糙皮侧耳pal2调控低温光照环境刺激下菌丝生长的分子机制

doi:10.16420/j.issn.0513-353x.2022-0001

园艺学报 ›› 2023, Vol. 50 ›› Issue (4): 831-841.doi: 10.16420/j.issn.0513-353x.2022-0001

糙皮侧耳pal2调控低温光照环境刺激下菌丝生长的分子机制

降玉清¹, 何琪¹^,², 侯潞丹³, 邬向丽¹, 黄晨阳¹, 赵梦然¹^,^*()

1.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
2.吉林农业大学食药用菌教育部工程研究中心,吉林长春130118
3.山西农业大学食品科学与工程学院，山西省食用菌工程技术研究中心，黄土高原食用菌山西省重点实验室，山西晋中 030801

收稿日期:2022-09-09 修回日期:2022-12-26 出版日期:2023-04-25 发布日期:2023-04-27
通讯作者: *（E-mail：zhaomengran@caas.cn）
基金资助:
中国农业科学院所长基金项目(1610132022030);国家现代农业产业技术体系建设专项资金项目(CARS-20)

The Molecular Mechanism of Phenylalanine Ammonia-Lyase Gene pal2 Regulating Pleurotus ostreatus Mycelial Growth Under Stimulation of Low-temperature and Light

JIANG Yuqing¹, HE Qi¹^,², HOU Ludan³, WU Xiangli¹, HUANG Chenyang¹, and ZHAO Mengran¹^,^*()

1. 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
2. Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi，Jilin Agricultural University，Jilin，Changchun 130118，China
3. Key Laboratory of Shanxi Province for Loess Plateau Edible Fungi，Collaborative Innovation Center of Quality and Efficiency of Loess Plateau Edible Fungi in Shanxi，College of Food Science and Engineering，Shanxi Agricultural University，Jinzhong，Shanxi 030801，China

Received:2022-09-09 Revised:2022-12-26 Online:2023-04-25 Published:2023-04-27
Contact: *（E-mail：zhaomengran@caas.cn）

摘要/Abstract

摘要：

以糙皮侧耳野生型菌株和pal2基因干扰菌株为材料，通过对比菌丝在28 ℃始终黑暗培养和28 ℃黑暗培养3 d后进行15 ℃12 h光照/12 h黑暗培养6 d的环境刺激这两种条件下苯丙氨酸解氨酶（phenylalanine ammonia-lyase，PAL）基因pal2转录水平、酶活性、底物L-苯丙氨酸和产物反式肉桂酸含量的变化，以及分析外源添加不同浓度底物和产物对菌丝表型的影响，初步解释了环境刺激下菌丝生长速度减慢的原因，发现苯丙氨酸解氨酶及其编码基因pal2均能响应环境刺激，但酶活性、产物和底物含量与基因表达量相比存在明显地响应滞后性；阐明环境刺激通过诱导pal2转录水平下调，总酶活性升高，使菌丝胞内L-苯丙氨酸减少，反式肉桂酸含量增加，从而负调控糙皮侧耳菌丝生长的分子通路。

关键词: 糙皮侧耳, 低温光照, 苯丙烷类代谢途径

Abstract:

Wild-type strain and pal2 gene interference strains of Pleurotus ostreatus were used as materials in this study. All the strains were incubated at 28 ℃ in the darkness for three days and then were treated with environmental stimulation（15 ℃，12 h light/12 h darkness）for six days. Strains incubated at 28 ℃ in the darkness without the former environmental stimulation were taken as control. The pal2 gene transcription level，enzyme activity，contents of enzyme substrate and product of all the strains treated with different environmental conditions were tested and compared. The effects of various concentrations of exogenous substrates and products on the mycelial phenotype were examined. The results preliminarily explained the reasons for the slowdown of mycelial growth under environmental stimulation. The results revealed that both phenylalanine ammonia-lyase and the gene pal2 responded to environmental stimulation，but there was an obivous response lag in enzyme activity，product and substrate content compared with the expression of pal2. It was further clarified that the environmental stimulation induced down-regulation of both pal2 gene transcription level，increased enzyme activity，which caused the reduction of L-phe content and the increase of t-CA content in the mycelia，and thus negatively regulated the mycelial growth of P. ostreatus.

Key words: Pleurotus ostreatus, stimulation of low-temperature and light, phenylpropanoid metabolic pathway

中图分类号:

S646.141

降玉清, 何琪, 侯潞丹, 邬向丽, 黄晨阳, 赵梦然. 糙皮侧耳pal2调控低温光照环境刺激下菌丝生长的分子机制[J]. 园艺学报, 2023, 50(4): 831-841.

JIANG Yuqing, HE Qi, HOU Ludan, WU Xiangli, HUANG Chenyang, and ZHAO Mengran. The Molecular Mechanism of Phenylalanine Ammonia-Lyase Gene pal2 Regulating Pleurotus ostreatus Mycelial Growth Under Stimulation of Low-temperature and Light[J]. Acta Horticulturae Sinica, 2023, 50(4): 831-841.

图/表 6

表1 RT-qPCR中用到的引物

Table 1 Premer sequences of RT-qPCR

用途Usage	基因Gene	引物序列（5′-3′）Primer sequence	参考文献 Reference
内参基因Reference gene	tublin	F：AGGCTTTCTTGCATTGGTACACGC； R：TATTCGCCTTCTTCCTCATCGGCA	侯志浩等,2019
qPCR	pal1	F：CTCCTTCACAATCGCATCTA；R：CTTCAGCCGCCTATGTTG	Hou et al.,2019
qPCR	pal2	F：CAACTGCTGCGTATGTCA；R：GATGTAGAGGTATGAGGAGATT	Hou et al.,2019

图1 环境刺激（15 ℃，12 h光照/12 h黑暗）对糙皮侧耳菌丝生长的影响

Fig. 1 Effects of environmental stimulation（15 ℃，12 h light/12 h dark）on the growth of Pleurotus ostreatus mycelium

图2 环境刺激（15 ℃，12 h光照/12 h黑暗）对糙皮侧耳菌丝pal2（A）、PAL（B）、L-phe（C）和t-CA（D）的影响不同小写字母表示处理间差异显著（P < 0.05）。下同。

Fig. 2 Effects of environmental stimulation(15 ℃，12 h light/12 h dark)on pal2（A），PAL（B），L-phe（C），and t-CA（D）of Pleurotus ostreatus mycelium Different lowercase letters indicate significant differences among different treatments at 0.05 level. The same below.

图3 外源添加L-phe和t-CA对糙皮侧耳菌丝生长的影响

Fig. 3 Effect of exogenous addition L-phe and t-CA on the growth of Pleurotus ostreatus mycelium

图4 干扰pal2有助于菌丝在环境刺激（15 ℃，12 h光照/12 h黑暗）下的生长 WT：野生型菌株；Ri-1、Ri-9：pal2干扰菌株。下同。

Fig. 4 Interfering with pal2 facilitates the growth of mycelium under environmental stimulation(15 ℃，12 h light/12 h dark) WT：wild type strain；Ri-1，Ri-9：pal2 interfering strains. The same below.

图5 干扰pal2对菌丝pal2（A）、PAL（B）、L-phe（C）和 t-CA（D）的影响 Effect of interfere with pal2 on pal2（A），PAL（B），L-phe（C）和t-CA（D）of Pleurotus ostreatus mycelium

Fig. 5

参考文献 33

[1]	Barros J, Dixon R A. 2020. Plant phenylalanine/tyrosine ammonia-lyases. Trends Plant Science, 25 (1):66-79. doi: 10.1016/j.tplants.2019.09.011 URL
[2]	Bernadeth B, Jacobo D A, Cisneros L, Luis B V. 2021. Physiological role of reactive oxygen species,ethylene,and jasmonic acid on UV light induced phenolic biosynthesis in wounded carrot tissue. Postharvest Biology and Technology, 172:111388. doi: 10.1016/j.postharvbio.2020.111388 URL
[3]	Cheng G X, Sun J T, Shang J P, Gong Z H. 2019. Virus-induced gene silencing for phenylalanine ammonia-lyase affects pepper adaption to low temperature. Biologia Plantarum, 63:601-609. doi: 10.32615/bp.2019.063
[4]	Cong Ying, Huang Mingqian, Liu Mingyue, Zang Ying, Zheng Zhaojuan, Ouyang Jia. 2015. Simultaneous determination of L-phenylalanine and trans-cinnamic acid in biocatalytic reaction by high performance liquid chromatography. Guangdong Chemical Industry, 42:157-159. (in Chinese)
	丛莹, 黄铭茜, 刘明月, 臧颖, 郑兆娟, 欧阳嘉. 2015. 高效液相色谱法同时测定生物转化液中L-苯丙氨酸和反式肉桂酸. 广东化工, 42:157-159.
[5]	Feldman D, Amedi N, Carmeli S, Yarden O, Hadar Y. 2019. Manipulating the expression of small secreted protein 1(Ssp1)alters patterns of development and metabolism in the white-rot fungus Pleurotus ostreatus. Applied and Environmental Microbiology, 85:e00761-19.
[6]	Guerra D, Anderson A J, Salisbury F B. 1985. Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps. Plant Physiology, 78 (1):126-130. doi: 10.1104/pp.78.1.126 URL
[7]	Hou L D, Wang L N, Wu X L, Gao W, Zhang J X, Huang C Y. 2019. Expression patterns of two pal genes of Pleurotus ostreatus across developmental stages and under heat stress. BMC Microbiology, 19:16-24. doi: 10.1186/s12866-019-1391-0
[8]	Hou Zhihao, Zhao Mengran, Chen Qiang, Zhang Yan, Huang Chenyang, Wu Xiangli. 2019. Selection of internal reference genes of Pleurotus ostreatus under heat stress by real-time fluorescent quantitative PCR. Acta Edulis Fungi, 26 (3):11-18,157. (in Chinese)
	侯志浩, 赵梦然, 陈强, 张妍, 黄晨阳, 邬向丽. 2019. 热胁迫下糙皮侧耳实时荧光定量PCR内参基因的选择. 食用菌学报, 26 (3):11-18,157.
[9]	Huang J, Gu M, Lai Z B, Fan B F, Shi K, Zhou Y H, Yu J Q, Chen Z X. 2010. Functional analysis of the Arabidopsis PAL gene family in plant growth,development,and response to environmental stress. Plant Physiology, 153 (4):1526-1138. doi: 10.1104/pp.110.157370 URL
[10]	Ji Qiangqiang, Chen Weida, Chen Jingdong, Wang Yuhang, Yu Yi, Zeng Changli. 2021. Family identification and photo regulation analysis of phenylalanine ammonia-lyase(AaPAL)gene of Artemisia annua. Molecular Plant Breeding, https://kns.cnki.net/kcms/detail/46.1068.S.20210705.1443.013.html. (in Chinese)
	戢强强, 陈伟达, 陈敬东, 王宇航, 余忆, 曾长立. 2021. 青蒿苯丙氨酸解氨酶(AaPAL)基因家族鉴定及光调控分析. 分子植物育种, https://kns.cnki.net/kcms/detail/46.1068.S.20210705.1443.013.html.
[11]	Jiang Li, Yuan Huaibo, Zhou Qiang, Jiang Hanhu. 2007. Effects of substrate and products on of phenylalanine ammonia-lyase(PAL)in fresh-cut yam. Food Science, 28 (10):35-38. (in Chinese)
	江力, 袁怀波, 周强, 江汉湖. 2007. 底物和产物对鲜切山药苯丙氨酸解氨酶活性的影响. 食品科学, 28 (10):35-38.
[12]	Joos H J, Hahlbrock K. 1992. Phenylalanine ammonia-lyase in potato(Solanum tuberosum L.):genomic complexity,structural comparison of two selected genes and modes of expression. European Journal of Biochemistry, 204:621-629. doi: 10.1111/j.1432-1033.1992.tb16675.x pmid: 1541277
[13]	Kavil S, Otti G, Bouvaine S, Armitage A, Maruthi M N. 2021. PAL1 gene of the phenylpropanoid pathway increases resistance to the cassava brown streak virus in cassava. Virology Journal, 18 (1):184-184. doi: 10.1186/s12985-021-01649-2 pmid: 34503522
[14]	Koukol J, Conn E E. 1961. The metabolism of aromatic compounds in higher plants:IV. Purification and properties of the phenylalanine deaminase of Hordeum vulgare. Journal of Biological Chemistry, 236:2692-2698. doi: 10.1016/S0021-9258(19)61721-7 URL
[15]	Leyva A, Jarillo J A, Salinas J, Martinez-Zapater J M. 1995. Low temperature induces the accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs of Arabidopsis thaliana in a light dependent manner. Plant Physiology, 108 (1):39-46. doi: 10.1104/pp.108.1.39 pmid: 12228452
[16]	Li N N, Yue C, Cao H L, Qian W J, Hao X Y, Wang Y C, Wang L, Ding C Q, Wang X C, Yang Y J. 2018. Transcriptome sequencing dissection of the mechanisms underlying differential cold sensitivity in young and mature leaves of the tea plant(Camellia sinensis). Journal of Plant Physiology, 224:144-155.
[17]	Li R R, Zheng Q W, Lu J L, Zou Y, Lin J F, Guo L Q, Ye S Q. 2021. Chemical composition and deterioration mechanism of Pleurotus tuoliensis during postharvest storage. Food Chemistry, 338:127731. doi: 10.1016/j.foodchem.2020.127731 URL
[18]	Li Tianzhu, Sun Yuwen, Wang Jitao, Gan Defang, Hu Keling. 2021. Effects of different storage temperature on quality and lignin accumulation in postharvest oyster mushrooms. Acta Agriculturae Zhejiangensis, 33 (2):248-258. (in Chinese) doi: 10.3969/j.issn.1004-1524.2021.02.08
	李甜竹, 孙玉文, 汪季涛, 甘德芳, 胡克玲. 2021. 不同贮藏温度对糙皮侧耳采后品质及木质素积累的影响. 浙江农业学报, 33 (2):248-258. doi: 10.3969/j.issn.1004-1524.2021.02.08
[19]	Lin W P, Liu A, Weng C H, Li H, Sun S W, Song A H, Zhu H. 2018. Cloning and characterization of a novel phenylalanine ammonia-lyase gene from Inonotus baumii. Enzyme and Microbial Technology, 112:52-58. doi: 10.1016/j.enzmictec.2017.10.010 URL
[20]	Lotfi M, Mars M, Werbrouck S. 2019. Optimizing pear micropropagation and rooting with light emitting diodes and trans-cinnamic acid. Plant Growth Regulation, 88 (2):173-180. doi: 10.1007/s10725-019-00498-y
[21]	Lü Sijia, Wu Yueyan, Jia Yonghong, He Fan, Jiang Baoxin, Yang Guoxia, Jia Yonghong. 2022. Cloning and functional analysis of the phenylalannine ammonia-lyase gene from Rhododendron fortunei. Chinese Journal of Biotechnology, 38 (1):374-385. (in Chinese) doi: 10.13345/j.cjb.210325 pmid: 35142143
	吕思佳, 吴月燕, 贾永红, 何凡, 蒋宝鑫, 杨国霞, 谢晓鸿. 2022. 云锦杜鹃苯丙氨酸解氨酶基因的克隆及功能分析. 生物工程学报, 38 (1):374-385.
[22]	Lü Xiaomeng, Liu Ao, Wang Qingji, Wang Xiangfeng, Chou Tiansheng, Wang Wei. 2021. Transcriptome and proteome analyses of Flammulina filiformis during stipe development. Mycosystema, 40 (12):3231-3245. (in Chinese)
	吕晓萌, 刘澳, 王庆佶, 王祥锋, 丑天胜, 王威. 2021. 金针菇菌柄发育的转录组与蛋白组分析. 菌物学报, 40 (12):3231-3245.
[23]	Pan H Y, Fang C M, Zhou T S, Wang Q Z, Chen J K. 2007. Accumulation of calycosin and its 7-O-b-D-glucoside and related gene expression in seedlings of Astragalus membranaceus Bge. var. Mongholicus(Bge.) Hsiao induced by low temperature stress. Plant Growth Regulation, 26 (7):1111-1120.
[24]	Qi Y C, Zhang R X, Zhang M K, Wen Q, Shen J W. 2021. Effects of exogenous ascorbic acid on the mycelia growth and primordia formation of Pleurotus ostreatus. Journal of Basic Microbiology, 61 (8):736-744. doi: 10.1002/jobm.v61.8 URL
[25]	Qi Yuancheng, Liu Yuandong, Sun Xiankai, Zhang Mengke, Zhang Qian, Wen Qing, Qiu Liyou, Shen Jinwen. 2017. Cloning and expression analysis of Pleurotus ostreatus aldehyde dehydrogenase gene PoALDH1 under abiotic stresses. Mycosystema, 36 (8):1121-1131. (in Chinese)
	戚元成, 刘元栋, 孙宪凯, 张梦珂, 张倩, 文晴, 邱立友, 申进文. 2017. 糙皮侧耳乙醛脱氢酶基因PoALDH1的克隆与非生物胁迫下的表达分析. 菌物学报, 36 (8):1121-1131.
[26]	Qian X C, Hou Q, Liu J N, Huang Q H, Jin Z L, Zhou Q L, Jiang T J, Zheng X L. 2021. Inhibition of browning and shelf life extension of button mushroom(Agaricus bisporus)by ergothioneine treatment. Scientia Horticulturae, 288:110385. doi: 10.1016/j.scienta.2021.110385 URL
[27]	Tasaki Y, Hayashi S, Kato M. 2018. L-phenylalanine supplementation increases the production of phenylalanine ammonia-lyase and methyl cinnamate in the mycelia of Tricholoma matsutake. Mycoscience, 59 (1):8-11. doi: 10.1016/j.myc.2017.07.001 URL
[28]	Wang Li, Shi Lingling, Liu Yujun. 2007. Effects of different light treatments on growth and PAL activity of the suspension-cultured cells of Rhodiola fastigiata. Scientia Silvae Sinicae, 43 (6):52-56. (in Chinese)
	王莉, 史玲玲, 刘玉军. 2007. 不同光质对长鞭红景天悬浮细胞生长及苯丙氨酸解氨酶活性的影响. 林业科学, 43 (6):52-56.
[29]	Wu Yuanhang, Liu Qing, Liu Pandao, Guo Pengfei, Li Ming, Jiang Lingyan, Luo Lijuan. 2019. Cloning of cassava phenylalanine ammonia lyase genes and their responses to low temperature stress. Chinese Journal of Tropical Crops, 40 (3):483-489. (in Chinese)
	吴远航, 刘秦, 刘攀道, 郭鹏飞, 李敏, 蒋凌雁, 罗丽娟. 2019. 木薯苯丙氨酸解氨酶基因的克隆及其对低温胁迫的响应. 热带作物学报, 40 (3):483-489.
[30]	Xiang L, Moore B S. 2005. Biochemical characterization of a prokaryotic phenylalanine ammonia lyase. Journal of Bacteriology, 187 (12):4286-4829. pmid: 15937191
[31]	Xiong Fei, Lu Qinhua, Fang Wanping, Yang Yajun, Wang Xinchao, Zhu Xujun, Wang Yuchun. 2020. Genome-wide identification and expression analyses of PAL genes under biotic and abiotic stress in Camellia sinensis. Acta Horticulturae Sinica, 47 (3):517-528. (in Chinese)
	熊飞, 卢秦华, 房婉萍, 杨亚军, 王新超, 朱旭君, 王玉春. 2020. 基于全基因组的茶树PAL家族基因鉴定及其在生物与非生物胁迫下的表达分析. 园艺学报, 47 (3):517-528. doi: 10.16420/j.issn.0513-353x.2019-0376
[32]	Zhang Yan-tong. 2020. Molecular mechanism of light factor affecting the synthesis of phlorizin from Lithocarpus polystachyus[M. D. Dissertation]. Tangshan: North China University of Science and Technology. (in Chinese)
	张妍彤. 2020. 光因子影响多穗柯中根皮苷合成的分子机制[硕士论文]. 唐山: 华北理工大学.
[33]	Zhao T, Li R, Yao W, Wang Y J, Zhang C H, Li Y. 2021. Genome-wide identification and characterisation of phenylalanine ammonia-lyase gene family in grapevine. The Journal of Horticultural Science and Biotechnology, 96 (4):456-468. doi: 10.1080/14620316.2021.1879685 URL

糙皮侧耳pal2调控低温光照环境刺激下菌丝生长的分子机制

The Molecular Mechanism of Phenylalanine Ammonia-Lyase Gene pal2 Regulating Pleurotus ostreatus Mycelial Growth Under Stimulation of Low-temperature and Light

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 33

相关文章 2

编辑推荐

Metrics

本文评价

访问总数:　当日访问总数:　当前在线人数:

[1]	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.
[2]	殷朝敏1，陈叶叶2，卢静2，马爱民2,. 糙皮侧耳凝集素基因Plectin*的克隆和表达分析[J]. 园艺学报, 2017, 44(1): 131-138.

糙皮侧耳pal2调控低温光照环境刺激下菌丝生长的分子机制

The Molecular Mechanism of Phenylalanine Ammonia-Lyase Gene pal2 Regulating Pleurotus ostreatus Mycelial Growth Under Stimulation of Low-temperature and Light

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 33

相关文章 2

编辑推荐

Metrics

本文评价

访问总数: 当日访问总数: 当前在线人数:

访问总数:　当日访问总数:　当前在线人数: