The Molecular Mechanism of Alternative Oxidase Enhancing High Temperature Tolerance of Pleurotus ostreatus

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

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (9): 1922-1934.doi: 10.16420/j.issn.0513-353x.2021-0622

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The Molecular Mechanism of Alternative Oxidase Enhancing High Temperature Tolerance of Pleurotus ostreatus

HOU Ludan, ZHAO Mengran, HUANG Chenyang, ZHANG Jinxia^*()

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,China

Received:2022-03-10 Revised:2022-05-06 Online:2022-09-25 Published:2022-10-08
Contact: ZHANG Jinxia E-mail:zhangjinxia@caas.cn

Abstract

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

CLC Number:

S646.141

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.

Figures/Tables 10

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

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

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.

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.

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

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.

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

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

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

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

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The Molecular Mechanism of Alternative Oxidase Enhancing High Temperature Tolerance of Pleurotus ostreatus

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