114	52.91	4.88
D-ldh2	1692/2459	61.34	4.84
D-ldh3	1776/2603	63.92	4.82
L-ldh4	711/820	25.18	5.05
L-ldh5	1485/2158	54.54	4.86
L-ldh6	1515/2360	56.02	4.83
L-ldh7	1497/2182	54.83	6.14
L-ldh8	957/1128	33.76	5.02

图1 LDH的氨基酸序列比对

Fig. 1 Amino acid sequence alignment of LDH

图2 LDH的三维结构预测

Fig. 2 Three-dimensional structure prediction of LDH A: D-LDH1; B: D-LDH2; C: D-LDH3; D: L-LDH4; E: L-LDH5; F: L-LDH6; G: L-LDH7; H: L-LDH8

图3 LDH的系统发育树

Fig. 3 Phylogenetic tree of LDH A: D-LDH; B: L-LDH

图4 糙皮侧耳菌丝高温处理下ldh的基因表达量、酶活性和LA含量不同字母代表差异显著，P<0.05，下同

Fig. 4 Gene expression of ldh, LDH activity and LA content in P. ostreatus mycelium treated with high temperature The different letters over the columns within the same graph denote significant differences. P<0.05, the same below

图5 不同浓度外源LA对菌丝形态与生长速率的影响

Fig. 5 Effects of exogenous LA with different concentrations on mycelial morphology and growth rate

图6 LDH抑制剂对LDH活性、菌丝生长速率和菌落形态的影响

Fig. 6 Effects of LDH inhibitor on LDH activity, mycelial growth rate and colony morphology

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糙皮侧耳乳酸脱氢酶鉴定及其菌丝高温胁迫下表达特征分析

Identification of Lactate Dehydrogenase in Pleurotus ostreatus and Heat Stress Expression Analysis of Mycelium

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