Analysis of XBP1 Transcription Factor in Pleurotus ostreatus by RNAi

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

Abstract

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

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.

Figures/Tables 11

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

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

Fig.2 Prediction of the secondary structure of PoXBP1 protein

Fig.3 Prediction of the tertiary structure of PoXBP1 protein

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

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

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

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

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

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

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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