微生物学报

Primers	Paired sequences (5′→3′)	Purpose
Underlined regions denote the sites of restriction enzyme for disruption of sakA (endonuclease Xma I/BamH I and Xho I/Bgl Ⅱ) through homogenous recombination of 5′ and 3′ fragments separated by hygR marker and for the expression cassette including the promoter and open reading frame of hygR (BamH I/Xho I).
sakA-F/R	ATGGCTGAGTTCTTGCGATC/CTAAATCTGGAGGTACTGCA	Cloning ORF of sakA
5′sakA-F/R	aaaaaCCCGGGGCCAACAACAAACCACCACG/	Cloning sakA 5′ (1 892 bp)
	aaaaaGGATCCTCTGACTGAAGGGGGAATCG
3′sakA-F/R	aaaaaCTCGAGCTATCTGATTACGGATGCTCTG/	Cloning sakA 3′ (1 969 bp)
	aaaaaAGATCTTGGGGAGTATTGGAAGTCTG
sakA-ID-F/R	GCTTTGCCACCTACAGTTGC/AAACAAAGTCGCCCAAGGAG	PCR detecting sakA
qsakA-F/R	GAGAATTGTGACTTGAAG/ATCTAGTTGAGACATATCC	qPCR detecting sakA gene
qβ-tubulin-F/R	TCAAGATGTCCTCTACCT/GGAACATAGCAGTGAACT	qPCR detecting β-tubulin gene

Primers	Paired sequences (5′→3′)	Purpose
Underlined regions denote the sites of restriction enzyme for disruption of sakA (endonuclease Xma I/BamH I and Xho I/Bgl Ⅱ) through homogenous recombination of 5′ and 3′ fragments separated by hygR marker and for the expression cassette including the promoter and open reading frame of hygR (BamH I/Xho I).
sakA-F/R	ATGGCTGAGTTCTTGCGATC/CTAAATCTGGAGGTACTGCA	Cloning ORF of sakA
5′sakA-F/R	aaaaaCCCGGGGCCAACAACAAACCACCACG/	Cloning sakA 5′ (1 892 bp)
	aaaaaGGATCCTCTGACTGAAGGGGGAATCG
3′sakA-F/R	aaaaaCTCGAGCTATCTGATTACGGATGCTCTG/	Cloning sakA 3′ (1 969 bp)
	aaaaaAGATCTTGGGGAGTATTGGAAGTCTG
sakA-ID-F/R	GCTTTGCCACCTACAGTTGC/AAACAAAGTCGCCCAAGGAG	PCR detecting sakA
qsakA-F/R	GAGAATTGTGACTTGAAG/ATCTAGTTGAGACATATCC	qPCR detecting sakA gene
qβ-tubulin-F/R	TCAAGATGTCCTCTACCT/GGAACATAGCAGTGAACT	qPCR detecting β-tubulin gene

Tag code	Gene	Annotation	Sequences of paired primers (5′→3′)
Gene08183	fluG	Upstream development activator	AAGATTGCTGCTGCTGATA/GTCGTAGGTAATCGTCTCATT
Gene03944	flbA	Upstream development activator	AGCAGCATCTCTACTTCAG/CTCCTTCGCCATCTTCAC
Gene04120	flbC	Upstream development activator	CTACTATTCCGCCTCTGT/GCCATTCTCGTAAGATTCC
Gene06270	flbB	Upstream development activator	CTCTTCCTTCCATCTCTA/TCATTCTAGTACGCTTTG
Gene04684	flbD	Upstream development activator	TGGACATGAGACAGCAATAC/AGAGATCGTGGTAGGCATA
Gene02514	flbE	Upstream development activator	TGGTGGATTGTGTATAAC/CTCATCATACCCATCATC
Gene00348	laeA	Developmental repressor	GGACTTGGAGAGAATCAG/AGATGTTGTAAGCGTGTAT
Gene02682	veA	Developmental activator	GTTCCTCTTACACTTCTCAT/GAGTTCCAGGTAGTTGTG
Gene00832	sfgA	Developmental repressor	CGAACAACCTGCTTATAGTC/GCTGCTTGAATCTCCATC
Gene04565	velB	Developmental repressor;	CCTCAGATGAACAATTAC/TAGTAAGGCTGATAATAGG
		Regulation for conidial
		maturation
Gene04761	abaA	Regulation for sterigmata	AACATCCTTTCCTTACCT/GTCATCCATCAACTTCAG
		formation
Gene04357	wetA	Regulation for conidial	CAGTGTGGATGGCAACAA/CGCTGGACAATGACTCAAG
		maturation
Gene10505	vosA	Regulation for conidial	ATGTGGATAATACTGATG/ATCTATCTGTGATGATTG
		maturation
Gene00550	brlA	Initiator for conidiogenesis	AGAACCTTCCGTCTATTATCA/ATGCTCTTGCCTCTTGAA
Gene05820	stuA	Modulator for conidiophore	ATATGATTAACGGCACAA/CTCACTCTTCAGAATACC
		formation
Gene00245	creA	Carbon catabolite repressor	TTCTTCTGATGAGGAGAG/CGGACTAACAACAACTTT
Gene08487	srgA	Secretion related GTPase	TACTGCTGATTGGTGACT/TAATGAACGACGGAGTGA
Gene08788	amyA	Alpha-amylase	GCGAAGAATATCCTCACAT/CATCCAAATGCTGTTCCT
Gene06696	amyC	Alpha-amylase	TCTAATGGTTCGGCTTAT/GTATAGTTGACGCAGTTC
Gene00277	amyD	Alpha-amylase	AACATCTTCTCTGATACATTAGT/GATTGTCCTGGTTGAGTG
Gene11416	amyE	Alpha-amylase	GCTTGTCTCTAACTACTC/TTCTTGACTGTGTCTATAC
Gene08787	amyF	Alpha-amylase	GAAGTGGATTATGTCTGT/ATAAACTCCCTCTGAAAC
Gene07580	amyG	Glucan 1, 4-alpha-glucosidase	TCAATCCATCTACTTCCT/ATAATTCCTTGCCAACTT
Gene10420	amyH	Alpha-amylase	GCTTGTCTCTAACTACTC/TTCTTGACTGTGTCTATAC
Gene05110	amyM	Alpha-amylase	TCCATTGTACTACATTCTCA/GCTGATAGACTCGGTAAT
Gene10422	amyR	Transcription regulator of	CAGTAGATGGAATGAATATAGAA/GCTTGACAGAGGTAGAAC
		maltose utilization
Gene00933	aglB	Alpha-galactosidase	GAGTTGAAGAGTAAGAATT/CACCTCATACTTATCCTT
Gene00399	lacA	Beta-galactosidase	ATTGCCATTATGAACTTGTA/TAGTCGTAGGAGGTGTAT
Gene03324	pexB	Pectinase	ACTCTTCAGCGACTTCCT/GCCTCTAATTGTATTCCATAACG
Gene04089	pecA	Pectinase	GAGACTCTTGACTTGACT/CTTCCATTCCTTGTAACC
Gene08774	pecB	Pectinase	CGTCAAGATCGAGAACAT/CGCAGAGGATGTAGTAATC
Gene06504	pecC	Pectinase	TTGACCTCACTGACCTTA/CTGTCCATTCCTCATAACC
Gene09643	endA	NADH dehydrogenase	AAGACCGTTTATGATGCTA/CGATGACAATACCGTAGT
Gene06504	endC	NADH dehydrogenase	TTGACCTCACTGACCTTA/CTGTCCATTCCTCATAACC
Tag code	Gene	Annotation	Sequences of paired primers (5′→3′)
Gene08115	poly	Polymerase	GGCTCTGATACCAATGTTAG/CTACCTCCGTTGCTCTTA
Gene05290	xlnR	Xylanolytic transcriptional	CAACTTTCCTTCTTTCAG/ATAATGGAGGCAATATGT
		activator
Gene01326	cbhA	1, 4-beta-d-glucan	AATAATGACAACACAGGTA/GAGATACTATTGGCTTCC
		cellobiohydrolase
Gene00450	cbhB	1, 4-beta-d-glucan	ATGTCTTCCTTTCAGATC/GTCTCAGTAGTGTAGGTT
		cellobiohydrolase
Gene08250	eglA	Endoglucanase	GAGCAGAAGACATATAGC/GTGAGATAGTTGAAGAAGT
Gene01363	eglB	Endoglucanase	TATCTCCTTCTATTGCTTCC/ATCGTTCTATCGTCACTAC
Gene11315	eglC	Endoglucanase	TCTCTACACCATGATTCAAG/GAAGTATCCTGGGCAATG
Gene09061	ena1	Sodium P-type ATPase	ATCTGTGCTGTCATTGTC/GTGAAGCGATTCCATAGT
Gene05606	ena2	Sodium P-type ATPase	CTAGTGGAGACGATGAAC/GGAAGGTAGTAGTAGATGATT
Gene03146	ena4	Sodium P-type ATPase	AGGCTATTGCTGCTCTTC/CTTGACACGCTTGGTAAC
Gene11257	sho1	High osmolarity signaling	TTACAACAATCTCCATCA/CCGAAGTAGAATATCCAA
		protein
Gene01120	fhlA	Sensor histidine kinase/response	CACACCAACATCAACATC/CCTTCTCCATATCATCCAA
		regulator
Gene03514	nik1	Two-component response	TGAACGGTAATACGATAG/TTGCTTCCATACTAACAT
		regulator
Gene10443	ypdl	Phosphotransmitter protein	ACGATAGCGACAGAGATT/AAGCATCCTCCATCTTGA
Gene09867	pkA	CAMP-dependent protein	CAGCAACTTCCTCTTCTC/GATAGGCTTCTCCACCAG
		kinase subunit
Gene02843	β-tubulin	Internal standard	TCAAGATGTCCTCTACCT/GGAACATAGCAGTGAACT

Tag code	Gene	Annotation	Sequences of paired primers (5′→3′)
Gene08183	fluG	Upstream development activator	AAGATTGCTGCTGCTGATA/GTCGTAGGTAATCGTCTCATT
Gene03944	flbA	Upstream development activator	AGCAGCATCTCTACTTCAG/CTCCTTCGCCATCTTCAC
Gene04120	flbC	Upstream development activator	CTACTATTCCGCCTCTGT/GCCATTCTCGTAAGATTCC
Gene06270	flbB	Upstream development activator	CTCTTCCTTCCATCTCTA/TCATTCTAGTACGCTTTG
Gene04684	flbD	Upstream development activator	TGGACATGAGACAGCAATAC/AGAGATCGTGGTAGGCATA
Gene02514	flbE	Upstream development activator	TGGTGGATTGTGTATAAC/CTCATCATACCCATCATC
Gene00348	laeA	Developmental repressor	GGACTTGGAGAGAATCAG/AGATGTTGTAAGCGTGTAT
Gene02682	veA	Developmental activator	GTTCCTCTTACACTTCTCAT/GAGTTCCAGGTAGTTGTG
Gene00832	sfgA	Developmental repressor	CGAACAACCTGCTTATAGTC/GCTGCTTGAATCTCCATC
Gene04565	velB	Developmental repressor;	CCTCAGATGAACAATTAC/TAGTAAGGCTGATAATAGG
		Regulation for conidial
		maturation
Gene04761	abaA	Regulation for sterigmata	AACATCCTTTCCTTACCT/GTCATCCATCAACTTCAG
		formation
Gene04357	wetA	Regulation for conidial	CAGTGTGGATGGCAACAA/CGCTGGACAATGACTCAAG
		maturation
Gene10505	vosA	Regulation for conidial	ATGTGGATAATACTGATG/ATCTATCTGTGATGATTG
		maturation
Gene00550	brlA	Initiator for conidiogenesis	AGAACCTTCCGTCTATTATCA/ATGCTCTTGCCTCTTGAA
Gene05820	stuA	Modulator for conidiophore	ATATGATTAACGGCACAA/CTCACTCTTCAGAATACC
		formation
Gene00245	creA	Carbon catabolite repressor	TTCTTCTGATGAGGAGAG/CGGACTAACAACAACTTT
Gene08487	srgA	Secretion related GTPase	TACTGCTGATTGGTGACT/TAATGAACGACGGAGTGA
Gene08788	amyA	Alpha-amylase	GCGAAGAATATCCTCACAT/CATCCAAATGCTGTTCCT
Gene06696	amyC	Alpha-amylase	TCTAATGGTTCGGCTTAT/GTATAGTTGACGCAGTTC
Gene00277	amyD	Alpha-amylase	AACATCTTCTCTGATACATTAGT/GATTGTCCTGGTTGAGTG
Gene11416	amyE	Alpha-amylase	GCTTGTCTCTAACTACTC/TTCTTGACTGTGTCTATAC
Gene08787	amyF	Alpha-amylase	GAAGTGGATTATGTCTGT/ATAAACTCCCTCTGAAAC
Gene07580	amyG	Glucan 1, 4-alpha-glucosidase	TCAATCCATCTACTTCCT/ATAATTCCTTGCCAACTT
Gene10420	amyH	Alpha-amylase	GCTTGTCTCTAACTACTC/TTCTTGACTGTGTCTATAC
Gene05110	amyM	Alpha-amylase	TCCATTGTACTACATTCTCA/GCTGATAGACTCGGTAAT
Gene10422	amyR	Transcription regulator of	CAGTAGATGGAATGAATATAGAA/GCTTGACAGAGGTAGAAC
		maltose utilization
Gene00933	aglB	Alpha-galactosidase	GAGTTGAAGAGTAAGAATT/CACCTCATACTTATCCTT
Gene00399	lacA	Beta-galactosidase	ATTGCCATTATGAACTTGTA/TAGTCGTAGGAGGTGTAT
Gene03324	pexB	Pectinase	ACTCTTCAGCGACTTCCT/GCCTCTAATTGTATTCCATAACG
Gene04089	pecA	Pectinase	GAGACTCTTGACTTGACT/CTTCCATTCCTTGTAACC
Gene08774	pecB	Pectinase	CGTCAAGATCGAGAACAT/CGCAGAGGATGTAGTAATC
Gene06504	pecC	Pectinase	TTGACCTCACTGACCTTA/CTGTCCATTCCTCATAACC
Gene09643	endA	NADH dehydrogenase	AAGACCGTTTATGATGCTA/CGATGACAATACCGTAGT
Gene06504	endC	NADH dehydrogenase	TTGACCTCACTGACCTTA/CTGTCCATTCCTCATAACC
Tag code	Gene	Annotation	Sequences of paired primers (5′→3′)
Gene08115	poly	Polymerase	GGCTCTGATACCAATGTTAG/CTACCTCCGTTGCTCTTA
Gene05290	xlnR	Xylanolytic transcriptional	CAACTTTCCTTCTTTCAG/ATAATGGAGGCAATATGT
		activator
Gene01326	cbhA	1, 4-beta-d-glucan	AATAATGACAACACAGGTA/GAGATACTATTGGCTTCC
		cellobiohydrolase
Gene00450	cbhB	1, 4-beta-d-glucan	ATGTCTTCCTTTCAGATC/GTCTCAGTAGTGTAGGTT
		cellobiohydrolase
Gene08250	eglA	Endoglucanase	GAGCAGAAGACATATAGC/GTGAGATAGTTGAAGAAGT
Gene01363	eglB	Endoglucanase	TATCTCCTTCTATTGCTTCC/ATCGTTCTATCGTCACTAC
Gene11315	eglC	Endoglucanase	TCTCTACACCATGATTCAAG/GAAGTATCCTGGGCAATG
Gene09061	ena1	Sodium P-type ATPase	ATCTGTGCTGTCATTGTC/GTGAAGCGATTCCATAGT
Gene05606	ena2	Sodium P-type ATPase	CTAGTGGAGACGATGAAC/GGAAGGTAGTAGTAGATGATT
Gene03146	ena4	Sodium P-type ATPase	AGGCTATTGCTGCTCTTC/CTTGACACGCTTGGTAAC
Gene11257	sho1	High osmolarity signaling	TTACAACAATCTCCATCA/CCGAAGTAGAATATCCAA
		protein
Gene01120	fhlA	Sensor histidine kinase/response	CACACCAACATCAACATC/CCTTCTCCATATCATCCAA
		regulator
Gene03514	nik1	Two-component response	TGAACGGTAATACGATAG/TTGCTTCCATACTAACAT
		regulator
Gene10443	ypdl	Phosphotransmitter protein	ACGATAGCGACAGAGATT/AAGCATCCTCCATCTTGA
Gene09867	pkA	CAMP-dependent protein	CAGCAACTTCCTCTTCTC/GATAGGCTTCTCCACCAG
		kinase subunit
Gene02843	β-tubulin	Internal standard	TCAAGATGTCCTCTACCT/GGAACATAGCAGTGAACT

黑曲霉RAF106 sakA基因的敲除及功能分析

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朱韵琦 ¹^,²^,^# , 周刚 ¹^,²^,^# , 刘通 ¹^,² , 王颖思 ¹ , 李素娟 ¹ , 彭如群 ¹ , 彭红 ¹ , 施庆珊 ¹ , 王洁 ²^,^* , 谢小保 ¹^,^*

微生物学报 | 研究报告 2024,64(8): 2684-2701

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微生物学报 | 研究报告 2024, 64(8): 2684-2701

黑曲霉RAF106 sakA基因的敲除及功能分析

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朱韵琦¹^,²^,^#, 周刚¹^,²^,^#, 刘通¹^,², 王颖思¹, 李素娟¹, 彭如群¹, 彭红¹, 施庆珊¹, 王洁²^,^*, 谢小保¹^,^*

作者信息

1 广东省科学院微生物研究所华南应用微生物国家重点实验室广东省菌种保藏与应用重点实验室, 广东广州 510070

2 华南农业大学食品学院, 广东广州 510642

Gene knockout reveals the roles of sakA in Aspergillus niger RAF106

Yunqi ZHU¹^,²^,^#, Gang ZHOU¹^,²^,^#, Tong LIU¹^,², Yingsi WANG¹, Sujuan LI¹, Ruqun PENG¹, Hong PENG¹, Qingshan SHI¹, Jie WANG²^,^*, Xiaobao XIE¹^,^*

Affiliations

1 Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, Guangdong, China

2 College of Food Science, South China Agricultural University, Guangzhou 510642, Guangdong, China

出版时间: 2024-03-28 doi: 10.13343/j.cnki.wsxb.20240012

文章导航

摘要

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【目的】黑曲霉(Aspergillus niger) sakA基因是应激丝裂原活化蛋白激酶家族(mitogen-activated protein kinase, MAPK)的重要成员，然而，在黑曲霉中，关于SakA的分子功能研究很少。本研究通过构建黑曲霉sakA缺失突变株探究SakA在黑曲霉中的功能。【方法】以黑曲霉RAF106为出发菌株，通过农杆菌介导法构建突变株ΔsakA菌株。监测ΔsakA菌株与野生菌株在3种不同培养基上生长与产孢情况；研究ΔsakA菌株对不同逆境胁迫的敏感性变化；测定ΔsakA菌株的胞内外淀粉酶、果胶酶和纤维素酶酶活差异；qRT-PCR分析ΔsakA菌株产孢相关基因、淀粉酶相关基因、果胶酶相关基因、纤维素酶相关基因及高渗调节相关基因的相对转录水平。【结果】成功获得3株sakA缺失突变株ΔsakA菌株；发现ΔsakA菌株较野生菌株生长缓慢、产孢延迟及分生孢子梗分化延迟；ΔsakA菌株在0.6 mol/L KCl、0.8 mol/L NaCl和1.2 mol/L NaCl胁迫条件下，菌落生长均比野生型缓慢；ΔsakA菌株胞外淀粉酶产量提高20.68%−21.43%，胞内淀粉酶产量显著下降19.18%−20.26%；胞外果胶酶产量显著下降36.71%−38.30%，胞内果胶酶产量显著上升35.68%−36.53%；与野生菌株相比，ΔsakA菌株胞外纤维素酶产量显著下降28.04%−33.82%，而胞内纤维素酶产量显著上升15.28%−18.19%；产孢相关基因(fluG、sfgA、flbA、flbB、flbD、laeA、brlA、abaA、vosA、stuA和velB)的转录水平相较于野生菌株下调8.53%−90.87%；淀粉酶相关基因(amyC、amyD、amyE、amyF、amyG和amyH)及转录因子(amyR)下调8.87%−87.50%，果胶酶相关基因(aglB、lacA、pexB、pecA、pecC、pecB、endA、endC和poly)下调23.23%−84.01%，纤维素酶相关基因(xlnR、chbA、chbB和eglB)下调3.75%−81.02%，高渗调节相关基因(ena1、ena2、sho1、nik1、ypdl、pkA和hAD)下调5.27%−94.36%。【结论】sakA基因正向调控黑曲霉RAF106的产孢能力，是产孢过程中的重要基因，其缺失影响黑曲霉分生孢子的产生；在参与渗透压胁迫应答的同时对淀粉酶、果胶酶与纤维素酶的合成与分泌有重要作用。

关键词

黑曲霉 / sakA基因 / 丝裂原活化蛋白激酶(MAPK)信号通路 / 产孢 / 渗透压 / 代谢

Abstract

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[Objective] The protein SakA encoded by sakA is a member of the mitogen-activated protein kinase (MAPK) family in Aspergillus niger. However, little is known about the roles of SakA in A. niger. In this study, we constructed the A. niger strains with knockout of sakA to investigate the roles of this gene. [Methods] The Agrobacterium-mediated method was utilized to construct ΔsakA strains from A. niger RAF106 (the wild type, WT). The growth and spore production of ΔsakA and WT were observed on three different media. The sensitivity of ΔsakA and WT to different stress conditions was studied. The intracellular and extracellular levels of amylase, pectinase, and cellulase were compared between ΔsakA and WT. Real-time quantitative polymerase chain reaction (qRT-PCR) was employed to determine the relative transcript levels of the genes associated with spore production, amylase, pectinase, cellulase, and hyperosmotic regulation. [Results] Three ΔsakA strains were successfully obtained and verified by PCR and qRT-PCR. The ΔsakA strains had slow growth, delayed spore production, and delayed conidiophore differentiation compared with WT. The ΔsakA strains showcased slower colony growth than WT under the stress conditions of 0.6 mol/L KCl, 0.8 mol/L NaCl, and 1.2 mol/L NaCl. Compared with WT, the knockout of sakA increased the extracellular amylase production by 20.68%–21.43% and decreased the intracellular amylase production by 19.18%–20.26%, decreased the extracellular pectinase production by 36.71%–38.30% and increased the intracellular pectinase production by 35.68%–36.53%, decreased the extracellular cellulase production by 28.04%–33.82% and increased the intracellular cellulase production by 15.28%–18.19%. Compared with WT, the knockout of sakA down-regulated the transcript levels of spore production-related genes (fluG, sfgA, flbA, flbB, flbD, laeA, brlA, abaA, vosA, stuA, and velB) by 8.53%–90.87%. Furthermore, it down-regulated the transcript levels of amylase-related genes (amyC, amyD, amyE, amyF, amyG, and amyH) and the transcription factor (amyR) by 8.87%–87.50%, the pectinase-related genes (aglB, lacA, pexB, pecA, pecC, pecB, endA, endC, and poly) by 23.23%–84.01%, the cellulase-related genes (xlnR, chbA, chbB, and eglB) by 3.75%–81.02%, and the hyperosmotic regulation-related genes (ena1, ena2, sho1, nik1, ypdl, pkA, and hAD) by 5.27%–94.36%. [Conclusion] The sakA gene of A. niger positively regulates spore production and is essential for spore production. The knockout of sakA affects the spore production of A. niger. Furthermore, SakA plays a crucial role in the synthesis and secretion of amylase, pectinase, and cellulase as well as osmotic stress response.

Key words

Aspergillus niger / sakA / mitogen-activated protein kinase (MAPK) signaling pathway / spore production / osmotic stress / metabolism

引用本文

朱韵琦, 周刚, 刘通, 王颖思, 李素娟, 彭如群, 彭红, 施庆珊, 王洁, 谢小保. 黑曲霉RAF106 sakA基因的敲除及功能分析. 微生物学报, 2024 , 64 (8) : 2684 -2701 . DOI: 10.13343/j.cnki.wsxb.20240012

Yunqi ZHU, Gang ZHOU, Tong LIU, Yingsi WANG, Sujuan LI, Ruqun PENG, Hong PENG, Qingshan SHI, Jie WANG, Xiaobao XIE. Gene knockout reveals the roles of sakA in Aspergillus niger RAF106[J]. Acta Microbiologica Sinica, 2024 , 64 (8) : 2684 -2701 . DOI: 10.13343/j.cnki.wsxb.20240012

正文

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真核生物在面对各种外界刺激时，需要利用一系列的蛋白质信号级联反应及时作出快速响应^[1]。应激丝裂原活化蛋白激酶家族(mitogen-activated protein kinase, MAPK)通路是真核生物中高度保守的信号级联典型通路之一^[2]，可调节真菌从有性生殖到应激反应的众多过程，是许多丝状真菌调节发育和次级代谢的关键途径，被广泛研究和利用^[3]。MAPK级联的特点是具有三级模块，由MAPKKK、MAPKK和MAPK三种蛋白激酶组成；它将细胞外刺激转化为真核生物的细胞内反应，3个蛋白激酶依次进行磷酸化激活，最终结果是激活不同的转录因子，启动相应的生物学过程，从而适应环境的变化^[4]。

MAPK信号通路参与真核生物对各种胁迫的响应和发育过程；通路中相关基因的突变导致病原菌的表型异常、在不同生长条件下的严重损伤、发育和分化的改变，以及病原菌的毒力降低或丧失^[5]。Kawasaki等^[6]鉴定并报道的sakA基因为编码应激MAPK的家族之一，其编码的SakA蛋白为上游MAPKK (MAPK激酶)家族中的底物^[7]。Miskei等^[8]对构巢曲霉、黄曲霉、黑曲霉、烟曲霉和米曲霉等8种曲霉的应激反应蛋白按应激类型进行了标注和分组，显示8种曲霉均含有与SakA直接关联的胁迫信号元件。许多研究也表明^[9-11]，SakA及其同源物也在许多真菌中与应激感知和发育或致病性调节有关。SakA参与了丝状真菌胁迫信号转导和有性发育抑制，是孢子抗胁迫和存活所必需的^[6]，在渗透和氧化胁迫下以及诱导无性孢子形成(分生)后的早期都发生了短暂的磷酸化，虽然sakA突变体对渗透胁迫不敏感，但它们产生的无性孢子逐渐失去生存能力，对氧化和热休克胁迫敏感；SakA对分生孢子维持其生存能力和在高浓度H₂O₂中存活也是至关重要的^[12]。然而，SakA在黑曲霉中的功能研究报道极少。黑曲霉因其代谢系统强大，广泛应用于生产有机酸和各种酶制剂等，是工业领域生产中常用的工程微生物之一。研究人员对黑曲霉代谢通路中某个或某些基因的敲除甚至过表达是改造野生型黑曲霉为工业化菌株的常用手段。

本课题组前期从普洱茶中分离到一株能降解黄曲霉毒素B1 (aflatoxin B1, AFB1)的黑曲霉RAF106，该菌株能合成α-淀粉酶、纤维素酶、果胶酶、单宁酶和柠檬酸等活性物质，不产生真菌毒素，可介导多酚、黄酮类化合物生物转化而提高生物可利用度和生理活性^[13-15]，因而颇具食品加工与工业发酵应用潜力。因此，本研究以黑曲霉RAF106为出发菌株，利用同源重组技术敲除sakA基因，通过分析对比野生株和sakA突变株之间的多表型变化、各种酶活及表型相关基因的转录表达情况，以期全面揭示黑曲霉中SakA的功能，为进一步探究黑曲霉MAPK信号途径基因的功能奠定基础。

1 材料与方法

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1.1 菌株和质粒

黑曲霉(Aspergillus niger) RAF106野生菌株(wide type, WT)由本课题组从普洱茶中自行分离，并保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC NO. 9608；大肠杆菌(Escherichia coli) DH5α用于质粒的扩增；根瘤农杆菌(Agrobacterium tumefaciens) AGL-1用于黑曲霉RAF106的遗传转化。

p0380-hygR为真菌敲除质粒并保存于本实验室，质粒构建参照文献[16]。

1.2 培养基

生长与产孢培养基为马铃薯葡萄糖琼脂(potato dextrose agar, PDA)固体培养基(g/L)：去皮马铃薯200.0，葡萄糖20.0，琼脂15.0；察氏(Czapek agar, CZA)固体培养基(g/L)：蔗糖30.0，NaNO₃ 30.0，K₂HPO₄ 10.0，KCl 5.0，MgSO₄ 5.0，FeSO₄ 0.1，琼脂15.0；萨氏(Sabouraud dextrose agar, SDAY)培养基(g/L)：葡萄糖40.0，蛋白胨10.0，酵母粉10.0，琼脂15.0。发酵培养基为麦麸-秸秆(WBS)液体培养基(g/L)：秸秆粉25.0，麦麸30.0，蛋白胨30.0，(NH₄)₂SO₄ 1.6，MgSO₄ 0.2，KH₂PO₄ 2.0，CaCl₂ 0.01，ZnCl₂ 0.002，尿素0.02，pH 5.0。

1.3 主要试剂和仪器

主要试剂：TRIzol^® Reagent，Invitrogen公司；RNase抑制剂和Taq酶，Promega公司；核酸内切酶，Biolabs公司；吐温80，天津市大茂化学试剂厂；潮霉素HYG和噻孢霉素，北京索莱宝科技有限公司；高保真聚合酶PrimeSTAR Max DNA Polymerase和DNA Ligation Kit连接酶，TaKaRa公司；ChamQ Universal SYBR qPCR Master Mix，Vazyme公司。

试剂盒：真菌基因组DNA提取试剂盒，BioFlux公司；质粒回收和胶回收试剂盒E.Z.N.A^® Gel Extraction Kit，Omega公司；HiScript^® II 1st Strand cDNA Synthesis Kit，Vazyme公司；SanPrep柱式质粒DNA小量抽提试剂盒，生工生物工程(上海)股份有限公司。

仪器：PCR扩增仪和荧光定量PCR仪，Bio-Rad公司；酶标仪，赛默飞世尔科技公司；生化培养箱，天津市泰斯特仪器有限公司；生物显微镜，麦克奥迪实业集团有限公司。

1.4 黑曲霉基因组的提取

黑曲霉在PDA上30 ℃培养5 d，在平板上加入吐温80，刮下孢子，用自制带棉花滤头过滤制得孢子悬液。将浓度为10⁶个/mL的黑曲霉RAF106孢子悬液100 μL涂布接种于铺有玻璃纸的PDA平板上，30 ℃静置培养24 h后，轻刮平板收集菌丝，在研钵中加入液氮研磨至粉末，按照BioFlux真菌基因组提取试剂盒提取黑曲霉基因组总DNA。

1.5 sakA基因的克隆及分析

使用NCBI数据库进行查询，在黑曲霉基因组中鉴定了一个MAP激酶SakA。用配对引物sakA-F/R (表1)从WT扩增基因序列，并送往Invitrogen进行测序验证。通过在线BLAST对推导的蛋白质序列进行结构分析(http://blast.ncbi.nlm.nih.gov/blast.cgi)，搜索并下载与SakA同源性较高的蛋白序列，随后使用MEGA 7.0软件进行系统聚类分析。

1.6 sakA基因缺失突变株的构建

提取黑曲霉RAF106总DNA，使用5′sakA-F/R和3′sakA-F/R (表1)以及高保真聚合酶PrimeSTAR Max DNA Polymerase分别扩增sakA的上、下游同源臂sakA-F (1 892 bp)和sakA-R (1 969 bp)的片段，对扩增产物进行胶回收。分别经Xma I/BamH I、Xho I/Bgl II双酶切后用DNA Ligation Kit连接酶依次插入p0380-hygR质粒hygR两端。经DH5α转化、PCR验证即获得正确的转化子，LB液体培养基中扩大培养，按照SanPrep柱式质粒DNA小量抽提试剂盒说明书提取质粒。通过酶切验证sakA缺失突变株的正确性。采用农杆菌介导的真菌转化法^[17]，将该敲除质粒导入黑曲霉RAF106中，在含有噻孢霉素(300 μg/mL)和潮霉素(50 μg/mL)的M-100抗性平板进行筛选，分别使用引物sakA-ID (表1)进行PCR及qRT-PCR鉴定，获得sakA缺失突变株。构建质粒所用引物见表1。

1.7 sakA缺失对黑曲霉生长与产孢的影响

将10⁶个/mL的孢子悬液点样1 μL在PDA、SDAY和CZA的平板上，在30 ℃条件下培养48 h，十字交叉法测量各菌落直径，计算相对生长抑制率(relative growth inhibition, RGI)，作为评估真菌生长的指标。为了评估ΔsakA菌株产孢能力，将10⁶个/mL孢子悬液均匀涂布于PDA、SDAY和CZA平板，置于30 ℃下培养7 d，从第3天起用打孔器从各平板上切取直径5 mm的菌片于1.5 mL离心管中，每管加入1 mL 0.02%吐温80，超声10 min洗下孢子，用血球计数板测定各管中孢子浓度，再转换为每板单位面积的产孢量(spores/cm²)。使用刀片切取PDA平板上培养3 d的黑曲霉RAF106野生株与ΔsakA菌株的菌丝块，在光学显微镜下观察分生孢子梗形态。

1.8 sakA缺失对黑曲霉逆境胁迫反应的影响

通过将10⁶个/mL的ΔsakA菌株分生孢子悬液点样1 μL在单独的PDA (对照)或添加刚果红(CR：2 mg/mL、3 mg/mL)、十二烷基硫酸钠(SDS：0.02%、0.03%)的平板上，评估分生孢子对细胞壁胁迫的敏感度。同时观察菌株在不同胁迫条件下的生长差异，计算相对生长抑制率，不同胁迫条件包含：氯化钾(KCl：0.6 mol/L)、氯化钠(NaCl：0.8 mol/L、1.2 mol/L)、氧化剂H₂O₂ (H₂O₂：6 mmol/L、9 mmol/L)、山梨醇(sorbitol：0.6、0.8、1.2 mol/L)、甲萘醌(MND：0.5 μmol/L、2.0 μmol/L)。

1.9 胞内外淀粉酶、果胶酶和纤维素酶酶活测定

采用DNS法测定淀粉酶、果胶酶、纤维素酶活性^[14]。首先，将10⁸个/mL的孢子悬液接种于WBS，置于30 ℃、180 r/min下培养3 d，取上清液用于胞外酶活测定；与此同时，取WBS培养基中菌丝于预冷的研钵中，液氮充分研磨后转移至离心管，加入1 mL pH 5.0的0.05 mol/L柠檬酸缓冲液，振荡5 min后于4 ℃、10 000 r/min离心10 min即为胞内酶粗提液，用于胞内酶活测定。

对于淀粉酶酶活测定，在0.05 mol/L柠檬酸钠缓冲液中加入0.45 mL 1%可溶性淀粉溶液于刻度管中，吸取0.05 mL发酵上清液混合均匀，50 ℃水浴30 min。对于果胶酶酶活测定，在0.05 mol/L柠檬酸钠缓冲液中加入0.45 mL 0.5%果胶酶溶液于刻度管中，吸取0.05 mL发酵上清液混合均匀，50 ℃水浴30 min。对于纤维素酶酶活测定，在0.05 mol/L柠檬酸钠缓冲液中加入0.95 mL 1%羧甲基纤维素钠(sodium carboxymethylcellulose, CMC-Na)溶液于刻度管中，吸取0.05 mL发酵上清液混合均匀，50 ℃水浴30 min。上述反应液分别加入0.75 mL DNS显色液，将刻度管放在沸水中加热10 min，反应停止。空白组中加入0.05 mL煮沸灭酶(灭活)的发酵上清液。一个酶活力单位(U)定义为在一定的pH条件下，每分钟水解底物产生1 mmol葡萄糖、1 mmol半乳糖醛酸和1 mmol纤维二糖所需的酶量。

1.10 RNA提取及反转录

将10⁶个/mL的孢子悬液接种于PDA和WSA (加入2%的琼脂粉到WBS中)平板表面的玻璃纸上，于30 ℃下培养1 d后收获菌丝。取适量菌丝于液氮预冷的研钵中研磨至粉末，参照文献[18]的方法提取RNA。根据反转录试剂盒说明书操作，反转录反应体系(20 μL)：5×PrimeScript^® Buffer 4 μL，PrimeScript^®RT Enzyme Mix I 1 μL，Oligo Dt Primer (50 μmol/L) 1 μL，Random 6 mers (100 μmol/L) 1 μL，RNA模板2 μL，RNase-free ddH₂O 11 μL。反应条件：37 ℃ 15 min，85 ℃ 5 s，即获得野生株的cDNA。

1.11 实时荧光定量PCR

以β-tubulin为内参，通过qRT-PCR和配对引物评估15个产孢基因、11个淀粉酶相关基因、9个果胶酶相关基因、6个纤维素酶相关基因及9个高渗调节相关基因的转录水平，采用Livak阈值循环(2^−ΔΔCt)方法进行相对定量分析，利用SPSS Statistics 23分析不同组间的差异性。实时荧光定量PCR所用引物见表2。

2 结果与分析

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2.1 sakA基因的生物学特征

使用NCBI数据库进行查询，在黑曲霉RAF106基因组中定位到单个sakA (基因库登录号：01755)，保守结构域如图1A所示。通过基因组DNA和cDNA序列，获得了8个内含子，分别由55、84、54、69、50、51、69和50个碱基组成，SakA的开放阅读框由1 191个碱基组成，这些碱基编码396个氨基酸，其分子量约为25.52 kDa，等电点为5.00。氨基酸序列比较和系统发育分析显示，与Aspergillus piperis的MAP激酶SakA相似性最高(99.64%)，与Aspergillus sclerotiicarbonarius的丝裂原活化蛋白激酶Mpkc相似性最低(72.13%，图1B)。将SakA蛋白进行结构域分析(https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)发现，SakA具有丝裂原活化蛋白激酶的保守位点和典型的ATP结合位点。ATP结合位点在保守结构域STKc_Sty1_Hog1上，构成此保守特征的22个残基中有22个序列已被查询(附图1A，数据已提交国家微生物科学数据中心，编号为NMDCX0000265)。通过与蛋白激酶的保守性比对分析(附图1B，数据已提交国家微生物科学数据中心，编号：NMDCX0000265)，发现SakA蛋白具有PKC蛋白激酶超家族(NCBI登录号为cl21453)。蛋白激酶超家族主要由丝氨酸/苏氨酸特异性和酪氨酸特异性蛋白激酶的催化结构域组成，它还包括RIO激酶，即非典型丝氨酸蛋白激酶、氨基糖苷磷酸转移酶和胆碱激酶^[19]。这些蛋白质催化γ-磷酸基团从ATP转移到特定底物(如蛋白质的丝氨酸、苏氨酸或酪氨酸残基)中的羟基；丝裂原活化蛋白激酶通过磷酸化途径在真菌逆境胁迫响应中发挥重要作用^[20]，因此推测SakA可能与发育过程中逆境胁迫响应有关。

2.2 sakA敲除质粒的构建及转化子筛选

利用同源重组的方法，以潮霉素的抗性基因hygR表达元件替换sakA基因中的部分序列(图2A)。如图2B所示，通过PCR鉴定，获得3株sakA缺失突变株(ΔsakA)。qRT-PCR结果(图2C)显示，在3株突变株中未检测到sakA的表达，进一步表明突变体中的sakA基因已被敲除。

2.3 sakA缺失影响黑曲霉的生长及产孢

在PDA、SDAY和CZA平板上培养24 h后，所有ΔsakA菌株的菌落直径与野生型相比无明显差异(图3A)。然而，sakA缺失后生长缓慢导致黑曲霉RAF106分生孢子产量降低，孢子梗发育迟缓。与野生菌株相比，ΔsakA菌株在第3−7天的平均孢子量在PDA平板上分别减少87.50%、66.80%、51.03%、38.86%和40.68% (图3B)，在SDAY平板上分别减少57.77%、73.77%、56.35%、49.68%和31.64% (图3C)，在CZA平板上分别减少90.38%、84.38%、73.68%、75.96%和62.72% (图3D)。通过光学显微镜观察野生株与ΔsakA菌株分生孢子梗发现：黑曲霉RAF106野生株分生孢子梗分化完全，顶端膨大，孢子囊呈圆形，而ΔsakA菌株的分生孢子梗分化延迟，孢子囊形态有变形(图3E)。

鉴于黑曲霉sakA基因可能在响应生长与产孢调控过程中发挥作用，采用qRT-PCR反应测得WT与ΔsakA菌株产孢基因的表达水平。通过测定黑曲霉中15个产孢相关基因的转录水平发现，它们在ΔsakA菌株中有不同程度的上调或下调(图3F)，ΔsakA菌株表现严重产孢缺陷，产孢相关基因(fluG、sfgA、flbA、flbB、flbD、laeA、brlA、abaA、vosA、stuA和velB)的转录水平下调8.53%−90.87%，仅flbC产孢基因有所上调。产孢相关基因转录水平的下调显然是其表现产孢缺陷的重要原因。

2.4 sakA基因对黑曲霉逆境胁迫反应的影响

为了探究SakA是否参与到黑曲霉对外界环境胁迫应答反应，分析了ΔsakA菌株在不同胁迫条件下的生长情况(图4A)。在0.6 mol/L KCl、0.8 mol/L NaCl和1.2 mol/L NaCl的胁迫条件下，ΔsakA菌株菌落生长均较野生型缓慢，与WT相比，ΔsakA菌株生长平均抑制率分别为12.46%、16.09%和55.35%。相比渗透压胁迫剂，在0.8 mol/L sorbitol、0.02% SDS、0.03% SDS、6 mmol/L H₂O₂和9 mmol/L H₂O₂胁迫条件下，WT与突变株的相对生长抑制率较小，但是ΔsakA菌株产孢量明显变少。以上结果表明，黑曲霉中sakA的缺失导致了菌株对渗透压更加敏感。与野生株相比，ΔsakA菌株在0.6 mol/L sorbitol、1.2 mol/L sorbitol、0.5 μmol/L MND、2 μmol/L MND、2 mg/mL CR和3 mg/mL CR胁迫条件下无差异。为分析SakA对黑曲霉抗渗透胁迫能力的影响，检测了突变株中9个高渗调节相关基因的表达(图4B)。除ena4和fhlA外，ena1、ena2、sho1、nik1、ypdl、pkA和hAD平均表达量均低于野生型。

2.5 sakA缺失影响淀粉酶、果胶酶与纤维素酶的合成与分泌

sakA基因缺失影响了黑曲霉RAF106在常温条件下淀粉酶、果胶酶与纤维素酶的合成与分泌。与野生菌株相比，ΔsakA菌株胞外淀粉酶产量上升20.68%−21.43% (图5A)，胞内淀粉酶产量显著下降19.18%−20.26% (图5B)，这表明sakA基因缺失影响了黑曲霉在常温条件下淀粉酶的合成。ΔsakA菌株胞外果胶酶产量显著下降36.71%−38.30% (图5C)，胞内果胶酶产量显著上升35.68%−36.53% (图5D)，这表明sakA基因缺失影响了黑曲霉果胶酶的分泌。与野生菌株相比，在30 ℃下ΔsakA菌株胞外纤维素酶产量显著下降28.04%−33.82% (图5E)，胞内纤维素酶产量显著上升15.28%−18.19% (图5F)。

2.6 淀粉酶、果胶酶与纤维素酶相关基因的转录水平分析

通过测定黑曲霉中11个淀粉酶、9个果胶酶与6个纤维素酶相关基因的转录水平。在sakA缺失突变株中，淀粉酶相关基因amyC、amyD、amyE、amyF、amyG、amyH及转录因子amyR下调8.87%−87.50% (图6A)，果胶酶相关基因aglB、lacA、pexB、pecA、pecC、pecB、endA、endC、poly下调23.23%−84.01% (图6B)，纤维素酶相关基因xlnR、chbA、chbB、eglB下调3.75%−81.02% (图6C)。从图6可以看到突变株的淀粉酶、果胶酶与纤维素酶的相关基因比野生菌株的表达量低，这可能是淀粉酶、果胶酶与纤维素酶产量降低的主要原因。

3 讨论与结论

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高渗透压甘油途径(high osmolarity glycerol pathway, HOGP)中的激酶MAPK是一种丝裂原活化的蛋白激酶，已在多种真菌物种中得到鉴定，它能够调节多种细胞过程，如渗透适应等^[21-22]。sakA是Hog1-MAPK信号通路中的核心基因，对它的研究主要集中在构巢曲霉与烟曲霉中，而在黑曲霉中鲜有报道。SakA在构巢曲霉中被报道为反应调节器、响应氧化或渗透应激信号^[23]。在马尔尼菲青霉(Penicillium marneffei)这种致病性真菌中，sakA基因不仅参与氧化和热应激反应，而且对分生孢子的形成发挥重要作用^[24]。本研究对ΔsakA菌株在3种不同培养基下生长和产孢情况与野生菌株进行比较，同时分析产孢关键基因的转录水平。从表型上看，ΔsakA菌株菌落直径与野生型相比无明显差异，但是产孢量明显下降。通过测定3−7 d在PDA、SDAY和CZA平板上的孢子量，发现ΔsakA菌株产孢量均明显降低，显微镜下观察到孢子囊变小且孢子梗分化延迟。在构巢曲霉中sakA/atfA基因敲除后同样会导致菌株生长迟缓且分生孢子形成受到抑制^[23]，同时，sakA基因也是烟曲霉生长所必需的因子^[25]。通过qRT-PCR分析发现，黑曲霉ΔsakA菌株中sfgA、flbA、laeA、brlA、abaA、stuA和velB基因的表达量明显下调，这与sakA基因的缺失导致马尔尼菲青霉产孢数量减少，且abaA、wetA等基因的转录受抑制结果相似，但brlA基因的调控并无显著性变化^[24]。黑曲霉中abaA、wetA和brlA基因是产孢调控的核心调节因子，组成了主要的产孢调控途径；这3个基因协同调节多个基因的表达，在孢子形成过程中起着重要的作用^[25-26]。黑曲霉ΔsakA菌株中sfgA、flbA、flbB、laeA、brlA、abaA、stuA和velB基因的表达量明显下调90%左右，由此我们推测sakA基因直接调控abaA和brlA产孢核心调节基因，从而影响下游基因的表达。

真菌利用与MAP激酶模块偶联的磷酸化系统来感知和处理环境信号，SakA信号在不同类型曲霉的应激反应中有重要作用^[8]。在烟曲霉中，向培养基中添加H₂O₂、甲萘醌或SDS时，sakA缺失突变株生长抑制率增加^[27]。在我们的研究中，0.8 mol/L sorbitol、0.02% SDS、0.03% SDS、6 mmol/L H₂O₂和9 mmol/L H₂O₂胁迫条件下，相对生长抑制率较小。sakA缺失突变株对H₂O₂、甲萘醌或SDS不敏感。我们的研究发现，ΔsakA菌株在渗透压胁迫剂0.6 mol/L KCl、0.8 mol/L NaCl与1.2 mol/L NaCl的胁迫条件下，较野生株生长缓慢，对渗透压敏感，揭示sakA基因在黑曲霉抵抗外界胁迫中有着重要的作用。通过qRT-PCR分析发现，其中sho1与nik1响应稳压调节的基因下调82.66%与82.51%左右，这可能是导致sakA缺失突变体对渗透压敏感的原因^[28-29]。

真菌具有适应环境不同渗透压的能力，从而引起酶系的变化^[30-31]。在本研究中，与野生菌株相比，ΔsakA菌株胞外淀粉酶产量上升，胞内淀粉酶产量显著下降，影响了黑曲霉淀粉酶的合成。果胶酶与纤维素酶结果相反，胞外酶产量显著下降，胞内酶产量上升，基因缺失影响了黑曲霉果胶酶与纤维素酶的分泌。通过qRT-PCR分析发现，黑曲霉ΔsakA菌株中淀粉酶转录因子amyR及6个α-淀粉酶基因转录下调，减少了调控水解淀粉的过程^[32]，这可能是引起胞内淀粉酶酶活下降的主要原因，CreA是黑曲霉碳源阻遏效应中的负调控因子，胞外淀粉酶产量上升可能与CreA基因表达量上调有关^[33-34]；值得关注的是，9个果胶酶相关基因均下调，这可能是引起胞外果胶酶活性变化的主要原因^[35-36]，1999年Benen等报道endA、endC为半乳糖醛酸酶，参与果胶分解，是果胶分解酶，胞内果胶酶酶活上升可能与endA、endC基因表达量下降有关^[37-38]。XlnR为纤维素酶分解的转录激活剂^[39]，ΔsakA菌株胞外纤维素酶酶活较野生株有显著差异，这可能是由于xlnR基因表达量降低41%所致。然而，ΔsakA菌株中eglA、eglC转录表达量上升，可能引起各种冗余机制^[40-41]，从而阻止胞内纤维素酶的分解。不仅如此，测定ΔsakA菌株生长与产孢能力时发现其分生孢子梗分化延迟，孢子囊形态有变形，说明菌株的生长代谢受到影响，导致内容物合成与分泌异常。

综上所述，本研究通过基因敲除技术证实sakA基因参与调控黑曲霉的生长与产孢，是影响黑曲霉产孢的关键基因；揭示了SakA在抵御环境渗透应激反应中发挥重要的调控作用；同时，SakA还参与调控淀粉酶、果胶酶与纤维素酶的合成与分泌。研究结果为进一步明确SakA在黑曲霉生长发育、逆境胁迫反应的生理功能奠定了基础。

基金

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广东省自然科学基金(2023A1515030059)
广东省自然科学基金(2023A1515012057)
广东省重点领域研发计划(2022B1111040002)
广东省重点领域研发计划(2020B020226008)
广东省重点领域研发计划(2022B0202040002)
广东省科学院打造综合产业技术创新中心行动资金(2022GDASZH-2022010101)

参考文献

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XING S, LI GL, SUN XL, MA S, CHEN GJ, WANG LS, GAO PJ.Dynamic changes in xylanases and β-1, 4-endoglucanases secreted by Aspergillus niger An-76 in response to hydrolysates of lignocellulose polysaccharide[J].Applied Biochemistry and Biotechnology,2013,171(4):832-846.

https://doi.org/10.1007/s12010-013-0402-0

2024年第64卷第8期

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doi: 10.13343/j.cnki.wsxb.20240012

接收时间：2024-01-05
首发时间：2026-03-19
出版时间：2024-03-28

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收稿日期：2024-01-05
录用日期：2024-03-21

基金

Natural Science Foundation of Guangdong Province(2023A1515030059)

广东省自然科学基金(2023A1515030059)

Natural Science Foundation of Guangdong Province(2023A1515012057)

广东省自然科学基金(2023A1515012057)

Research and Development Plan in Key Areas of Guangdong Province(2022B1111040002)

广东省重点领域研发计划(2022B1111040002)

Research and Development Plan in Key Areas of Guangdong Province(2020B020226008)

广东省重点领域研发计划(2020B020226008)

Research and Development Plan in Key Areas of Guangdong Province(2022B0202040002)

广东省重点领域研发计划(2022B0202040002)

GDAS' Project of Science and Technology Development(2022GDASZH-2022010101)

广东省科学院打造综合产业技术创新中心行动资金(2022GDASZH-2022010101)

作者信息

1 广东省科学院微生物研究所华南应用微生物国家重点实验室广东省菌种保藏与应用重点实验室, 广东广州 510070

2 华南农业大学食品学院, 广东广州 510642

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Primers	Paired sequences (5′→3′)	Purpose
Underlined regions denote the sites of restriction enzyme for disruption of sakA (endonuclease Xma I/BamH I and Xho I/Bgl Ⅱ) through homogenous recombination of 5′ and 3′ fragments separated by hygR marker and for the expression cassette including the promoter and open reading frame of hygR (BamH I/Xho I).
sakA-F/R	ATGGCTGAGTTCTTGCGATC/CTAAATCTGGAGGTACTGCA	Cloning ORF of sakA
5′sakA-F/R	aaaaaCCCGGGGCCAACAACAAACCACCACG/	Cloning sakA 5′ (1 892 bp)
	aaaaaGGATCCTCTGACTGAAGGGGGAATCG
3′sakA-F/R	aaaaaCTCGAGCTATCTGATTACGGATGCTCTG/	Cloning sakA 3′ (1 969 bp)
	aaaaaAGATCTTGGGGAGTATTGGAAGTCTG
sakA-ID-F/R	GCTTTGCCACCTACAGTTGC/AAACAAAGTCGCCCAAGGAG	PCR detecting sakA
qsakA-F/R	GAGAATTGTGACTTGAAG/ATCTAGTTGAGACATATCC	qPCR detecting sakA gene
qβ-tubulin-F/R	TCAAGATGTCCTCTACCT/GGAACATAGCAGTGAACT	qPCR detecting β-tubulin gene

Primers

Paired sequences (5′→3′)

Purpose

Underlined regions denote the sites of restriction enzyme for disruption of sakA (endonuclease Xma I/BamH I and Xho I/Bgl Ⅱ) through homogenous recombination of 5′ and 3′ fragments separated by hygR marker and for the expression cassette including the promoter and open reading frame of hygR (BamH I/Xho I).

sakA-F/R

ATGGCTGAGTTCTTGCGATC/CTAAATCTGGAGGTACTGCA

Cloning ORF of sakA

5′sakA-F/R

aaaaaCCCGGGGCCAACAACAAACCACCACG/

Cloning sakA 5′ (1 892 bp)

aaaaaGGATCCTCTGACTGAAGGGGGAATCG

3′sakA-F/R

aaaaaCTCGAGCTATCTGATTACGGATGCTCTG/

Cloning sakA 3′ (1 969 bp)

aaaaaAGATCTTGGGGAGTATTGGAAGTCTG

sakA-ID-F/R

GCTTTGCCACCTACAGTTGC/AAACAAAGTCGCCCAAGGAG

PCR detecting sakA

qsakA-F/R

GAGAATTGTGACTTGAAG/ATCTAGTTGAGACATATCC

qPCR detecting sakA gene

qβ-tubulin-F/R

TCAAGATGTCCTCTACCT/GGAACATAGCAGTGAACT

qPCR detecting β-tubulin gene

Tag code	Gene	Annotation	Sequences of paired primers (5′→3′)
Gene08183	fluG	Upstream development activator	AAGATTGCTGCTGCTGATA/GTCGTAGGTAATCGTCTCATT
Gene03944	flbA	Upstream development activator	AGCAGCATCTCTACTTCAG/CTCCTTCGCCATCTTCAC
Gene04120	flbC	Upstream development activator	CTACTATTCCGCCTCTGT/GCCATTCTCGTAAGATTCC
Gene06270	flbB	Upstream development activator	CTCTTCCTTCCATCTCTA/TCATTCTAGTACGCTTTG
Gene04684	flbD	Upstream development activator	TGGACATGAGACAGCAATAC/AGAGATCGTGGTAGGCATA
Gene02514	flbE	Upstream development activator	TGGTGGATTGTGTATAAC/CTCATCATACCCATCATC
Gene00348	laeA	Developmental repressor	GGACTTGGAGAGAATCAG/AGATGTTGTAAGCGTGTAT
Gene02682	veA	Developmental activator	GTTCCTCTTACACTTCTCAT/GAGTTCCAGGTAGTTGTG
Gene00832	sfgA	Developmental repressor	CGAACAACCTGCTTATAGTC/GCTGCTTGAATCTCCATC
Gene04565	velB	Developmental repressor;	CCTCAGATGAACAATTAC/TAGTAAGGCTGATAATAGG
		Regulation for conidial
		maturation
Gene04761	abaA	Regulation for sterigmata	AACATCCTTTCCTTACCT/GTCATCCATCAACTTCAG
		formation
Gene04357	wetA	Regulation for conidial	CAGTGTGGATGGCAACAA/CGCTGGACAATGACTCAAG
		maturation
Gene10505	vosA	Regulation for conidial	ATGTGGATAATACTGATG/ATCTATCTGTGATGATTG
		maturation
Gene00550	brlA	Initiator for conidiogenesis	AGAACCTTCCGTCTATTATCA/ATGCTCTTGCCTCTTGAA
Gene05820	stuA	Modulator for conidiophore	ATATGATTAACGGCACAA/CTCACTCTTCAGAATACC
		formation
Gene00245	creA	Carbon catabolite repressor	TTCTTCTGATGAGGAGAG/CGGACTAACAACAACTTT
Gene08487	srgA	Secretion related GTPase	TACTGCTGATTGGTGACT/TAATGAACGACGGAGTGA
Gene08788	amyA	Alpha-amylase	GCGAAGAATATCCTCACAT/CATCCAAATGCTGTTCCT
Gene06696	amyC	Alpha-amylase	TCTAATGGTTCGGCTTAT/GTATAGTTGACGCAGTTC
Gene00277	amyD	Alpha-amylase	AACATCTTCTCTGATACATTAGT/GATTGTCCTGGTTGAGTG
Gene11416	amyE	Alpha-amylase	GCTTGTCTCTAACTACTC/TTCTTGACTGTGTCTATAC
Gene08787	amyF	Alpha-amylase	GAAGTGGATTATGTCTGT/ATAAACTCCCTCTGAAAC
Gene07580	amyG	Glucan 1, 4-alpha-glucosidase	TCAATCCATCTACTTCCT/ATAATTCCTTGCCAACTT
Gene10420	amyH	Alpha-amylase	GCTTGTCTCTAACTACTC/TTCTTGACTGTGTCTATAC
Gene05110	amyM	Alpha-amylase	TCCATTGTACTACATTCTCA/GCTGATAGACTCGGTAAT
Gene10422	amyR	Transcription regulator of	CAGTAGATGGAATGAATATAGAA/GCTTGACAGAGGTAGAAC
		maltose utilization
Gene00933	aglB	Alpha-galactosidase	GAGTTGAAGAGTAAGAATT/CACCTCATACTTATCCTT
Gene00399	lacA	Beta-galactosidase	ATTGCCATTATGAACTTGTA/TAGTCGTAGGAGGTGTAT
Gene03324	pexB	Pectinase	ACTCTTCAGCGACTTCCT/GCCTCTAATTGTATTCCATAACG
Gene04089	pecA	Pectinase	GAGACTCTTGACTTGACT/CTTCCATTCCTTGTAACC
Gene08774	pecB	Pectinase	CGTCAAGATCGAGAACAT/CGCAGAGGATGTAGTAATC
Gene06504	pecC	Pectinase	TTGACCTCACTGACCTTA/CTGTCCATTCCTCATAACC
Gene09643	endA	NADH dehydrogenase	AAGACCGTTTATGATGCTA/CGATGACAATACCGTAGT
Gene06504	endC	NADH dehydrogenase	TTGACCTCACTGACCTTA/CTGTCCATTCCTCATAACC
Tag code	Gene	Annotation	Sequences of paired primers (5′→3′)
Gene08115	poly	Polymerase	GGCTCTGATACCAATGTTAG/CTACCTCCGTTGCTCTTA
Gene05290	xlnR	Xylanolytic transcriptional	CAACTTTCCTTCTTTCAG/ATAATGGAGGCAATATGT
		activator
Gene01326	cbhA	1, 4-beta-d-glucan	AATAATGACAACACAGGTA/GAGATACTATTGGCTTCC
		cellobiohydrolase
Gene00450	cbhB	1, 4-beta-d-glucan	ATGTCTTCCTTTCAGATC/GTCTCAGTAGTGTAGGTT
		cellobiohydrolase
Gene08250	eglA	Endoglucanase	GAGCAGAAGACATATAGC/GTGAGATAGTTGAAGAAGT
Gene01363	eglB	Endoglucanase	TATCTCCTTCTATTGCTTCC/ATCGTTCTATCGTCACTAC
Gene11315	eglC	Endoglucanase	TCTCTACACCATGATTCAAG/GAAGTATCCTGGGCAATG
Gene09061	ena1	Sodium P-type ATPase	ATCTGTGCTGTCATTGTC/GTGAAGCGATTCCATAGT
Gene05606	ena2	Sodium P-type ATPase	CTAGTGGAGACGATGAAC/GGAAGGTAGTAGTAGATGATT
Gene03146	ena4	Sodium P-type ATPase	AGGCTATTGCTGCTCTTC/CTTGACACGCTTGGTAAC
Gene11257	sho1	High osmolarity signaling	TTACAACAATCTCCATCA/CCGAAGTAGAATATCCAA
		protein
Gene01120	fhlA	Sensor histidine kinase/response	CACACCAACATCAACATC/CCTTCTCCATATCATCCAA
		regulator
Gene03514	nik1	Two-component response	TGAACGGTAATACGATAG/TTGCTTCCATACTAACAT
		regulator
Gene10443	ypdl	Phosphotransmitter protein	ACGATAGCGACAGAGATT/AAGCATCCTCCATCTTGA
Gene09867	pkA	CAMP-dependent protein	CAGCAACTTCCTCTTCTC/GATAGGCTTCTCCACCAG
		kinase subunit
Gene02843	β-tubulin	Internal standard	TCAAGATGTCCTCTACCT/GGAACATAGCAGTGAACT

Tag code

Gene

Annotation

Sequences of paired primers (5′→3′)

Gene08183

fluG