Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (23): 4802-4812.doi: 10.3864/j.issn.0578-1752.2020.23.007

• PLANT PROTECTION • Previous Articles     Next Articles

The Structure Characteristics and Biological Functions on Regulating Trehalose Metabolism of Two NlTret1s in Nilaparvata lugens

YU WeiDong1,2(),PAN BiYing1,QIU LingYu1,HUANG Zhen2,ZHOU Tai2,YE Lin2,TANG Bin1,WANG ShiGui1()   

  1. 1College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036
    2Zhejiang Dingyi Biotechnology Co., LTD, Quzhou 324100, Zhejiang
  • Received:2020-07-18 Accepted:2020-09-03 Online:2020-12-01 Published:2020-12-09
  • Contact: ShiGui WANG E-mail:hzyuweidong@hotmail.com;sgwang@hznu.edu.cn

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Abstract:

【Objective】 Trehalose is the main blood sugar substance in insects and plays an important role in insect development and physiological activities. Among them, trehalose transporter (Tret) plays a key role in the transportation of trehalose from trehalose-producing tissues (such as fat body) to trehalose-consuming tissues. The objective of this study is to explore the biological functions of these two NlTret1s in brown planthopper (Nilaparvata lugens) by analyzing the sequence structure of two NlTret1s and further suppressing the expression of NlTret1s. 【Method】Taking these two NlTret1 sequences as the research object, the protein structure and homology with other insects were analyzed by bioinformatics technology. The RNA interference (RNAi) technology was used to inject the synthetic exogenous dsRNA (double-stranded RNA) into the laboratory feeding population of N. lugens, to inhibit the expression of the NlTret1s. The total RNA was extracted to synthesize the first-strand cDNA using reverse transcription Kit, qRT-PCR (quantitative real-time PCR) technology was used to detect the RNAi effect of dsNlTret1s and the expression of related genes in the trehalose metabolism pathway in N. lugens after RNAi, and finally glucose, trehalose and glycogen content as well as trehalase enzyme activity were determined. 【Result】 Bioinformatics analysis showed that the open reading frames of NlTret1-like X1 and NlTret1-2 X1 are 1 920 and 1 578 bp in length, encoding 639 and 525 amino acids, respectively. The predicted protein molecular weights are 69.29 and 58.71 kD, and the isoelectric points are 8.32 and 8.36, respectively. The secondary structure of NLTret1-like X1 and NLTret1-2 X1 is mainly composed of helix and coil. Conservative domain analysis showed that they all belong to the MFS family. The results of evolutionary analysis showed that Tret1 of different insects had high homology, and N. lugens was closely related to other hemiptera insects. Compared with the dsGFP group, the target gene was inhibited significantly after injection with dsNlTret1-like X1 or dsNlTret1-2 X1. Furthermore, the content of glycogen and glucose in N. lugens did not change significantly, but unlike the dsNlTret1-2 X1 group, the trehalose content of N. lugens was significantly increased with the injection of dsNlTret1-like X1. Meanwhile, the expression of the TPS1, TPS2, TRE1-1, TRE1-2 and TRE2 in N. lugens was significantly down-regulated after the NlTret1-like X1 knocked down for 48 h. The expression of TPS1, TPS2 and TRE1-1 in N. lugens also decreased significantly after injection with dsNlTret1-2 X1 for 48 h, while TRE1-2 and TRE2 showed a very significant upward trend. Moreover, after injection of dsNlTret1-like X1, the activities of soluble trehalase and membrane-bound trehalase were significantly reduced, but there was no significant change after injection with dsNlTret1-2 X1. 【Conclusion】These two Tret1s of N. lugens play different functions in different tissues, among which NlTret1-like X1 plays a more significant role in the specific transport of trehalose involved in energy supply. The results are helpful to explore the regulatory mechanism of Tret1 regulating the balance of trehalose metabolism in insects or invertebrates, and provide a theoretical basis for the future control of pests by regulating blood sugar balance, such as N. lugens.

Key words: brown planthopper (Nilaparvata lugens), trehalose transporter (Tret), structure, trehalose metabolism, RNAi

Table 1

Primer sequence used for qRT-PCR and dsNlTret1s, dsGFP"

引物名称 Primer name 正向引物 Forward primer (5′-3′) 反向引物 Reverse primer (5′-3′) 产物长度 Length (bp)
dsNlTret1-like X1 GCAAACAACAGCGAGCAA CCAAGAGGCACCCATCC 472
dsNlTret1-like X1-T7 T7-GCAAACAACAGCGAGCAA T7-CCAAGAGGCACCCATCC 552
dsNlTret1-2 X1 CTTCGTTCACCAGCACCT CAAATGGCACTTATTATCGTC 547
dsNlTret1-2 X1-T7 T7-CTTCGTTCACCAGCACCT T7-CAAATGGCACTTATTATCGTC 597
dsGFP AAGGGCGAGGAGCTGTTCACCG CAGCAGGACCATGTGATCGCGC 657
dsGFP-T7 T7-AAGGGCGAGGAGCTGTTCACCG T7-CAGCAGGACCATGTGATCGCGC 707
qNl18S CGCTACTACCGATTGAA GGAAACCTTGTTACGACTT 165
qNlTret1-like X1 GTGGGAATCGTGAACATGGG ATGGTCATGAGTGTGCTGGA 101
qNlTret1-2 X1 TATGTGTCGGCTGGGTTCTT CTCTGAGCCAGCACAATGAC 190
qNlTPS1 AAGACTGAGGCGAATGGT AAGGTGGAAATGGAATGTG 154
qNlTPS2 AGAGTGGACCGCAACAACA TCAACGCCGAGAATGACTT 161
qNlTRE1-1 GCCATTGTGGACAGGGTG CGGTATGAACGAATAGAGCC 132
qNlTRE1-2 GATCGCACGGATGTTTA AATGGCGTTCAAGTCAA 178
qNlTRE2 TCACGGTTGTCCAAGTCT TGTTTCGTTTCGGCTGT 197

Fig. 1

Amino acid sequences and structural analysis of NlTret1s GenBank登录号为XP_022183984.1（NlTret1-like X1）和XP_022195528.1（NlTret1-2 X1）Initiation and termination GenBank accession numbers: XP_022183984.1 (NlTret1-like X1) and XP_022195528.1 (NlTret1-2 X1)。A：NlTret1二级结构预测Prediction of the secondary structure of NlTret1s;B：NlTret1结构域分析（蓝色方块表示跨膜结构,粉色方块表示低复杂区域）Analysis of domain of NlTret1s (Blue blocks represent transmembrane region and pink blocks represent low complexity region);C：NlTret1三级结构预测Prediction of the tertiary structure of NlTret1s"

Fig. 2

Phylogenetic tree of NlTret1s and Tret1 proteins from other insect species based on the amino acid sequence (neighbor- joining method) Tret1蛋白来源物种及其GenBank登录号 Source species of Tret1 proteins and their GenBank accession numbers。豌豆蚜Acyrthosiphon pisum：ApTret1 (XP_001943832.1)、ApTret1 X1 (XP_003247868.1);桃蚜Myzus persicae：MpTret1-like (XP_022175298.1)、MpTret1-like X1 (XP_022168826.1);高粱蚜Melanaphis sacchari：MsTret1-like (XP_025205275.1)、MsTret1-like X1 (XP_025193454.1);玉米缢管蚜Rhopalosiphum maidis：RmTret1-like (XP_026817841.1)、RmTret1-like X1 (XP_026807124.1);棉蚜Aphis gossypii：AgTret1-like (XP_027847026.1)、AgTret1-like X1 (XP_027852240.1);黑豆蚜Aphis craccivora：AcTret1-like (KAF0773727.1);黄蔗蚜Sipha flava：SfTret1-like (XP_025415984.1)、SfTret1-like X1 (XP_025419752.1);褐飞虱Nilaparvata lugens：NlTret1-2 X1 (XP_022195528.1)、NlTret1-like X1 (XP_022183984.1);臭虫Cimex lectularius：ClTret1-like (XP_014254493.1);茶翅蝽Halyomorpha halys：HhTret1-like (XP_014285740.1);湿木白蚁Zootermopsis nevadensis：ZnTret1-like (XP_021920751.1);第二隐白蚁Cryptotermes secundus：CsTret1 X1 (XP_023724429.1);埃及伊蚊Aedes aegypti：AaTret1 (XP_001654366.1);猫虱Ctenocephalides felis：CfTret1-like (XP_026477563.1);菜粉蝶Pieris rapae：PrTret1-like (XP_022131116.1);麦茎蜂Cephus cinctus：CcTret1 X1 (XP_015587164.1);瘿蜂Belonocnema treatae：BtTret1-like (XP_033208929.1);汗蜂Dufourea novaeangliae：DnTret1 (KZC12919.1);彩带蜂Nomia melanderi：NmTret1-like X1 (XP_031847112.1);银额果蝇Drosophila albomicans：DaTret1 X1 (XP_034103480.1);铜绿蝇Lucilia cuprina：LcTret1 X1 (XP_023292603.1);德国小蠊Blattella germanica：BgTret1 (PSN42188.1);柑橘木虱Diaphorina citri：DcTret1-like (XP_026682851.1)"

Fig. 3

Relative expression level of NlTret1-like X1 and NlTret1-2 X1 after injection for 48 h in N. lugens"

Fig. 4

Relative expression level of regulated genes of trehalose metabolic pathway in N. lugens after RNAi"

Fig. 5

Contents of trehalose, glucose and glycogen in N. lugens after RNAi N.S. indicates no significant difference. The same as below"

Fig. 6

Enzyme activity of trehalase in N. lugens after RNAi"

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