中国农业科学 ›› 2020, Vol. 53 ›› Issue (19): 4092-4102.doi: 10.3864/j.issn.0578-1752.2020.19.021
• 畜牧·兽医·资源昆虫 • 上一篇
收稿日期:
2020-01-17
接受日期:
2020-03-10
出版日期:
2020-10-01
发布日期:
2020-10-19
通讯作者:
苏松坤,聂红毅
作者简介:
高艳,E-mail: 基金资助:
GAO Yan(),ZHU YaNan,LI QiuFang,SU SongKun(
),NIE HongYi(
)
Received:
2020-01-17
Accepted:
2020-03-10
Online:
2020-10-01
Published:
2020-10-19
Contact:
SongKun SU,HongYi NIE
摘要:
【目的】意大利蜜蜂(Apis mellifera ligustica)哺育行为在维护蜂群稳定和生产蜂王浆方面发挥重要作用。本研究通过人工组建蜂群获得相同日龄的哺育蜂和采集蜂,筛选出排除日龄因素干扰后哺育蜂脑部与哺育行为密切相关的差异表达基因,揭示哺育蜂脑部调控哺育行为的分子网络。【方法】通过人工组建蜂群的方法获得3日龄工蜂、10日龄哺育蜂和采集蜂、21日龄哺育蜂和采集蜂,解剖各组工蜂头部获得脑组织样本,应用RNA-seq技术对5组脑部样本(3日龄工蜂、10日龄哺育蜂、10日龄采集蜂、21日龄哺育蜂、21日龄采集蜂)中基因表达量进行转录组测序的全面分析,筛选出哺育蜂脑部哺育行为密切相关的差异表达基因,并对这些差异表达基因进行GO和KEGG富集分析。同时利用实时荧光定量PCR(qPCR)对随机选取的4个差异表达基因的表达模式进行验证。【结果】RNA-seq分析筛选得到32个与哺育蜂哺育行为密切相关的差异表达基因,这些基因在10日龄哺育蜂脑中表达量均显著高于3日龄工蜂、10日龄采集蜂、21日龄采集蜂,且在21日龄哺育蜂脑中的表达量也显著高于21日龄采集蜂。GO富集分析发现上调差异表达基因主要参与氧化还原酶活性、气味结合、跨膜运输等功能。KEGG富集结果显示,上调的差异表达基因主要参与蛋白质代谢(核糖体)、能量代谢(氧化磷酸化、碳代谢、三羧酸循环、淀粉和蔗糖代谢、氮代谢、其他聚糖降解通路)、信号转导(Toll和Imd信号通路、光传导、鞘脂代谢)、消化作用(溶酶体),其中显著性富集在鞘脂代谢和其他聚糖降解通路。qPCR结果显示3个上调差异表达基因(LOC409709、LOC551813、LOC409708)和1个下调差异表达基因(LOC551165)表达模式的检测结果与测序数据一致。【结论】通过对3日龄工蜂、10日龄哺育蜂、10日龄采集蜂、21日龄哺育蜂、21日龄采集蜂5组样本脑部进行全面分析,获得相同日龄哺育蜂和采集蜂脑部基因的转录组图谱,分析得到了脑部哺育行为相关的32个上调差异表达基因。哺育蜂脑部的差异表达基因主要通过信号转导和能量代谢等途径调控哺育蜂的哺育行为。
高艳,朱雅楠,李秋方,苏松坤,聂红毅. 转录组学分析意大利蜜蜂脑部哺育行为相关基因[J]. 中国农业科学, 2020, 53(19): 4092-4102.
GAO Yan,ZHU YaNan,LI QiuFang,SU SongKun,NIE HongYi. Transcriptomic Analysis of Genes Related to Nursing Behavior in the Brains of Apis mellifera ligustica[J]. Scientia Agricultura Sinica, 2020, 53(19): 4092-4102.
表1
qPCR引物信息"
基因 Gene | 登录号 Accession number | 引物序列 Primer sequence (5′-3′) | 产物大小 Amplicon length (bp) |
---|---|---|---|
LOC409709 | 409709 | F: TTGGTCAGTTGGATGGGTAGA | 236 |
R: CCGACGGTGTAAGAAAGGC | |||
LOC551813 | 551813 | F: CAAAATTCAACAGCTCCTGC | 210 |
R: TCATCGCTACCGAAATCATAA | |||
LOC409708 | 409708 | F: TTGTTGATCGCAATCCTGTC | 480 |
R: CGTCGCATCGTCATCGTAA | |||
LOC551165 | 551165 | F:TGCCTGTTGTATTCTCGTAT | 304 |
R:TCTGACCTTGCCCTCCTC | |||
Actin | NM_001185146.1 | F: CCTAGCACCATCCACCATGAA | 87 |
R: GAAGCAAGAATTGACCCACCAA |
表2
RNA-seq数据总览"
样品 Sample | 原始读数 Number of raw reads | 有效读数 Number of clean reads | 有效匹配数(匹配率) Clean map (Map rate) | 单一匹配数(匹配率)Unique map (Map rate) | 多匹配数(匹配率)Multiple map (Map rate) | 99.9%的碱基正确率99.9% base accuracy (%) |
---|---|---|---|---|---|---|
3 d_1 | 67515798 | 66708824 | 56766708 (85.10%) | 55951632 (83.87%) | 815076 (1.22%) | 88.25 |
3 d_2 | 83920302 | 81832028 | 70899666 (86.64%) | 69821133 (85.32%) | 1078533 (1.32%) | 90.59 |
3 d_3 | 81546254 | 80442702 | 69502321 (86.40%) | 68471536 (85.12%) | 1030785 (1.28%) | 89.66 |
10 dN_1 | 57288572 | 56348822 | 49321933 (87.53%) | 48015717 (85.21%) | 1306216 (2.32%) | 90.51 |
10 dN_2 | 83787622 | 82964416 | 63408429 (76.43%) | 60124557 (72.47%) | 3283872 (3.96%) | 90.66 |
10 dN_3 | 72837334 | 71663646 | 56922446 (79.43%) | 56028890 (78.18%) | 893556 (1.25%) | 91.00 |
10 dF_1 | 67610626 | 66671128 | 55057317 (82.58%) | 54208585 (81.31%) | 848732 (1.27%) | 90.37 |
10 dF_2 | 81258562 | 80246018 | 63725456 (79.41%) | 62620480 (78.04%) | 1104976 (1.38%) | 90.06 |
10 dF_3 | 66021926 | 65365544 | 52522944 (80.35%) | 51744723 (79.16%) | 778221 (1.19%) | 91.00 |
21 dN_1 | 71882296 | 71020506 | 56181047 (79.11%) | 54846870 (77.23%) | 8223024 (13.18%) | 92.19 |
21 dN_2 | 72018192 | 70803290 | 60295752 (85.16%) | 58523770 (82.66%) | 11236986 (15.51%) | 92.33 |
21 dN_3 | 72458810 | 71460376 | 59684702 (83.52%) | 57902404 (81.03%) | 8742852 (13.63%) | 92.50 |
21 dF_1 | 72168956 | 70890254 | 59385891 (83.77%) | 58485427 (82.50%) | 900464 (1.27%) | 91.94 |
21 dF_2 | 69418794 | 68335020 | 55559809 (81.31%) | 54623413 (79.93%) | 936396 (1.37%) | 88.98 |
21 dF_3 | 81670548 | 80692702 | 67432186 (83.57%) | 66368993 (82.25%) | 1063193 (1.32%) | 91.86 |
表3
脑部41个DEG基于日龄依赖性在5组样本中的显著性趋势"
基因数目 Gene number | 10 dN vs 3 d显著性比较Significant comparison of 10 dN vs 3 d | 10 dN vs 21 dF显著性比较Significant comparison of 10 dN vs 21 dF | 10 dN vs 10 dF显著性比较Significant comparison of 10 dN vs 10 dF | 21 dN vs 21 dF显著性比较Significant comparison of 21 dN vs 21 dF | 10 dN vs 21 dN显著性比较 Significant comparison of 10 dN vs 21 dN |
---|---|---|---|---|---|
32 | ↑ | ↑ | ↑ | ↑ | ns |
6 | ↓ | ↓ | ↓ | ↓ | ns |
3 | ↑ | ↓ | ↓ | ↓ | ns |
表4
脑部前11个上调DEG在5组样本中的表达信息"
基因登录号 Gene_ID | 基因 Gene | 基因描述 Gene description | 平均FPKM值 Average FPKM value | log2 FC (10 dN vs 10 dF) | log2 FC (21 dN vs 21 dF) | ||||
---|---|---|---|---|---|---|---|---|---|
3 d | 10 dN | 10 dF | 21 dN | 21 dF | |||||
551813 | LOC551813 | 王浆主蛋白1 Major royal jelly protein 1 | 0.40 | 1564.78 | 2.27 | 510.71 | 9.64 | 9.44 | 5.71 |
725215 | LOC725215 | 毒酸磷酸酶Acph-1样 Venom acid phosphatase Acph-1-like | 0.31 | 29.97 | 0.66 | 23.80 | 0.73 | 5.52 | 5.01 |
411186 | LOC411186 | 突触小泡糖蛋白2C Synaptic vesicle glycoprotein 2C | 0.22 | 12.81 | 0.47 | 11.96 | 0.60 | 4.78 | 4.29 |
410149 | LOC410149 | 羧肽酶Q样 Carboxypeptidase Q-like | 1.59 | 31.09 | 1.82 | 26.29 | 2.81 | 4.10 | 3.21 |
409709 | LOC409709 | 假定的葡萄糖基神经酰胺酶4 Putative glucosylceramidase 4 | 2.83 | 75.03 | 7.81 | 50.90 | 8.63 | 3.27 | 2.54 |
552478 | Obp17 | 气味结合蛋白17 Odorant binding protein 17 | 1.77 | 6.43 | 0.73 | 4.05 | 0.80 | 3.14 | 2.33 |
551935 | Obp21 | 气味结合蛋白21 Odorant binding protein 21 | 4.50 | 34.70 | 4.43 | 22.20 | 7.71 | 2.97 | 1.51 |
677673 | Obp14 | 气味结合蛋白14 Odorant binding protein 14 | 1.00 | 7.70 | 1.01 | 3.32 | 0.69 | 2.92 | 2.24 |
409708 | LOC409708 | 葡糖基神经酰胺酶样 Glucosylceramidase-like | 13.84 | 132.92 | 22.42 | 113.84 | 27.93 | 2.57 | 2.01 |
726798 | LOC726798 | Sn1特异性二酰基甘油脂肪酶β Sn1-specific diacylglycerol lipase beta | 0.53 | 2.67 | 0.49 | 3.46 | 0.85 | 2.45 | 2.01 |
408383 | CYP6AQ1 | 细胞色素P450 6AQ1 Cytochrome P450 6AQ1 | 0.49 | 1.97 | 0.42 | 1.41 | 0.39 | 2.22 | 1.84 |
表5
上调DEG富集的GO条目"
GO条目 GO term | 基因 Gene |
---|---|
氧化还原酶活性 Oxidoreductase activity | CYP6AQ1/LOC726418/ LOC408734/LOC413590 |
气味结合 Odorant binding | Obp21/Obp14/Obp17 |
跨膜运输 Transmembrane transport | LOC411186/LOC725462 |
蛋白质水解 Proteolysis | LOC725215/LOC725154 |
翻译 Translation | LOC550715 |
脂质代谢 Lipid metabolism | LOC726798 |
DNA复制 DNA replication | LOC725238 |
细胞器组分Organelle part | LOC408444 |
图3
转录组数据的qPCR验证 黑色柱状图表示RNA-seq数据中基因的FPKM值,灰色表示qPCR数据中基因的相对表达量,纵坐标均为相应取过以2为底的对数值The black bar graph represents the FPKM value of the gene in the RNA-seq data, and the gray represents the relative expression of genes in the qPCR data; the ordinates are the corresponding logarithmic values with a base of 2。图中数据为平均值±标准误,采用单因素方差分析 Data in the figure are the mean±SE, One-way ANOVA. *P<0.05; **P<0.01; ***P<0.001"
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