Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (19): 3357-3366.doi: 10.3864/j.issn.0578-1752.2019.19.007

• PLANT PROTECTION • Previous Articles     Next Articles

Regulation Function of Trehalose-6-phosphate Synthase Genes on Chitin Synthesis in Sogatella furcifera

ZHANG DaoWei1,YU YaYa2,PAN BiYing3,KANG Kui1,ZENG BoPing1,CHEN Jing2,TANG Bin1,3()   

  1. 1 College of Biology and Agriculture, Zunyi Normal University/Key Laboratory of Protection and Utilization of Animal Resource in Chishui River Basin, Zunyi 563006, Guizhou
    2 College of Basic Medical Science, Zunyi Medical University, Zunyi 563006, Guizhou
    3 College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036
  • Received:2019-05-05 Accepted:2019-06-10 Online:2019-10-01 Published:2019-10-11
  • Contact: Bin TANG E-mail:tbzm611@163.com

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Abstract:

【Background】 It is well known that trehalose-6-phosphate synthase (TPS) plays an important role in trehalose synthesis, which can mediate trehalose metabolism to regulate chitin synthesis and insect development. 【Objective】 In this study, the effect of silencing SfTPS was detected, the molting status of Sogatella furcifera was observed and the content of chitin and the quantitative expression of chitin synthase (CHS) gene were determined through inhibiting the expression of TPS by RNAi in S. furcifera. The purpose is to explore the potential regulatory effects of SfTPS on the chitin synthesis in S. furcifera.【Method】 The S. furcifera population, which has been fed in laboratory for many years, was used as experimental material. The double-stranded RNA (dsRNA) of SfTPS and GFP were synthesized in vitro in order to study the potential function of TPSs, the RNA of two dsSfTPSs (dsSfTPS1 and dsSfTPS2) was injected into S. furcifera, respectively. Firstly, after dsRNA injection, the total RNA of S. furcifera was extracted by Trizol method at 48 h, and the first strand DNA was synthesized as the template of quantitative real-time PCR (qRT-PCR). As well as qRT-PCR was used to detect the silencing of SfTPS expression for the effect of RNAi. Secondly, the whole chitin of S. furcifera was detected at 48 and 72 h after dsRNA injection, and photographs were taken of the winged developmental malformations. Finally, the relative expression level of SfCHS in S. furcifera was detected by qRT-PCR, and the roles of SfTPS1 and SfTPS2 in the regulation of chitin synthesis were analyzed.【Result】 Compared with dsGFP injection, the expression of SfCHS increased after dsSfTPS1 and dsSfTPS2 injection, as well as the chitin content increased, which lead to the wing deformity of S. furcifera. The qRT-PCR results showed that the expression of SfTPS was significantly inhibited after dsRNA injection of a single SfTPS, which was less than 30% of that in the control group injected with dsGFP. In addition, the expression of another SfTPS also decreased significantly after a single SfTPS dsRNA injection. The adult wings of S. furcifera were all long wings after dsSfTPS1 and dsSfTPS2 injection. Some deformities such as wing curl in a certain ratio had been found in these long wings, and a certain ratio of mortality was found at 48 and 72 h. The chitin content increased significantly at 72 h after these two dsSfTPSs RNA injection. Compared with the control group, the expression of SfCHS1 and SfCHS1a increased significantly at 72 h after dsSfTPS1 injection, followed it increased significantly at 48 and 72 h after dsSfTPS2 injection. In the same time, the expression of SfCHS1b increased significantly after dsSfTPS1 and dsSfTPS2 injection.【Conclusion】 The SfTPS can control the synthesis of chitin through the regulation of chitin synthase gene in S. furcifera. The results are helpful to evaluate the regulatory role of SfTPS in S. furcifera and other insects and as the potential pest control target. It provides a theoretical basis for further development and screening of effective trehalose-6-phosphate synthase inhibitors to control S. furcifera pests and so on.

Key words: white-backed planthopper (Sogatella furcifera), trehalose-6-phosphate synthase (TPS), RNA interference, chitin synthesis, quantitative real-time PCR (qRT-PCR)

Table 1

The primer sequences for dsRNA synthesis"

引物名称 Primer name 上游引物 Forward primer (5′-3′) 下游引物 Reverse primer (5′-3′) 产物长度 Length (bp)
dsSfTPS1 CCCGTTGTGGTGAGAAATA CAAGGTGGGAATGGAATG 473
dsSfTPS1-T7 T7-CCCGTTGTGGTGAGAAATA T7-CAAGGTGGGAATGGAATG 523
dsSfTPS2 CGCATAGACCGCAACAAC TCGCAACGGAGTAACCAG 459
dsSfTPS2-T7 T7-CGCATAGACCGCAACAAC T7-TCGCAACGGAGTAACCAG 509
dsGFP AAGGGCGAGGAGCTGTTCACCG CAGCAGGACCATGTGATCGCGC 688
dsGFP-T7 T7-AAGGGCGAGGAGCTGTTCACCG T7-CAGCAGGACCATGTGATCGCGC 738

Fig. 1

Comparison of nucleotide sequence of TPS1 and TPS2 in S. furcifera"

Table 2

The primer sequences for qRT-PCR detection"

引物名称 Primer name 上游引物 Forward primer (5′-3′) 下游引物 Reverse primer (5′-3′) 产物长度 Length (bp)
Q-SfTPS1 CCGATTCGCTACATCTACG GACAAACTCTTTCGCCACTAA 123
Q-SfTPS2 GATGCTGAGGGCAAAGAC TGTGGAAGCCGACAAAGT 226
Q-SfCHS1 GATTGGTCATTGGCTTCAGA GTAATGTCTTGCTTCGTCAG 151
Q-SfCHS1a CTTCGGTGTTTGGTTTCTT TGGGTAACATCATCATAGGA 136
Q-SfCHS1b GAGAAGGCGAGAATAGCA GCAGCAAGAACACGATTA 103
Q-18S rRNA GCCCCGTAATCGGAATGAGT GACAAGACGTCCCGCAAAAC 205

Fig. 2

The relative expression of two TPSs and moulting of adults of S. furcifera after RNAi"

Fig. 3

Changes of chitin content in S. furcifera after RNAi"

Fig. 4

The relative expression of SfCHS and variants transcripts after SfTPS RNAi"

Fig. 5

Deformity rate and mortality of S. furcifera after SfTPS RNAi The deformity rate of dsGFP control group is 0"

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