Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (1): 56-64.doi: 10.3864/j.issn.0578-1752.2019.01.006

• PLANT PROTECTION • Previous Articles     Next Articles

RNA Interference of Vitellogenin Receptor Gene in Beet Armyworm (Spodoptera exigua)

ZHAO Jing1,2(),TAO Rong1,HAO DeJun1(),XIAO LiuBin2(),TAN YongAn2   

  1. 1Co-Innovation Center for the Sustainable Forestry in Southern China/College of Forestry, Nanjing Forestry University, Nanjing 210037
    2 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2018-08-13 Accepted:2018-10-02 Online:2019-01-01 Published:2019-01-12
  • Contact: DeJun HAO,LiuBin XIAO E-mail:jingzhao0126@126.com;dejunhao@163.com;xlbwll@sohu.com

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Abstract:

【Objective】Vitellogenin receptor (VgR) is the main receptor that mediates the endocytosis of insect vitellin. The objective of this study is to clarify the function of VgR of beet armyworm (Spodoptera exigua) through RNA interference (RNAi) method, and to provide a basis for further understanding the molecular mechanism of reproductive physiology and developing effective new methods for prevention and control.【Method】The fragment of VgR was amplified from the cDNA of female adults abdomen tissues of S. exigua by PCR which included the ligand-binding domain region. The green fluorescent protein gene (GFP) fragment was amplified from the GFP plasmid stored in the laboratory by specific primers. The fragment of VgR and GFP was then inserted into the pMD-19T for sequencing. The nucleic acid sequence was analyzed by DNAMAN software. The correct plasmid confirmed by sequencing acted as the DNA template. PCR amplification was performed using primers with T7 promoter. VgR and GFP dsRNA were synthesized with T7 RiboMAX TM Express RNAi System synthesis kit. The abdomens of S. exigua female pupae on 2nd and 6th day were injected with 3 μL double RNA by 10 μL microsyringe (2 μg·μL -1). RT-qPCR was used to detect the changes of VgR expression in 0-, 24-, 48-hour-old female adults. Meanwhile, eclosion rate and eggs per female were evaluated in control groups (blank control, GFP-dsRNA injection) and treatment group (VgR-dsRNA injection). 【Result】 The VgR and GFP gene fragments obtained by amplification were 327 and 417 bp, respectively. The VgR expression level of 0-, 24-, 48-hour-old female adults in the VgR-dsRNA group decreased by 79.35%, 84.22% and 67.68% compared with the GFP-dsRNA group, respectively. Through anatomical observation of the ovary of 0-, 24-, 48-hour-old female adults, it was found that compared with the GFP-dsRNA group, the ovary development of the VgR-dsRNA group was significantly delayed. Compared with the GFP-dsRNA group, the length of the ovary tube in the VgR-dsRNA group decreased by 23.92% for the 24-hour-old female adults. The GFP-dsRNA group has more mature eggs in the ovary with larger average diameter of (0.46±0.05) mm while the number of mature eggs in the VgR-dsRNA group was small with an average diameter of (0.23±0.02) mm. There was no significant difference of eclosion rate between GFP-dsRNA group and VgR-dsRNA group. In the VgR-dsRNA group, the average number of eggs per female was 170, while in the control groups (blank group, GFP-dsRNA group), the average number of eggs per female was 451 and 420, respectively. There was a significant difference in the amount of oviposition between control groups and treatment group. 【Conclusion】 The function of VgR was studied by dsRNA injection in vitro, which could significantly reduce the expression of VgR. VgR plays an irreplaceable role in the reproduction of S. exigua, which directly affects the ovary development and spawning capacity, and can be used as a potential target for controlling S. exigua.

Key words: beet armyworm (Spodoptera exigua), vitellogenin receptor gene (VgR), RNA interference, ovary development

Table 1

Primers used in this study"

引物名称Primer name 引物序列 Primer sequence (5′ to 3′) 产物长度Product length (bp)
dsRNA VgR-F CACAATCAAGACCGATACCA 327
VgR-R GTCCAGTCACTCCAGAACACT
VgR-TF TAATACGACTCACTATAGGG CACAATCAAGACCGATACCA
VgR-TR TAATACGACTCACTATAGGG GTCCAGTCACTCCAGAACACT
GFP-F CACAAGTTCAGCGTGTCCG 417
GFP-R CACCTTGATGCCGTTC
GFP-TF TAATACGACTCACTATAGGG CACAAGTTCAGCGTGTCCG
GFP-TR TAATACGACTCACTATAGGG CACCTTGATGCCGTTC
RT-qPCR VgR-QF GAAGGGAGGGAAGTGTCCTGAG 104
VgR-QR TGATGGTGAAAGAAACGCTGTG
β-actin-F CCAGCCTTCCTTCTTGGGTAT 93
β-actin -R AGGTCCTTACGGATGTCAACG

Fig. 1

PCR amplification of VgR and GFP"

Fig. 2

Production of VgR transcripted in vitro"

Fig. 3

The relative expression level of VgR in S. exigua after dsRNA injection Different lowercases on the columns indicate significantly different (P<0.05). The same as Fig. 5"

Fig. 4

The ovary development of S. exigua after dsRNA injection"

Fig. 5

Eclosion rate and fecundity after VgR-dsRNA injection"

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