Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (6): 1091-1101.doi: 10.3864/j.issn.0578-1752.2024.06.006

• PLANT PROTECTION • Previous Articles     Next Articles

Effects of Extreme High Temperature on Trehalose Content and Trehalose Transporter Gene in Tetranychus truncatus

LUO LiDan(), CHEN JiaMing, AN Qi, LIU Lei, SUN QinZhe, LIU Huan, WANG SenShan, SONG LiWen()   

  1. College of Plant Protection, Gansu Agricultural University/Biological Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070
  • Received:2023-11-01 Accepted:2023-12-08 Online:2024-03-25 Published:2024-03-25
  • Contact: SONG LiWen

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Abstract:

【Objective】 The objective of this study is to clarify the effect of high temperature on the trehalose transporter gene of Tetranychus truncatus, and to provide a theoretical basis for the green control of pests.【Method】 According to the transcriptome data of T. truncatus induced by high temperature, the CDS sequences and protein amino acid sequences of two trehalose transporter genes TtTret1-like and TtTret1 were obtained. Bioinformatics analysis of TtTret1-like and TtTret1 was carried out using analytical tools such as ExPASy, ProScale and MEGA. The expression characteristics of TtTret1-like and TtTret1 at different development stages and different high temperatures (38, 42, 46 and 50 ℃) were analyzed by qRT-PCR. The trehalose content of T. truncatus at different high temperatures was determined by microanalysis. RNA interference (RNAi) technique was used to silence TtTret1-like and TtTret1, and to explore their functions in coping with high temperature.【Result】 Bioinformatics analysis showed that the full-length CDS of TtTret1-like and TtTret1 was 1 389 and 1 569 bp, respectively, encoding 463 and 523 amino acids. The predicted protein molecular weights were 50 189.03 and 57 358.10 Da, and the isoelectric points were 8.87 and 8.70, respectively. TtTret1-like and TtTret1 are both basic amino acids and hydrophobic amino acids, belonging to unstable proteins. The secondary structure is a spiral and coiled structure. Both trehalose transporters have conserved domains and 12 transmembrane domains of major facilitator superfamily (MFS). The amino acid sequence similarity and phylogenetic analysis showed that TtTret1-like and TtTret1 were highly consistent with the trehalose transporter sequences of other arachnids, especially with T. urticae. The expression level of TtTret1-like was higher in egg, larva and adult stages. TtTret1 was highly expressed in larva stage. With the increase of treatment temperature, the expression level of TtTret1-like increased, and reached the highest level at 50 ℃. However, with the increase of treatment temperature, the expression level of TtTret1 increased first and then decreased, reaching the maximum at 42 ℃. The trehalose content in T. truncatus increased significantly after high temperature stress. After silencing TtTret1-like and TtTret1 for 48 h, the trehalose content in T. truncatus increased, but the trehalose content in hemolymph decreased. The survival rates of T. truncatus were 11% and 46.67% after treated at 50 ℃ for 2 h and then recovered for 96 h, which were significantly lower than those of the control group.【Conclusion】 It is hypothesized that TtTret1-like and TtTret1 in T. truncatus play an important role in their responses to high temperature stress.

Key words: Tetranychus truncatus, high temperature stress, trehalose, trehalose transporter, RNA interference (RNAi)

Table 1

Primers used in this study"

基因Gene 引物序列Primer sequence (5′-3′) 引物用途Usage of primers
TtTret1-like F: CGGAACAGCACTTGGTTACT 实时荧光定量PCR
qRT-PCR
TtTret1-like R: GCGACAAATGGGACAGAGAG
TtTret1 F: GGCAACCAATGATTCTCAGC
TtTret1 R: AGCACTTCCTACGGTCACA
Actin F: GCCATCCTTCGTTTGGATTTGGCT 内参基因
Internal reference gene
Actin R: TCTCGGACAATTTCTCGCTCAGCA
RPS18 F: ACGTGCTGGTGAACTTACCGAAGA
RPS18 R: TGCCTATTCAAGAACCAAAGTGGG
dsTtTret1-like-T7 F: T7-TCGGAACAGCACTTGGTTAC dsRNA合成
dsRNA synthesis
dsTtTret1-like-T7 R: T7-CTGGAGGAGTTTGTCGGAAA
dsTtTret1-T7 F: T7-TGTGACCGTAGGAAGTGCTT
dsTtTret1-T7 R: T7-CTGGAAGGACGAACGAGGTA
dsGFP-T7 F: T7-CAGTTCTTGTTGAATTAGATG
dsGFP-T7 R: T7-TTTGGTTTGTCTCCCATGATG
T7 sequence TAATACGACTCACTATAGGG

Fig. 1

Bioinformatics analysis of TtTret1-like and TtTret1"

Fig. 2

Phylogenetic analysis of TtTret1-like and TtTret1"

Fig. 3

Relative expression level of TtTret1-like and TtTret1 at different developmental stages of T. truncates Single-factor ANOVA test was used. Different lowercase letters on the column indicated significant difference (P<0.05). The same as below"

Fig. 4

Relative expression level of TtTret1-like and TtTret1 at different high temperatures"

Fig. 5

Trehalose content of T. truncatus at different high temperatures"

Fig. 6

Effects of interference with TtTret1-like and TtTret1 on gene expression and trehalose content in T. truncates"

Fig. 7

Effects of 50 ℃ extreme temperature on the survival of T. truncatus adults after silencing TtTret1-like and TtTret1"

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