Differentiate Transcriptomes of Aerial and Creepy Agaricus bisporus Hyphae
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摘要:
目的 挖掘双孢蘑菌落形态变化可能涉及的重要基因,为双孢蘑菇种质资源评价、新品种选育提供科学依据。 方法 以双孢蘑菇主栽品种As2796为试验菌株,收集同一平板上生长的贴生与气生菌丝,提取样品的RNA进行转录组测序分析。 结果 通过转录组分析,A601(As2796气生组)与A602(As2796贴生组)共鉴定出965个差异表达基因(DEGs)。GO富集分析结果表明,在真菌细胞壁组成方面有疏水蛋白相关的DEGs富集,其中AGABI2DRAFT_13655、AGABI2DRAFT_136569、AGABI2DRAFT_193057属于ABH3蛋白基因家族,在气生菌丝的表达水平显著高于贴生菌丝,可能与气生菌丝的产生有关。研究还发现了疏水表面结合蛋白A(HsbA)相关基因AGABI2DRAFT_194662在气生菌丝中FPKM表达水平为0,在贴生菌丝中为10.24。通过对DEGs进一步分析,富集到8个可能与脂酶同工酶相关的DEGs,气生菌丝中这些基因的表达水平显著高于贴生菌丝。SNP/InDel分析结果表明,As2796气生组SNP数为110 601,贴生组SNP数为111 188,单核苷酸变异中,C:G>T:A的变异次数较多,共筛选出2 374个共同InDel。 结论 成功获得双孢蘑菇不同形态菌丝的转录组数据,并从中挖掘出潜在的相关基因和SNP位点,为双孢蘑菇相关分子标记开发及辅助育种提供参考。 Abstract:Objective Key genes associated with Agaricus bisporus of different mycelial morphology were analyzed. [Methods] As 2796, the main cultivated variety of A. bisporus, with aerial or creepy hyphae grown on same plate were collected for RNA transcriptome sequencing and comparison with reference. Results There were 965 differentially expressed genes (DEGs) between the aerial hyphae-A601 and the creepy hyphae-A602. According to the GO enrichment analysis, the hydrophobic DEGs were enriched in the fungal cell wall, such as AGABI2DRAFT_13655, AGABI2DRAFT_136569, and AGABI2DRAFT_193057 that belonged to the ABH3 protein gene family, with expressions significantly higher in A601 than in A602. Thus, they were postulated to be related to the production of aerial hyphae. In addition, the hydrophobic surface binding protein A (HsbA)-related gene, AGABI2DRAFT_194662, showed a FPKM expression of 0 in A601, but 10.24 in A602. Furthermore, 8 enriched DEGs related to lipase isoenzymes exhibited a FPKM expression significantly higher in A601 than in A602. The SNP/InDel analysis indicated that the SNPs in A601 was 110 601 as opposed to 111 188 in A602, the most frequent mutation in SNPs was C:G>T:A, and that 2 374 common InDels were identified. Conclusion The transcriptomes and possibly associated genes and SNP sites in A. bisporus with different mycelial morphology obtained in this study would be of value for the mushroom germplasm evaluation and breeding. -
Key words:
- Agaricus bisporus /
- As 2796 /
- creepy hypha /
- aerial hypha /
- transcriptome sequencing /
- differentially expressed genes
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表 1 样品测序数据及与参考基因组的序列比对结果
Table 1. Statistics of experimental data and alignment with reference on genome sequences
编号
ID样品
Sample总Read数
Total clean reads比对到参考基因组的Reads数目
Mapped reads比对到参考基因组的Reads百分比
Mapped reads/%GC含量
GC content/%≥Q30的百分比
≥Q30/%A601 As2796气生 42 155 674 25 410 008 60.28 49.12 92.00 A602 As2796贴生 45 225 288 28 324 702 62.63 49.28 92.37 表 2 新基因功能注释结果统计
Table 2. Number of new genes with functional annotation
用于功能注释的数据库
Annotated Database获得注释的新基因数目
Annotated Gene Number长度在≥300~ 1000 的新基因数目
Gene Number (300≤gene length<1000 )长度≥ 1000 的新基因数目
Gene Number (Gene length≥1000 )COG 15 12 3 GO 21 13 7 KEGG 38 21 14 KOG 20 12 8 Pfam 34 20 12 Swissprot 24 15 8 nr 132 70 57 All 139 76 57 表 3 部分差异表达基因
Table 3. Partial DEGs
编号
NO基因ID
Gene ID相对表达量FPKM
Relative expression FPKMLog2FC Pfam蛋白注释
Pfam protein annotation基因注释
Gene annotationA601 A602 1 AGABI2DRAFT_194497 15.6653 0.0000 −6.78 — — 2 AGABI2DRAFT_177536 20.5777 0.0000 −4.47 — 假设蛋白OH76DRAFT_1359727 3 AGABI2DRAFT_120985 1.0452 0.0000 −3.64 — 假设蛋白AN958_00580 4 AGABI2DRAFT_203489 2.3380 0.0001 −3.85 — 假设蛋白 AN958_02967 5 AGABI2DRAFT_44066 6.2585 0 −3.75 HhH-GPD超家族碱基
切除DNA修复蛋白假设蛋白 AN958_02739 6 AGABI2DRAFT_113583 0.0000 4.2489 4.73 — 假设蛋白 AN958_04089 7 AGABI2DRAFT_117617 0.0008 6.9437 3.30 — 假设蛋白 AN958_03520 8 AGABI2DRAFT_179131 0.0010 2.5569 4.28 — 假设蛋白 AN958_00324 9 AGABI2DRAFT_194662 0 10.2374 4.66 疏水表面结合蛋白A 假设蛋白AN958_09554 10 AGABI2DRAFT_136569 196.301 32.0293 −2.58 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 11 AGABI2DRAFT_193057 999.26 189.163 −2.38 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 12 AGABI2DRAFT_136557 19733 3086.17 −2.67 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 13 AGABI2DRAFT_143464 100.031 896.356 3.21 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 14 AGABI2DRAFT_178707 31.7555 666.848 4.35 真菌疏水蛋白 假定的疏水蛋白 [双孢蘑菇] 表 5 SNP突变统计表
Table 5. SNP mutation statistics
编号 ID T∶A>G∶C C∶G>G∶C T∶A>A∶T C∶G>T∶A C∶G>A∶T T∶A>C∶G 转化 Ts 颠换 Tv 转换/颠换比 ts/tv A601 6312 5221 7641 42864 6405 42158 85022 25579 3.32 A602 6276 5239 7586 43273 6338 42476 85749 25439 3.37 表 6 全基因组和编码区InDel长度分布表
Table 6. Length distribution of genome-wide and coding region InDel sites
编号
ID区域
Region−10 −9 −8 −7 −6 −5 −4 −3 −2 −1 1 2 3 4 5 6 7 8 9 10 A601 基因组
Genome11 2 0 0 1 2 0 6 106 1235 1559 121 1 0 0 2 0 0 0 0 A601 外显子
Exonic7 1 0 0 1 0 0 3 2 99 221 14 1 0 0 2 0 0 0 0 A602 基因组
Genome14 3 0 0 1 2 0 11 100 1080 1434 121 3 0 0 3 0 0 0 0 A602 外显子
Exonic9 1 0 0 1 0 0 7 2 100 229 15 3 0 0 3 0 0 0 0 -
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