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毛果杨丝氨酸蛋白酶抑制子PtrSPI的抗虫能力分析

遇文婧 杨帅 黄颖 刁桂萍

遇文婧, 杨帅, 黄颖, 刁桂萍. 毛果杨丝氨酸蛋白酶抑制子PtrSPI的抗虫能力分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20210163
引用本文: 遇文婧, 杨帅, 黄颖, 刁桂萍. 毛果杨丝氨酸蛋白酶抑制子PtrSPI的抗虫能力分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20210163
Yu Wenjing, Yang Shuai, Huang Ying, Diao Guiping. The analysis of insect resistance ability of the serine protease inhibitor PtrSPI from Populus tomentosa[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20210163
Citation: Yu Wenjing, Yang Shuai, Huang Ying, Diao Guiping. The analysis of insect resistance ability of the serine protease inhibitor PtrSPI from Populus tomentosa[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20210163

毛果杨丝氨酸蛋白酶抑制子PtrSPI的抗虫能力分析

doi: 10.12171/j.1000-1522.20210163
基金项目: 中央高校基本科研业务费专项资金项目(2572020BA08)
详细信息
    作者简介:

    遇文婧,博士,副研究员。主要研究方向:森林保护。Email:ywjlinda2008@163.com 地址:150040黑龙江省哈尔滨市南岗区哈平路134号

    责任作者:

    刁桂萍,博士,副教授。主要研究方向:森林保护。Email:dgp2003@126.com 地址:150040 黑龙江省哈尔滨市香坊区和兴路26号

  • 中图分类号: S763.1

The analysis of insect resistance ability of the serine protease inhibitor PtrSPI from Populus tomentosa

  • 摘要:   目的  研究毛果杨丝氨酸蛋白酶抑制子PtrSPI的抗虫功能,为开发新型林木抗虫生物农药奠定基础。  方法  本研究通过研究毛果杨丝氨酸蛋白酶抑制子基因PtrSPI的启动子及其在舞毒蛾幼虫取食胁迫下的表达模式,分析PtrSPI基因功能;利用真核重组蛋白PtrSPI饲喂舞毒蛾幼虫,观察记录幼虫的取食量、体质量和死亡率,并测定取食后的幼虫体内丝氨酸蛋白酶活性,探讨PtrSPI蛋白的抗虫能力。  结果  结果表明毛果杨丝氨酸蛋白酶抑制子基因PtrSPI的启动子区域共包含5个与植物抗病虫相关的元件;经舞毒蛾幼虫取食后,毛果杨叶片的PtrSPI基因呈先降后升的表达模式,且在取食30 h达到峰值,为空白对照的2.03倍;高浓度重组蛋白PtrSPI(300和500 mg/mL)对舞毒蛾幼虫的取食量和体质量有显著的抑制作用,而且分别在取食6和4 h后舞毒蛾幼虫死亡率均达到60%以上,而幼虫体内丝氨酸蛋白酶的活性在取食初期显著上升。  结论  本研究验证了毛果杨丝氨酸蛋白酶抑制子PtrSPI的抗虫能力,可为进一步研发新型无公害抗虫生物农药提供理论基础和研究材料。

     

  • 图  1  PtrSPI基因的启动子功能元件分析

    Figure  1.  The cis elements of PtrSPI gene promoter analysis

    图  2  毛果杨叶片PtrSPI基因在舞毒蛾幼虫取食胁迫下的表达特性

    Ck. 以未取食叶片为对照;T. 舞毒蛾幼虫取食后的叶片。大写字母表示同一处理下不同时间点间的差异显著性(P < 0.05);小写字母表示同一时间点不同处理间的差异显著性(P < 0.05)。Ck, the unfed leaves were used as the control; T, the leaves were fed by L. dispar larvae. Capital letters mean the significance of the different time in the same treatments (P < 0.05); lowercase letters mean the significance of the difference treatments at the same times (P < 0.05).

    Figure  2.  Expression feature of PtrSPI gene of P. trichocarpa under feeding stress of L. dispar larvaes

    图  3  重组蛋白PtrSPI对舞毒蛾幼虫取食量的影响

    A. 50 mg/mL质量浓度重组蛋白及对照;B. 100 mg/mL质量浓度重组蛋白及对照;C. 300 mg/mL质量浓度重组蛋白及对照;D. 500 mg/mL质量浓度重组蛋白及对照。大写字母表示同一处理下不同时间点间的差异显著性(P < 0.05);小写字母表示同一时间点不同处理间的差异显著性(P < 0.05)。下同。A: 50 mg/mL recombinant protein and the control; B: 100 mg/mL recombinant protein and the control; C: 300 mg/mL recombinant protein and the control; D: 500 mg/mL recombinant protein and the control. Capital letters mean the significance of the different time in the same treatments (P < 0.05); lowercase letters mean the significance of the difference treatments at the same times (P < 0.05). The same below.

    Figure  3.  The effect of recombinant protein PtrSPI on the consumption of L. dispar larvaes

    图  4  重组蛋白PtrSPI对舞毒蛾幼虫体质量的影响

    A:50 mg/mL质量浓度重组蛋白及对照;B:100 mg/mL质量浓度重组蛋白及对照;C:300 mg/mL质量浓度重组蛋白及对照;D:500 mg/mL浓度重组蛋白及对照。A: 50 mg/mL recombinant protein and the control; B: 100 mg/mL recombinant protein and the control; C: 300 mg/mL recombinant protein and the control; D: 500 mg/mL recombinant protein and the control.

    Figure  4.  The effect of recombinant protein PtrSPI on the body mass of L. dispar larvaes

    图  5  重组蛋白PtrSPI对舞毒蛾幼虫死亡率的影响

    A:50 mg/mL浓度重组蛋白及对照;B:100 mg/mL浓度重组蛋白及对照;C:300 mg/mL浓度重组蛋白及对照;D:500 mg/mL浓度重组蛋白及对照。A: 50 mg/mL recombinant protein and the control; B: 100 mg/mL recombinant protein and the control; C: 300 mg/mL recombinant protein and the control; D: 500 mg/mL recombinant protein and the control.

    Figure  5.  The effect of recombinant protein PtrSPI on the mortality rate of L. dispar larvaes

    图  6  不同处理下舞毒蛾幼虫体内丝氨酸蛋白酶活性

    A:50 mg/mL质量浓度重组蛋白及对照;B:100 mg/mL质量浓度重组蛋白及对照;C:300 mg/mL质量浓度重组蛋白及对照;D:500 mg/mL质量浓度重组蛋白及对照。A: 50 mg/mL recombinant protein and the control; B: 100 mg/mL recombinant protein and the control; C: 300 mg/mL recombinant protein and the control; D: 500 mg/mL recombinant protein and the control.

    Figure  6.  The activities of serine protease in L. dispar larvaes under different treatments

    表  1  RT-qPCR引物

    Table  1.   RT-qPCR primers

    基因 Gene基因全称 Total name引物 Primer序列(5′—3′) Sequence (5′−3′)
    PtrSPI Serine proteinase inhibitor gene Ptr1 AAGGAATGGGTGGTCAGCAA
    Ptr2 GGGACTAGGATTGGTGTCGC
    Actin Reference gene Act1 AACATGGGATTGTTAGCAACTGG
    Act2 TCCATCACCAGAATCCAGCACA
    18S rRNA Reference gene R1 CGAAGACGATCAGATACCGTCCTA
    R2 TTTCTCATAAGGTGCTGGCGGAGT
    下载: 导出CSV
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  • 收稿日期:  2021-04-30
  • 修回日期:  2021-06-17
  • 网络出版日期:  2021-07-14

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