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西花蓟马几丁质合成酶UAP基因克隆及功能分析

陆承聪 李恒 张祥琴 王谅 陈艺欣 田厚军 林硕 张洁 陈勇 魏辉

陆承聪,李恒,张祥琴,等. 西花蓟马几丁质合成酶 UAP基因克隆及功能分析 [J]. 福建农业学报,2019,34(12):1419−1425. doi: 10.19303/j.issn.1008-0384.2019.12.009
引用本文: 陆承聪,李恒,张祥琴,等. 西花蓟马几丁质合成酶 UAP 基因克隆及功能分析 [J]. 福建农业学报,2019,34(12):1419−1425. doi: 10.19303/j.issn.1008-0384.2019.12.009
LU C C, LI H, ZHANG X Q, et al. Molecular Cloning and Functional Analysis of the UDP N-acetylglucosamine Pyrophosphorylases Gene in the Frankliniella occidentalis [J]. Fujian Journal of Agricultural Sciences,2019,34(12):1419−1425. doi: 10.19303/j.issn.1008-0384.2019.12.009
Citation: LU C C, LI H, ZHANG X Q, et al. Molecular Cloning and Functional Analysis of the UDP N-acetylglucosamine Pyrophosphorylases Gene in the Frankliniella occidentalis [J]. Fujian Journal of Agricultural Sciences,2019,34(12):1419−1425. doi: 10.19303/j.issn.1008-0384.2019.12.009

西花蓟马几丁质合成酶UAP基因克隆及功能分析

doi: 10.19303/j.issn.1008-0384.2019.12.009
基金项目: 国家重点研发计划项目(2018YFD0201102、2018YFD0201209);国家自然科学基金项目(31871936、31560499);福建省科技重大专项(2017NZ0003-1-4);福建省科技计划公益类专项(2017R1025-9、2018R1025-7、2019R1024-2、2019R1024-3);福建省农业科学院科技项目(STIT2017-3-2、AGY2018-5、AC2017-6、YC2019003);云南省应用基础研究计划面上项目(2016FB063);云南省应用基础研究计划重点项目(2018FA020);云南省中青年学术技术带头人后备人才项目(2015HB081)
详细信息
    作者简介:

    陆承聪(1990−),男,硕士,研究方向:昆虫生态与害虫综合治理(E-mail:470258916@qq.com

    通讯作者:

    魏辉(1972−),男,博士,研究员,研究方向:昆虫化学生态学(E-mail:weihui@faas.cn

  • 中图分类号: S 435

Molecular Cloning and Functional Analysis of UDP N-acetylglucosamine Pyrophosphorylases Gene in Frankliniella occidentalis

  • 摘要:   目的  阐明几丁质合成通路中的关键基因UDP-N-乙酰氨基葡萄糖焦磷酸化酶(UDP N-acetylglucosamine pyrophosphorylases, UAP)对西花蓟马Frankliniella occidentalis生长发育的影响。  方法  基于西花蓟马转录组数据,克隆验证了UAP基因的开放阅读框(Open Reading Frame, ORF)全长序列,命名为FoccUAP。利用RT-qPCR方法分析FoccUAP在各个组织和不同龄期的表达情况。最后通过向西花蓟马蜕皮24 h的2龄若虫显微注射dsUAP,检测沉默效率,观察其对西花蓟马生长发育的影响。  结果  FoccUAP基因开放阅读框全长1 545 bp,编码514个氨基酸,预测蛋白分子量为56.738 kDa,具有Glyco - tranf - GTA - type Super family家族的保守结构域,与等翅目、蜚蠊目、直翅目和半翅目昆虫的UAP基因亲缘关系较近。FoccUAP基因在西花蓟马各个组织都有表达,在头部和腹部相对表达量最高,触角、胸部和足相对表达量较低。该基因在虫体生长发育的各个阶段均有表达,其中2龄幼虫、蛹以及羽化后24、48、168、240 h的成虫表达量分别是1龄幼虫的1.30、2.50、1.56、2.0、2.43和2.56倍。RNAi结果表明,注射dsUAP的西花蓟马羽化率(36.3%)和注射后120 h的存活率(5.0%)均显著低于dsGFP对照组,且注射dsUAP的西花蓟马成虫出现翅膀发育不完整、胸腹部皱缩不规则等畸形现象。  结论  FoccUAP基因对西花蓟马生长发育具有重要的作用。
  • 图  1  FoccUAP基因的核苷酸及预测的氨基酸序列

    注:下划线部分为信号肽氨基酸序列;双下划线为蛋白保守结构域氨基酸序列;*为终止密码子。

    Figure  1.  Nucleotide and deduced amino acid sequence of FoccUAP

    Note: The signal peptide sequence (MEHQYENLRN) is marked with underline. The conserved amino acid sequences of Glyco-tranf-GTA-type superfamily are marked with double underline. The TAA stop codon is marked with *.

    图  2  FoccUAP的进化树分析

    Figure  2.  Phylogenetic tree of FoccUAP

    图  3  FoccUAP基因在西花蓟马不同发育时期(A)和不同组织(B)的mRNA表达分析

    注:数据上不同的字母表示差异显著(Tukey HSD test, P<0.05)

    Figure  3.  Relative expression levels of FoccUAP mRNA in different developmental stages (A) and tissues (B) of F. occidentalis

    Note: Different lowercase letters means significant difference (Tukey HSD test, P<0.05).

    图  4  注射dsUAP对西花蓟马死亡率及生长发育的影响

    注:*或者不同的字母表示差异显著(Tukey HSD test, P<0.05)

    Figure  4.  Effects of dsUAP on growth and mortality rate of F. occidentalis

    Note:* or different lowercase letters means significant difference (Tukey HSD test, P<0.05).

    表  1  引物信息

    Table  1.   Primers used

    引物名称
    Primer name
    序列
    Sequence
    FoccUAP-ORF-F ATGGAGCACCAGTACGAAAACCTC
    FoccUAP-ORF-R TTAGTGAACACCATTTGATATCCCA
    dsFoccUAP-F TAATACGACTCACTATAGGGTACAAATGCTACCTGCACTGAAAGTAAGCT
    dsFoccUAP-R TAATACGACTCACTATAGGGTGGCAACATCATCCTGGATTCTCC
    qFoccUAP-F TCCGTTTACTGATAGTCTTGTTGTCTGGG
    qFoccUAP-R GCTGTGCGTGGGCAATCCTTTT
    qβ-actin-F CACCACCGCTGAGCGTGAAATCG
    qβ-actin-R GTGATGACCTGACCGTCGGGAAGC
    dsGFP-F TAATACGACTCACTATAGGGCGAGGAGCTGTTCACCGG
    dsGFP-R TAATACGACTCACTATAGGGTCCTCGATGTTGTGGCGG
    下载: 导出CSV
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  • 收稿日期:  2019-10-12
  • 修回日期:  2019-11-29
  • 刊出日期:  2019-12-01

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