RNAi法分析糙皮侧耳XBP1转录因子

doi:10.13560/j.cnki.biotech.bull.1985.2021-1068

摘要/Abstract

摘要：

转录因子XBP1是真菌发育和其他生物过程的关键调节因子。利用RNAi法探究其在糙皮侧耳生长发育过程中的功能。以糙皮侧耳CCMSSC00389为材料,克隆获得PoXBP1基因,全长1 326 bp,编码441个氨基酸,生物信息学分析结果表明XBP1蛋白含有HTH APSES结构域,无信号肽,是非跨膜结构蛋白。在野生型菌株的不同发育阶段的表达显示,PoXBP1基因在菌丝、原基、孢子、菌盖、菌柄中的表达依次降低。通过农杆菌介导的遗传转化法筛选获得PoXBP1干扰菌株,其中,干扰菌株RNAiPoXBP1-1和RNAiPoXBP1-4的生长速度慢于野生型菌株,表明了PoXBP1表达量降低,延缓了糙皮侧耳菌丝和子实体的生长速度。

关键词: 糙皮侧耳, 转录因子, XBP1, 生长发育

Abstract:

Transcription factor XBP1 is a key regulatory factor for fungi development and other biological processes. In this study,RNA interference method was used to explore its function in the growth and development of Pleurotus ostreatus. The PoXBP1 gene was cloned from the CCMSSC00389（Pleurotus ostreatus）. The full-length of PoXBP1 was 1 326 bp and it encoded a protein of 441 amino acids. Bioinformatics analysis showed that XBP1 protein contained HTH APSES domain and had no signal peptide. It was a non transmembrane structural protein.The expression of PoXBP1 gene in different developmental stages of wild type strains decreased in turn in hyphae,primordium,spores,cap and stalk. PoXBP1 interfering strain were screened by Agrobacterium tumefaciens-mediated genetic transformation. Among them,the growth rates of interfering strain RNAiPoXBP1-1 and RNAiPoXBP1-4 were slower than that of wild-type strains,indicating that the expression of PoXBP1 decreased and the growth rate of hyphae and fruiting bodies of P. ostreatus delayed.

Key words: Pleurotus ostreatus, transcription factor, XBP1, growth and development

齐雨慧, 黄晨阳, 张利姣. RNAi法分析糙皮侧耳XBP1转录因子[J]. 生物技术通报, 2021, 37(11): 65-71.

QI Yu-hui, HUANG Chen-yang, ZHANG Li-jiao. Analysis of XBP1 Transcription Factor in Pleurotus ostreatus by RNAi[J]. Biotechnology Bulletin, 2021, 37(11): 65-71.

图/表 11

表1 引物序列

Table 1 Primer sequence

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Purpose
PoXBP1-Spel-F1	ATGATTGACTCACAGCCGGACT	克隆
PoXBP1-Apal-R1	TCAGTTGAGGATATTTGACCAA
PoXBP1-Spe1-R	CGACTAGTCCGTATGGAGAAT- ATCGGCTAT
PoXBP1-Apa1-F	CCGGGCCCATGATTGACTCA- CAGCCGGA
PoXBP1-intro-Spe1-R	GGACTAGTGGGAATATGAT- GCGCCGCTGGC
PoXBP1-Bg111-F	GAAGATCTTCATGATTGAC- TCACAGCCGGA
PoXBP1-qrt-SF1	ATACAGGGAACATGGATGCC	qPCR引物
PoXBP1-qrt-SR1	ACTTGACCATAACCAGGCTG	qPCR引物
β-actin-F	AGTCGGTGCCTTGGTTAT	内参基因
β-actin-R	ATACCGACCATCACACCT	内参基因
hyg-F	AGAAGATGTTGGCGACCTC	检测转化子
hyg-R	AGCGAGAGCCTGACCTATTG	检测转化子

图1 已知真菌XBP1和poXBP1的蛋白序列比对

Fig. 1 Protein sequence alignment of known fungus XBP1 and poXBP1

图2 PoXBP1蛋白的二级结构预测

Fig.2 Prediction of the secondary structure of PoXBP1 protein

图3 PoXBP1蛋白的三级结构预测

Fig.3 Prediction of the tertiary structure of PoXBP1 protein

图4 野生型菌株不同组织中PoXBP1基因相对表达量比较不同小写字母代表差异显著（P<0.05）,下同

Fig.4 Comparison of the relative expression of PoXBP1 gene in different tissues of wild-type strains Different lowercase letters represent significant differences（P<0.05）. The same below

图5 RNAi-PoXBP1转化子筛选 M:DL2000 marker;1-18:抗性菌块DNA;19:阳性对照:质粒RNAi-PoXBP1;20:阴性对照:389 DNA

Fig.5 Screening of RNAi-PoXBP1 transformants M:DL2000 marker;1-18:fast DNA of resistant bacteria;19:plasmid RNAi-PoXBP1;20:389 DNA

图6 RNAi-PoXBP1转化子中PoXBP1基因相对表达量

Fig.6 Relative expression levels of PoXBP1 gene in RNAi-PoXBP1 transformants

图7 干扰子与野生型菌落直径差异

Fig.7 Difference of colony diameter between RNAi strains and wild type

表2 菌丝显微形态对比

Table 2 Comparison of mycelial morphology

菌株 Strain	菌丝顶端细胞长度 Length of mycelial apical cell/μm	菌丝顶端细胞直径 Diameter of mycelial apical cell/μm	菌丝顶端细胞表面积 Surface area of mycelial apical cells/μm²
389	105 ± 17^a	4.90 ± 0.95	500 ± 187^a
RNAipoXBP1-4	72 ± 16^b	4.91 ± 1.09	348 ± 164^b
RNAipoXBP1-1	69 ± 13^b	4.97 ± 0.88	321 ± 117^b

图8 干扰子与野生型子实体发育对比

Fig.8 Comparison of the development of of fruiting body between RNAi strains and wild type

图9 干扰子与野生型不同时期、部位PoXBP1基因相对表达量比较 *代表差异显著（P<0.05）

Fig.9 Comparison of relative expressions of PoXBP1 gene in different periods and tissues between RNAi strains and wild type * represents significant difference（P<0.05）

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