中国农业科学 ›› 2022, Vol. 55 ›› Issue (12): 2461-2471.doi: 10.3864/j.issn.0578-1752.2022.12.016
收稿日期:
2021-12-19
接受日期:
2022-01-21
出版日期:
2022-06-16
发布日期:
2022-06-23
通讯作者:
李红亮
作者简介:
张莉,E-mail: 基金资助:
ZHANG Li(),ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang(
)
Received:
2021-12-19
Accepted:
2022-01-21
Online:
2022-06-16
Published:
2022-06-23
Contact:
HongLiang LI
摘要:
【背景】 作为我国本土重要的资源昆虫,中华蜜蜂(Apis cerana cerana,简称中蜂)对我国初冬季低温开花植物的传粉具有重要的生态价值,其传粉习性与嗅觉系统密切相关。前期对中蜂采集蜂高、低温处理后的触角转录组数据分析发现,与昆虫嗅觉相关的尼曼匹克C2型蛋白(Niemann-Pick type C2 protein,NPC2)基因家族在低温时表达量上升。【目的】 以中蜂NPC2家族基因为研究对象,克隆并分析其结构特征和表达谱以及高、低温处理下表达量的差异,为深入研究中蜂NPC2基因家族在中蜂低温适应的嗅觉感受功能提供参考。【方法】 基于中蜂高、低温转录组测序结果,利用RT-PCR克隆获得中蜂NPC2基因ORF序列,进行系统进化树分析和三维结构预测,然后通过实时荧光定量PCR分析中蜂NPC2基因在不同发育时期、组织的时空表达谱,以及高、低温时的表达量变化。【结果】 获得4个中蜂NPC2基因——AcNPC2a、AcNPC2b、AcNPC2c、AcNPC2d的ORF全长,分别为447、480、459和465 bp,编码148、159、152和154个氨基酸,预测蛋白分子量为16.12—18.53 kD,等电点分别为7.98、7.57、6.56和6.34。进化树分析显示AcNPC2与意大利蜜蜂的NPC2同源序列亲缘关系最近,与其他膜翅目昆虫NPC2也有一定相似性。实时荧光定量PCR结果显示,AcNPC2a在新出房蜂的腹部表达量最高,其次是在哺育蜂的腹部以及幼虫期;AcNPC2b在新出房蜂的胸部表达最高,在采集蜂的头部、胸部和后足也有表达;AcNPC2c在哺育蜂和采集蜂的触角中呈高丰度表达;AcNPC2d在采集蜂的头部表达量最高。经低温处理后,4个AcNPC2基因在采集蜂触角中的表达量均有所上升,但差异不显著。【结论】 AcNPC2具有NPC2蛋白的保守结构,其基因家族成员在中蜂的时空表达谱中呈现多样性,其中AcNPC2c在触角中呈高丰度表达,表明其与中蜂嗅觉感受功能关系密切。AcNPC2基因家族在低温时采集蜂触角中表达量均有所上升,表明该基因家族有可能参与中蜂的低温适应性,或与初冬季访花行为有关。
张莉,张楠,江虎强,吴帆,李红亮. 中华蜜蜂NPC2基因家族克隆及表达模式分析[J]. 中国农业科学, 2022, 55(12): 2461-2471.
ZHANG Li,ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang. Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana[J]. Scientia Agricultura Sinica, 2022, 55(12): 2461-2471.
表1
NPC2基因引物信息列表"
引物名称 Primer name | 引物序列 Primer sequence (5′-3′) | 产物长度 Product length (bp) | 退火温度 Tm (℃) | |
---|---|---|---|---|
用于基因克隆 For gene cloning | ||||
NPC2a | NPC2e-Fa | CCGAATTCATGGCAATCCTCACCTATGTT | 463 | 55 |
NPC2e-Ra | GGCTCGAGTTAGTCGACAACTTTAGCTGG | |||
NPC2b | NPC2e-Fb | CCGAATTCATGAATCGAAACGGTATGTTC | 496 | 58 |
NPC2e-Rb | GGCTCGAGTTAGTCGACAACTTTAGCTGG | |||
NPC2c | NPC2e-Fc | CCGAATTCATGTATCGAATGATTGCTGTAA | 475 | 60 |
NPC2e-Rc | GGCTCGAGCTATTTAATTCGAGCCGGAAT | |||
NPC2d | NPC2e-Fd | CCGAATTCATGAGGGGAACCATTCTCGTT | 481 | 61 |
NPC2e-Rd | GGCTCGAGTCAATTGGTGATCTTCGCTG | |||
用于实时荧光定量PCR For qRT-PCR | ||||
NPC2a | NPC2q-Fa | ACGTTGACTTTAAGGCTGTTGC | 129 | 51 |
NPC2q-Ra | ACTGTCCGCTCACCAGATTCTT | |||
NPC2b | NPC2q-Fb | TCCATTATACGCCTGCCTTCTCCT | 116 | 53 |
NPC2q-Rb | GGAAGTTGACAAGCAAGCATCGG | |||
NPC2c | NPC2q-Fc | GATGCTTGCCAAACTCCTGATTCT | 116 | 55 |
NPC2q-Rc | ACAGTAACACTCACCTTAGGATACG | |||
NPC2d | NPC2q-Fd | TTCGTAGCTCTGCTCGTAGTTGTCT | 138 | 54 |
NPC2q-Rd | CGCCTTCGTCCTTCGTTTCAGTT | |||
内参 Reference | β-actinq-F | TGCCAACACTGTCCTTTCTGGA | 96 | 53 |
β-actinq-R | TTCATGGTGGATGGTGCTAGGG | |||
酶切位点用下划线表示The underlines represent the restriction sites |
表2
中蜂NPC2基因家族序列特征和理化性质"
基因 Gene | GenBank登录号 GenBank accession number | 开放阅读框全长 ORF full length (bp) | 编码蛋白质全长 Length of encoding protein (aa) | 分子量 Molecular weight (kD) | 等电点 Isoelectric point (pI) | 与意蜂核苷酸序列的相似性 Similarity to the nucleotide sequence of A. m. ligustica (%, E value) |
---|---|---|---|---|---|---|
AcNPC2a | KJ633823.1 | 447 | 148 | 16.12 | 7.98 | 94.59%, 1e-98 |
AcNPC2b | OL741688 | 480 | 159 | 18.53 | 7.57 | 96.86%, 2e-113 |
AcNPC2c | OL741689 | 459 | 152 | 16.96 | 6.56 | 98.03%, 8e-103 |
AcNPC2d | OL741690 | 465 | 154 | 16.78 | 6.34 | 99.35%, 2e-108 |
图2
基于中蜂NPC2基因家族构建的系统发育进化树 膜翅目:Hymenoptera;鞘翅目:Coleoptera;双翅目:Diptera;蚤目:Siphonaptera;寄螨目:Parasitiformes;鳞翅目:Lepidoptera;缨翅目:Thysanoptera;半翅目:Hemiptera。分支长度代表遗传距离,各分支上数字为“Bootstrap”为1000 时循环不检验的置信度。前方标为三角的为目的序列,前方标为圆圈的为意蜂NPC2序列Branch length represented the distance of evolution. Bootstrap support values (%) based on 1000 replicates were indicated. The target sequences were labeled by triangles, the sequences of AmNPC2s were labeled by circles"
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