蜜蜂球囊菌菌丝和孢子中长链非编码RNA的比较及潜在功能分析

doi:10.3864/j.issn.0578-1752.2021.02.018

Abstract

Abstract:

【Background】Ascosphaera apis is a fungal pathogen that exclusively infects honeybee larvae, causing a sharp decrease of the population of adult honeybees and colony population. Long non-coding RNA (lncRNA), a recently discovered non-coding RNA (ncRNA), plays a vital biological role in various activities such as epigenetics, cell cycle and dose compensation.【Objective】This study aimed to clarify the differences of number, type and expression profile of lncRNAs between A. apis mycelium and spore, and investigate the potential role of the common lncRNAs, specific lncRNAs and differentially expressed lncRNAs (DElncRNAs). 【Method】The purified mycelia (AaM) and purified spores (AaS) of A. apis were respectively sequenced using strand specific library-based lncRNA-seq technology. The expression levels of lncRNAs in AaM and AaS were calculated using FPKM (Fragment Per Kilobase of per Million mapped reads) method. Common lncRNAs and specific lncRNAs were filtered out following Venn analysis. DElncRNAs within AaM vs AaS comparison group were screened out following the standard of P≤0.05 and |log₂fold change|≥1. Upstream and downstream genes of common lncRNAs, specific lncRNAs and DElncRNAs were aligned against GO and KEGG databases to obtain function and pathway annotations. The competing endogenous RNA (ceRNA) regulation networks of common lncRNAs, specific lncRNAs and DElncRNAs were constructed following target binding relationships, followed by visualization using Cytoscape software. RT-qPCR was performed to verify the reliability of the sequencing data.【Result】In total, 108 614 646 and 105 675 408 raw reads were gained from AaM and AaS, and after strict filtering, 107 780 032 and 104 621 402 clean reads were obtained, respectively, with Q20 of 98.76% and 98.72%, and Q30 of 95.84% and 95.78%. A total of 850 lncRNAs were identified. Seven hundred and one lncRNAs were shared by AaM and AaS, and there were 39 and 110 specific lncRNAs. Via cis function, these shared lncRNAs could regulate 3 992 upstream and downstream genes involving in 42 functional terms such as cellular process, metabolism process and catalytic activity; and 117 pathways such as metabolism pathway, biosynthesis of secondary metabolites and biosynthesis of antibiotics. Specific lncRNAs for AaM and AaS could respectively regulate 243 and 672 upstream and downstream genes. In AaM vs AaS comparison group, 255 DElncRNAs were identified and found to regulate 1 479 upstream and downstream genes, which were associated with 41 functional terms including cellular process, metabolism process and catalytic activity; and 107 pathways including metabolism pathway, biosynthesis of secondary metabolites and biosynthesis of antibiotics. Forty one, five and 13 miRNA precursors were predicted from common lncRNAs, specific lncRNAs and DElncRNAs. The result of regulatory network analysis showed the formation of ceRNA networks of mycelium lncRNAs and spore lncRNAs; ten lncRNAs in mycelium could bind to eight miRNAs, further targeting 77 mRNAs; while eight lncRNAs in spore could link to seven miRNAs, further targeting 87 mRNAs; two DElncRNAs including TCONS_00008630 and TCONS_00009302 could simultaneously target miR-4968-y, further regulating ten mRNAs. The result of RT-qPCR suggested the differential expression trend of four DElncRNAs were in accordance of that in sequencing result, indicating the reliability of our sequencing data.【Conclusion】Common lncRNAs, specific lncRNAs and DElncRNAs are likely to affect material and energy metabolisms, autophagy, transcription, MAPK signaling pathway, ubiquitin-mediated proteolysis, proteasome and biosynthesis of secondary metabolites, by regulating the expression of upstream and downstream genes, serving as miRNA precursors or ceRNAs, thus regulating the growth, development, reproduction and pathogenicity of A. apis.

Key words: Ascosphaera apis, long non-coding RNA (lncRNA), mycelium, spore, competing endogenous RNA (ceRNA)

CHEN HuaZhi,WANG Jie,ZHU ZhiWei,JIANG HaiBin,FAN YuanChan,FAN XiaoXue,WAN JieQi,LU JiaXuan,ZHENG YanZhen,FU ZhongMin,XU GuoJun,CHEN DaFu,GUO Rui. Comparison and Potential Functional Analysis of Long Non-Coding RNAs Between Ascosphaera apis Mycelium and Spore[J].Scientia Agricultura Sinica, 2021, 54(2): 435-448.

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References 64

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LncRNA名称 LncRNA ID	引物名称 Primer name	引物序列 Primer sequence
TCONS_00006988	F	CTCCAGTTGTGCGTTCAT
TCONS_00006988	R	GTTGTCACCGTCTCTTCC
TCONS_00003707	F	GGAATGAATGATGCCAACTT
TCONS_00003707	R	GTAGACCGAGGAAGAACAG
TCONS_00001814	F	ACAAGGAGGAAGTCAAGGA
TCONS_00001814	R	CGAGCATAAGCAGTAGAGAT
TCONS_00007359	F	CCATCGCCACGGATATTC
TCONS_00007359	R	CCAGAGCACATCAACATCA
actin	F	CAGGAAAGGCTATGTTCGC
actin	R	ATTACCGAGGAGCAAGACG

Comparison and Potential Functional Analysis of Long Non-Coding RNAs Between Ascosphaera apis Mycelium and Spore

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样品 Sample	原始读段 Raw reads	有效读段 Clean reads	99.9%碱基正确率Q20	99.99%碱基正确率Q30
AaM	108 614 646	107 780 032	98.76%	95.84%
AaS	105 675 408	104 621 402	98.72%	95.78%

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[15]	. Genetic analysis of distorted segregation ratio of mating types in Lentinula edodes [J]. Scientia Agricultura Sinica, 2007, 40(10): 2296-2302 .