极端高温对截形叶螨体内海藻糖含量及海藻糖转运蛋白基因的影响

doi:10.3864/j.issn.0578-1752.2024.06.006

摘要/Abstract

摘要：

【目的】明确高温对截形叶螨（Tetranychus truncatus）海藻糖转运蛋白基因的影响，为有害生物的绿色防控提供理论依据。【方法】根据高温诱导下的截形叶螨转录组数据获取两条海藻糖转运蛋白基因TtTret1-like和TtTret1的CDS序列和蛋白氨基酸序列；利用ExPASy、ProScale、MEGA等分析工具对TtTret1-like和TtTret1进行生物信息学分析；利用实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）技术分析TtTret1-like和TtTret1在截形叶螨不同发育阶段及不同高温（38、42、46、50 ℃）处理下的表达特性；采用微量法测定不同高温处理下截形叶螨的海藻糖含量；利用RNA干扰（RNAi）技术沉默TtTret1-like和TtTret1，探究其在应对高温中的功能。【结果】生物信息学分析表明，TtTret1-like和TtTret1的CDS全长分别为1 389和1 569 bp，分别编码463和523个氨基酸，预测的蛋白分子量分别为50 189.03和57 358.10 Da，等电点分别为8.87、8.70。TtTret1-like和TtTret1均为碱性氨基酸和疏水性氨基酸且同属于不稳定蛋白；二级结构预测为螺旋和卷曲结构；两个海藻糖转运蛋白均具有协同转运蛋白超家族（major facilitator superfamily，MFS）的保守结构域及12个跨膜结构域。氨基酸序列相似性和系统发育分析表明，TtTret1-like和TtTret1与蛛形纲其他物种海藻糖转运蛋白序列的一致性较高，尤其与二斑叶螨（T. urticae）的亲缘关系最近。TtTret1-like在卵期、幼螨期和成螨期的表达量较高；TtTret1在幼螨期高表达。随处理温度的升高，TtTret1-like的表达量大致呈上升趋势，在50 ℃时表达量最高；而TtTret1的表达量随处理温度的升高先上升后下降，在42 ℃时表达量达到最高；高温胁迫后截形叶螨体内的海藻糖含量显著升高。沉默TtTret1-like和TtTret1 48 h后，截形叶螨体内整体海藻糖含量上升，但血淋巴海藻糖含量下降；用50 ℃高温处理截形叶螨2 h，然后恢复至96 h时，截形叶螨存活率分别为11%和46.67%，显著低于对照组。【结论】截形叶螨TtTret1-like和TtTret1在其抵御高温胁迫过程中具有重要的作用。

关键词: 截形叶螨, 高温胁迫, 海藻糖, 海藻糖转运蛋白, RNA干扰

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)

罗丽丹, 陈嘉明, 安琪, 刘磊, 孙勤哲, 刘欢, 王森山, 宋丽雯. 极端高温对截形叶螨体内海藻糖含量及海藻糖转运蛋白基因的影响[J]. 中国农业科学, 2024, 57(6): 1091-1101.

LUO LiDan, CHEN JiaMing, AN Qi, LIU Lei, SUN QinZhe, LIU Huan, WANG SenShan, SONG LiWen. Effects of Extreme High Temperature on Trehalose Content and Trehalose Transporter Gene in Tetranychus truncatus[J]. Scientia Agricultura Sinica, 2024, 57(6): 1091-1101.

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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