一个影响糙皮侧耳生长发育的功能基因的发现与验证

doi:10.13560/j.cnki.biotech.bull.1985.2024-1257

摘要/Abstract

摘要：

目的研究功能未知基因g13394对糙皮侧耳菌丝到原基及子实体发育的影响。方法通过PCR技术从糙皮侧耳CCMSSC00389菌株中扩增g13394基因，并进行生物信息学分析。利用同源重组方法构建g13394基因的过表达载体和RNAi载体，并通过根癌农杆菌介导的方法对糙皮侧耳进行遗传转化，通过RT-qPCR检测基因表达量筛选g13394过表达菌株和RNAi菌株。对转化菌株和野生型菌株（WT）在菌丝生长阶段和子实体发育阶段的表型进行比较。结果生物信息学分析显示，g13394基因编码的蛋白无明确的已知结构域，亚细胞定位预测为细胞核。成功构建并获得了g13394基因的过表达菌株和RNAi菌株各两株。与野生型菌株相比，过表达菌株在菌丝生长和子实体发育阶段生长速度显著加快，而RNAi菌株菌丝生长速度显著减慢，原基和子实体发育时间延缓。结论基因g13394促进菌丝生长、加速子实体发育。

关键词: 糙皮侧耳, 基因克隆, 遗传转化, 子实体发育

Abstract:

Objective This study is aimed to investigate the role of the functionally unknown gene g13394 in the growth and development of Pleurotus ostreatus, specifically in the transition from mycelium to primordium and fruiting body formation. Method The g13394 gene was cloned from the P. ostreatus strain CCMSSC00389 using Polymerase Chain Reaction (PCR), and its biological characteristics were analyzed through bioinformatic tools. A homologous recombination approach was used to construct both overexpression and RNA interference (RNAi) vectors, which were then introduced into P. ostreatus via Agrobacterium tumefaciens-mediated transformation. Gene expressions in the transformed strains were validated by quantitative real-time PCR (qRT-PCR) to select the g13394-overexpressed and RNAi strains. The phenotypic differences between the overexpression, RNAi, and wild-type strains were assessed at the mycelial growth stage and during fruiting body development. Result Bioinformatic analysis revealed that g13394 encoded a nuclear-localized protein with unknown functional domains and the subcellular localization was predicted in the nucleus. Two strains were successfully constructed and obtained for g13394 overexpression and RNAi, respectively. Overexpressed strains showed significantly faster mycelial growth and accelerated fruiting body development compared to the wild-type. In contrast, RNAi strains showed slower mycelial growth, delayed primordium formation, and extended fruiting body development time. These results suggest that g13394 positively regulates both mycelial growth and the timing of fruiting body formation. Conclusion The gene g13394 promotes mycelial growth and accelerates the formation of fruiting bodies.

Key words: Pleurotus ostreatus, gene cloning, genetic transformation, fruiting body development

裴景琪, 赵梦然, 黄晨阳, 邬向丽. 一个影响糙皮侧耳生长发育的功能基因的发现与验证[J]. 生物技术通报, 2025, 41(6): 327-334.

PEI Jing-qi, ZHAO Meng-ran, HUANG Chen-yang, WU Xiang-li. Discovery and Verification of a Functional Gene Influencing the Growth and Development of Pleurotus ostreatus[J]. Biotechnology Bulletin, 2025, 41(6): 327-334.

图/表 7

表1 引物列表

Table 1 List of primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Purpose
g13394-F	ATGAACGAAGTCTCGGG	克隆
g13394-R	TCACGCGCCCTCTGCATTTA
OE13-F	ACTGACCTGGGGATCCATGAACGAAGTCTCGGGGCG
OE13-R	GCATGCCAATTCTAGATCACGCGCCCTCTGCATTTAACACG
Ri-sense-g13394 F	ACTGACCTGGGGATCCTCACGCGCCCTCTGCATT
Ri-sense-g13394 R	GCATGCCAATTCTAGAACGAAAGAGAGAGCATCCAAGGC
Ri-anti-g13394 F	TCTCTTTCGTTCTAGACGGATGTCCTCTACGAGGGA
Ri-anti-g13394 R	GGCCAGTGCCAAGCTTTCACGCGCCCTCTGCATT
q13-F	CGCTCACCTGCTCAATACG	RT-qPCR引物
q13-R	TCCATGACCCTGCCAATTG	RT-qPCR引物
tub-F	AGGCTTTCTTGCATTGGTACACGC	内参引物
tub-R	TATTCGCCTTCTTCCTCATCGGCA	内参引物
S10-F	CCACACAAATCCTCTCACTCACG	验证引物
S10-R	GCTTGCATGCCTGCAGTAATT
Hyg-F	CGACAGATCCGGTCGGCATCTACTCTATTTCTT
Hyg-R	TCTCGTGCTTTCAGCTTCGATGTAGGAGGG

图1 g13394的生物信息学分析A：基因结构；B：跨膜结构域分析；C：蛋白结构域预测；D：磷酸化位点预测；E：信号肽预测

Fig. 1 Bioinformatics analysis of g13394A: Gene structure; B: transmembrane structural domain analysis; C: signal peptide prediction; D: phosphorylation site prediction; E: protein structural domain prediction

图2 g13394 DNA和CDS的PCR扩增M：DL2000 marker；1、2：CDS；3、4：DNAM: DL2000 marker; 1 and 2: CDS; 3 and 4: DNA

Fig. 2 PCR amplification of g13394 DNA and CDS

图3 g13394过表达和干扰质粒图谱A：质粒Po-gpdOE-g13394；B：质粒RNAi-g13394

Fig. 3 g13394 overexpression and RNAi plasmid profilesA: Plasmid Po-gpdOE-g13394; B: plasmid RNAi-g13394

图4 g13394转化子PCR验证及相对表达水平A：过表达转化子；B：RNAi转化子；M：DL2000 marker； 1-20：g13394转化子；CK：阳性对照（g13394过表达/RNAi 质粒）；NC1：阴性对照（野生型）；NC2：阴性对照（ddH2O）；C：过表达阳性转化子；D：RNAi阳性转化子；WT：野生型；OE-2、OE-7、OE-9、OE-10、OE-13在图A中对应泳道1、6、8、9、12；Ri-12、Ri-13、Ri-23、Ri-32、Ri-34在图B中对应泳道1、2、7、16、18。不同字母表示不同菌株之间存在显著差异，P<0.05。下同

Fig. 4 PCR verification of g13394 putative transformants and the relative expressionA: Overexpression positive transformants; B: RNAi positive transformants; M: DL2000 marker; 1-20: g13394 transformants; CK: positive control (g13394 overexpression/RNAi plasmid); NC1: negative control (WT); NC2: negative control (ddH2O); C: Overexpressing positive transformants; D: RNAi transformants; WT: wild type; OE-2, OE-7, OE-9, OE-10, and OE-13 correspond to lane 1, 6, 8, 9, 12 in Fig. 4-A. Ri-12, Ri-13, Ri-23, Ri-32, and Ri-34 correspond to lane 1, 2, 7, 16, 18 in Fig. 4-B. Diﬀerent letters indicate signiﬁcant diﬀerences among strains at P<0.05. The same below

图5 野生型菌株和g13394转化菌株在PDA培养基上的菌丝生长A：菌落形态；B：菌丝生长速率

Fig. 5 Mycelial growth of wild-type strain and g13394 transformed strain on PDA mediumA: Colony morphology. B: Mycelial growth rate

图6 野生型菌株和g13394转化菌株的子实体形成情况A：子实体形态；M：菌丝期（12 d）；P：原基期（25 d）；F：子实体期（29 d）；B：原基形成时间；C：子实体形成时间

Fig. 6 Fruiting body development of the wild-type strain and g13394 transformantsA: Fruiting body morphology; M: mycelium (12 d); P: primordium (25 d); F: fruiting body (29 d). B: Primordium formation time. C: Fruiting body formation time

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