香菇ALDH家族基因鉴定、表达及功能分析

doi:10.16420/j.issn.0513-353x.2023-0200

摘要/Abstract

摘要：

在已发表的香菇（Lentinula edodes）4个基因组W1-26、NBRC111202、Bin0899和B17中分别鉴定到17、20、19和18个ALDH家族成员，其编码蛋白分属于11个家族，其中家族1成员数量最多。这些ALDH基因分别分布于17、16、16和10个scaffold中，在除W1-26外的其他3个基因组中均存在1 ~ 2对串联重复ALDH基因。香菇与模式真菌裂褶菌（Schizophyllum commune）之间仅存在4对共线性ALDH基因。启动子分析发现香菇W1-26基因组中17个ALDH基因具有光、激素、氧及干旱等10种响应元件，其中光响应元件数量显著多于其他元件。转录组和蛋白质组数据显示，LeW1-26ALDH1N1、LeW1-26ALDH3L1及LeW1-26ALDH6C1对热胁迫不同时间具有显著响应。LeW1-26ALDH3L1基因RNAi转化子与野生型S606菌株相比，在40 ℃热胁迫24 h条件下菌丝IAA含量显著下降了55%，25 ℃下菌丝恢复生长速度显著下降了71%，菌丝生物量显著下降了45%，LeW1-26ALDH1N1和LeW1-26ALDH6C1基因RNAi转化子与野生型相比表型无显著改变。以上结果说明LeW1-26ALDH3L1基因表达水平的下降可能导致热胁迫下菌丝IAA含量降低进而影响菌丝耐热能力。

关键词: 香菇, ALDH基因家族, IAA生物合成, 耐热性, 全基因组鉴定

Abstract:

In this study，17，20，19，and 18 ALDH family members were identified in the four published genomes W1-26，NBRC111202，Bin0899，and B17 of Lentinula edodes，respectively. ALDH encoded proteins belong to 11 families，among which family 1 has the largest number of genes. These ALDH genes were distributed in 17，16，16，and 10 scaffolds of four genomes，respectively，and one or two pairs of tandem duplication ALDH genes were found except W1-26 genome. Only four pairs collinear ALDH genes existed between L. edodes and the model fungus Schizophyllum commune. The promoter regions of 17 ALDH genes in the W1-26 genome of L. edodes contained ten kinds of response elements such as light，hormone，oxygen and drought，among which the number of light response elements was significantly more than other elements. The transcriptome and proteome data showed that LeW1-26ALDH1N1，LeW1-26ALDH3L1 and LeW1-26ALDH6C1 had significant responses to different time periods of heat stress. Compared with wild-type S606 strain，LeW1-26ALDH3L1 gene RNAi transformant had 55% decrease of IAA content of mycelia after heat stress at 40 ℃ for 24 h，and had 71% and 45% decrease of mycelial regrowth rate and biomass separately at 25 ℃. These phenotypes of LeW1-26ALDH1N1 and LeW1-26ALDH6C1 gene RNAi transformants did not change compared with wild-type S606 strain. Above all，it is indicated that the decreased gene expression level of LeW1-26ALDH3L1 may lead to mycelia’s lower IAA content under heat stress，and then reduced the thermotolerance of mycelia.

Key words: Lentinula edodes, aldehyde dehydrogenase gene family, IAA biosynthesis, thermotolerance, genome-wide identification

陈佳悦, 段英明, 周雁, 肖扬, 边银丙, 龚钰华. 香菇ALDH家族基因鉴定、表达及功能分析[J]. 园艺学报, 2024, 51(5): 1033-1046.

CHEN Jiayue, DUAN Yingming, ZHOU Yan, XIAO Yang, BIAN Yinbing, GONG Yuhua. Identification，Expression and Function Analysis of ALDH Gene Family in Lentinula edodes[J]. Acta Horticulturae Sinica, 2024, 51(5): 1033-1046.

图/表 9

表1 qRT-PCR 引物序列

Table 1 qRT-PCR primer sequence

基因号 ID	基因名称 Gene name	引物序列（5'-3'） Primer sequence
LE01Gene01050	LeW1-26Actin	F：AAGAGCGAAACCCTCGTAGATG；R：GCATCCTGTCCTTCTTACCGAG
LE01Gene12649	LeW1-26ALDH1N1	F：CCACGCCCAAAGATGTAGATA；R：CCTGCTGATGTTCCTCCATAA
LE01Gene12738	LeW1-26ALDH3L1	F：ACACTCCCGCCACTACGA；R：CAGCCATCTTCAGGTCACATT
LE01Gene12316	LeW1-26ALDH6C1	F：AGGGCACTAGCGTAATTGG；R：CACTCGTCCTACTCCATGATTT
LE01Gene03960	LeW1-26ALDH1T1	F：TGAGACCAACGAGCAGAAAC；R：CAGGACACCATAGTCAAAGGATAG
LE01Gene08279	LeW1-26ALDH1U1	F：GAAAGGGATGCGCAAGAATTAG；R：CCTCAAACACTGGATGCTATCT
LE01Gene07214	LeW1-26ALDH10D1	F：CTGAAATAGCCCGATTGGAGAG；R：CTGAGCAAGGCCGCATAATA
LE01Gene07676	LeW1-26ALDH5H1	F：GTCGCCTCATAACTCTGGAAA；R：GTTCGCCATAGGTTCGGATAG
LE01Gene05839	LeW1-26ALDH7E1	F：GGCTGTGTATGGATGGAATCT；R：GACAGCACCTCAGCGATAAT
LE01Gene12494	LeW1-26ALDH15B1	F：GGAACGATCCTCGCAGATAAC；R：GAATGACACGTCTCCTCTGAAC
LE01Gene06992	LeW1-26ALDH4C1	F：GGAATGATCGTGCTGCTATCT；R：CTTTCCTTGACCCAGCATAGT
LE01Gene04469	LeW1-26ALDHnew2A1	F：GAATGCACCGAGGCTAACA；R：GCCTCCATAGCGATTACTCTTC
LE01Gene06747	LeW1-26ALDH3M1	F：CTATCGCTGCTGGTTGTACTAT；R：GCGTCCTGGTCAAGGTATTT
LE01Gene01306	LeW1-26ALDHnewA1	F：CCCTTGACGGATACCGATAAAC；R：TTGCGGAGATGGTTTGAGTAG
LE01Gene03575	LeW1-26ALDH18E1	F：CTTACGACACGGAGCATCTT；R：TCGGTGTGATGAGACGAATG
LE01Gene04343	LeW1-26ALDH1N3	F：TCAATCCGGGAAAGATCAAGG；R：GGTGTGGTGTCCGTAAAGATAG
LE01Gene13422	LeW1-26ALDH1N2	F：CTCTATAAGCTCGCTACCCTAATG；R：CCATCCTTTCACGACTCCAA
LE01Gene10357	LeW1-26ALDHψ	F：CTGGGCAATGGACGTTGATA；R：GTGAACATGAGGACGAAGAGAG

图1 香菇W1-26基因组中已鉴定的17个ALDH蛋白结构域的序列比对方框表示保守基序，星号表示保守活性位点谷氨酸和半胱氨酸残基。

Fig. 1 Sequence alignment of the 17 ALDH domain of all the identified in the W1-26 genome of Lentinula edodes The boxes represent the conserve motifs，and the star indicates the conserved active site glutamate and cysteine residues.

表2 香菇W1-26基因组中鉴定的17个醛脱氢酶信息

Table 2 Sequences for 17 aldehyde dehydrogenases identified in the W1-26 genome of Lentinula edodes

家族 Family	释义 Definition	基因号 ID	命名 Name	酶号 EC
ALDH1	醛脱氢酶Aldehyde dehydrogenase（NAD+）	LE01Gene12649	LeW1-26ALDH1N1	1.2.1.3
		LE01Gene13422	LeW1-26ALDH1N2	1.2.1.3
		LE01Gene03960	LeW1-26ALDH1T1	1.2.1.3
		LE01Gene04343	LeW1-26ALDH1N3	1.2.1.3
		LE01Gene08279	LeW1-26ALDH1U1	1.2.1.3
	甜菜碱醛脱氢酶Betaine-aldehyde dehydrogenase	LE01Gene10357	LeW1-26ALDHψ	1.2.1.8
ALDH3	醛脱氢酶Aldehyde dehydrogenase（NAD+）	LE01Gene12738	LeW1-26ALDH3L1	1.2.1.3
		LE01Gene06747	LeW1-26ALDH3M1	1.2.1.3
ALDH4	L-谷氨酸γ-半醛脱氢酶L-glutamate gamma-semialdehyde dehydrogenase	LE01Gene06992	LeW1-26ALDH4C1	1.2.1.88
ALDH5	琥珀酸半醛脱氢酶Succinate-semialdehyde dehydrogenase	LE01Gene07676	LeW1-26ALDH5H1	1.2.1.16
ALDH6	甲基丙二酸半醛脱氢酶Methylmalonate-semialdehyde dehydrogenase	LE01Gene12316	LeW1-26ALDH6C1	1.2.1.27
ALDH7	醛脱氢酶Aldehyde dehydrogenase（NAD+）	LE01Gene05839	LeW1-26ALDH7E1	1.2.1.3
ALDH10	醛脱氢酶Aldehyde dehydrogenase[NAD（P）+]	LE01Gene07214	LeW1-26ALDH10D1	1.2.1.5
ALDH15	醛脱氢酶Aldehyde dehydrogenase（NAD+）	LE01Gene12494	LeW1-26ALDH15B1	1.2.1.3
ALDH18	谷氨酸-5-半醛脱氢酶Glutamate-5-semialdehyde dehydrogenase	LE01Gene03575	LeW1-26ALDH18E1	1.2.1.41
ALDHnew	醛脱氢酶Aldehyde dehydrogenase [NAD（P）+]	LE01Gene01306	LeW1-26ALDHnewA1	1.2.1.5
ALDHnew2	甜菜碱醛脱氢酶Betaine-aldehyde dehydrogenase	LE01Gene04469	LeW1-26ALDHnew2A1	1.2.1.8

图2 利用香菇LeALDH和其他植物、真菌、动物等19个物种283个ALDH蛋白序列构建的系统发育树紫色圆圈大小代表Bootstrap值。

Fig. 2 Phylogenetic tree construction by using 283 ALDH protein sequences from 19 species including plants，fungi，animals and Lentinula edodes The size of the purple circle represents the Bootstrap value.

图3 香菇ALDH系统发育关系，菌株S606和DnaJ- OE-S606在40 ℃ 0 ~ 24 h基因和蛋白表达水平及基因上游顺式作用元件基因和蛋白表达数据取对数处理；1 ~ 10分别代表生长素相关响应元件、赤霉素相关响应元件、水杨酸相关响应元件、茉莉酸甲酯相关响应元件、脱落酸相关响应元件、光相关响应元件、缺氧相关诱导元件、参与防御和应激反应的顺式作用元件、参与干旱诱导的MYB结合位点元件和低温响应元件。

Fig. 3 Phylogenetic relationship，gene and protein expression level of S606 and DnaJ-OE-S606 strains after 0-24 h at 40 ℃ and gene upstream cis-acting elements of ALDHs in Lentinula edodes Gene and protein expression data is processed logarithmically；1-10 represents auxin related response elements，gibberellin related response elements，salicylic acid related response elements，methyl jasmonate related response elements，abscisic acid related response elements，light related response elements，hypoxia related induction elements，cis-acting elements involved in defense and stress responses，MYB binding site elements involved in drought induction，and low-temperature response elements，respectively.

图4 qRT-PCR检测S606菌株在40 ℃热胁迫不同时间ALDH家族基因表达水平不同小写字母表示组间差异显著（P < 0.05）。

Fig. 4 The gene expression levels of ALDH gene family in S606 strain were detected by qRT-PCR after different time periods of heat stress at 40 ℃ Different lowercase letters indicate significant differences between groups（P < 0.05）.

图5 qRT-PCR检测DnaJ-OE-S606菌株在40 ℃热胁迫不同时间下ALDH家族基因表达水平不同小写字母表示组间差异显著（P < 0.05）。

Fig. 5 The gene expression levels of ALDH gene family in DnaJ-OE-S606 strain were detected by qRT-PCR after different time periods of heat stress at 40 ℃ Different lowercase letters indicate significant differences between groups（P < 0.05）.

图6 常温（25 ℃）和热胁迫（40 ℃）24 h香菇ALDH的RNAi转化子菌丝的IAA含量

Fig. 6 Determination of IAA content in mycelia of LeALDH RNA interference transformants under 25 ℃ and 40 ℃ 24 h * P < 0.05.

图7 热胁迫后香菇ALDH的RNAi转化子菌丝生长速度及生物量

Fig. 7 Determination of mycelial growth rate and mycelial biomass of LeALDH RNA interference transformants after heat stress ** P < 0.01，* P < 0.05.

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