交替氧化酶增强糙皮侧耳高温耐受性的分子机制

doi:10.16420/j.issn.0513-353x.2021-0622

摘要/Abstract

摘要：

为探索交替氧化酶（Alternative oxidase,aox）基因在大型真菌高温胁迫响应中的功能,以糙皮侧耳（Pleurotus ostreatus）CCMSSC00389菌株为出发菌株,克隆得到1个aox基因,其gDNA全长1 845 bp,cDNA全长1 152 bp,编码383个氨基酸,具2个跨膜螺旋区,蛋白分子量43.53 kD。以野生型和aox转化菌株为材料,验证了aox在高温胁迫下的功能及调控通路。结果表明,32 ℃高温胁迫下,菌丝生长速率下降了42.27%。aox基因转录水平的升高可能通过介导逆行信号通路影响抗氧化酶基因的表达,从而促进活性氧的清除,提高高温胁迫下菌丝生长速率,揭示aox在提高糙皮侧耳菌丝高温耐受性方面发挥正调控作用。

关键词: 糙皮侧耳, 交替氧化酶, 高温胁迫, 活性氧, 生物功能

Abstract:

In order to explore the function of alternative oxidase（aox）gene in high temperature stress response of macrofungi,the aox gene was cloned from Pleurotus ostreatus CCMSSC00389 strain. The total length of gDNA and cDNA were 1 845 bp and 1 152 bp respectively,encoding 383 amino acids and two transmembrane helix regions. The molecular weight of the protein was 43.53 kD. The function and regulatory pathway of aox under high temperature stress were verified by wild-type strain and transfor- mation strains of aox gene. The results showed that the mycelial growth rate decreased by 42.27% under 32 ℃ high temperature stress. The increase of aox gene transcription level may up-regulate the expression of antioxidant enzyme gene by mediating retrograde signal pathway,and then promote the clearance of ROS and improve the mycelial growth rate under high temperature stress. The results showed that aox gene played a positive regulatory role in improving high temperature tolerance of P. ostreatus mycelia.

Key words: Pleurotus ostreatus, alternative oxidase, heat stress, ROS, biological function

中图分类号:

S646.141

侯潞丹, 赵梦然, 黄晨阳, 张金霞. 交替氧化酶增强糙皮侧耳高温耐受性的分子机制[J]. 园艺学报, 2022, 49(9): 1922-1934.

HOU Ludan, ZHAO Mengran, HUANG Chenyang, ZHANG Jinxia. The Molecular Mechanism of Alternative Oxidase Enhancing High Temperature Tolerance of Pleurotus ostreatus[J]. Acta Horticulturae Sinica, 2022, 49(9): 1922-1934.

图/表 10

表1 荧光定量PCR引物

Table 1 Primers list for qPCR

引物	正向序列（5′-3′）	反向序列（5′-3′）
Primer	Forward sequence	Reverse sequence
qPCR_aox	ATGCTTCGCGCGCAACTC	TCAATTACTACCTTTCTGCTTCTCT
qPCR_cat1	TGTGCATTGGTTGAGAGAGG	TACGACGCTACAACTTCCG
qPCR_cat2	CGGACTTTCTTGCCCACAG	GACTTGCTCGCCCATTTCG
qPCR_gsh-px	AAGGTCTTCAGGCATTGTAT	GTGGTTACGCTCACAGAA
qPCR_trxr	GACGGCTACATCATCACC	AATGAGCTTCTCGACCTC
qPCR_sod1	CCTCATCTTCAACTACGCAAGT	GCAGACGAACCACAGGAATC
qPCR_sod2	ACACGAAGCATCATCAGACCTA	GAAGAGCGAGTGGTTGATATGG
qPCR_sod3	GTTCTCCTAGCAGCGAAGA	CATTCCCCGTTTTAAGTGAC
qPCR_sod4	TTGAACGAGACTTTGGCACC	ATGATCGGCGCGTGAGTTATC
β-actin	AGTCGGTGCCTTGGTTAT	ATACCGACCATCACACCT
tubulin	AGGCTTTCTTGCATTGGTACACGC	TATTCGCCTTCTTCCTCATCGGCA

图1 aox基因结构（A）及AOX系统发育树（B）

Fig. 1 aox gene structure features（A）and AOX phylogenetic tree（B）

图2 糙皮侧耳CCMSSC00389菌株AOX蛋白序列的生物信息 A：3D结构;B：跨膜预测;C：跨膜结构预测;D：保守结构域预测。

Fig. 2 Bioinformatics of AOX protein sequence of Pleurotus ostreatus CCMSSC00389 strain A：3D structure;B：Transmembrane prediction;C：Prediction of transmembrane structure;D：Prediction of conserved domain.

图3 糙皮侧耳CCMSSC00389与其他物种的AOX氨基酸序列同源性比较序列中保守氨基酸残基的位置下面用圆点表示。

Fig. 3 Homology of AOX amino acid sequence of Pleurotus ostreatus CCMSSC00389 with other species Positions with identical amino acid residues are indicated by circles below the sequence.

图4 高温32 ℃胁迫6 d对糙皮侧耳菌丝生长、H2O2含量和aox表达量的影响

Fig. 4 Effects of high temperature stress at 32 ℃ for 6 d on mycelial growth,H2O2 content and aox expression of Pleurotus ostreatus

图5 糙皮侧耳野生型（WT）aox过表达（OE-aox）和RNA干扰（RNAi-aox）菌株在32 ℃高温胁迫下的表型红色直线表示菌落半径。

Fig. 5 The growth phenotype of wild-type（WT）,aox overexpression（OE-aox）and RNA interference（RNAi-aox）strains of Pleurotus ostreatus under high temperature stress at 32 ℃ The red line indicates the colony radius.

图6 糙皮侧耳野生型（WT）、aox过表达（OE-aox）和干扰（RNAi-aox）菌株32 ℃高温胁迫下抗氧化酶基因的表达

Fig. 6 The expression of antioxidant enzyme genes of Pleurotus ostreatus wild type（WT）,aox overexpression（OE-aox）and interference（RNAi-aox）strains under 32 ℃ high temperature stress

图7 aox过表达增强了糙皮侧耳菌丝对H2O2的耐受性

Fig. 7 aox overexpression enhanced the tolerance of Pleurotus ostreatus mycelia to H2O2

图8 外源添加H2O2对aox转化菌株中关键抗氧化酶基因表达量的影响

Fig. 8 Effect of exogenous H2O2 on the expression of key antioxidant enzyme genes in aox transformed strains

图9 aox参与糙皮侧耳菌丝对32 ℃高温胁迫响应调控途径示意图

Fig. 9 The regulatory pathway of aox involved in the response of Pleurotus ostreatus mycelia to 32 ℃ high temperature stress

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