Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (20): 4057-4070.doi: 10.3864/j.issn.0578-1752.2024.20.011

• SPECIAL FOCUS: OCCURRENCE AND CONTROL OF MIGRATORY PESTS • Previous Articles     Next Articles

AQPs Characteristics of Megoura crassicauda and Their Expression Changes in Response to High Relative Humidity Stress

SUN SiSi1,3(), MA Wu2, SI HuiRu2, WANG XianZhong2, LIU Qiang4, LUO YanLin2, CHEN XiaoYuLong1(), TANG Bin2()   

  1. 1 College of Agriculture/College of Life Sciences, Guizhou University, Guiyang 550025
    2 College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121
    3 Guizhou Mountainous Meteorological Science Research Institute, Guiyang 550002
    4 Guizhou Tobacco Company Qiandongnan Tobacco Branch Company, Kaili 556000, Guizhou
  • Received:2024-07-02 Accepted:2024-08-22 Online:2024-10-16 Published:2024-10-24
  • Contact: CHEN XiaoYuLong, TANG Bin

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Abstract:

【Objective】At present, crop pests are more adaptable to high relative humidity. Aquaporin (AQP) is a membrane protein that is necessary for insects to maintain water balance in the body. The objectives of this study are to screen two McAQP sequence structures by transcriptome sequencing through high relative humidity stress, clarify the AQP characteristics of Megoura crassicauda and the expression changes in response to high relative humidity stress combined with real-time fluorescence quantitative PCR (qRT-PCR) verification, and to provided a theoretical basis for further exploring the gene function of AQP.【Method】The environmental relative humidity (RH) of 60%, 75% and 90% were set to cultivate M. crassicauda. The growth and development were observed, the relative expression of genes and the content of related substances in the body were detected. Based on two McAQP sequences (named as McAQP X1 and McAQP X2, respectively) obtained from comparative transcriptome sequencing of M. crassicauda, the physicochemical property, sequence structure, and the homology with other insects were analyzed by bioinformatics methods. Finally, qRT-PCR was used to determine the relative expression of AQP X1, AQP X2, Vg and VgR under long-term stable high relative humidity stress and 24 h emergency high relative humidity stress.【Result】Compared with RH 60% and RH 75%, the fecundity of M. crassicauda under RH 90% was significantly reduced, but there was no significant difference in development duration and survival rate. With the increase of relative humidity, the water content of M. crassicauda decreased, and the special phenotype phenomena such as body color change and long wing appeared. The molecular weights of McAQP X1 and McAQP X2 are 33.89 and 28.94 kDa, the theoretical isoelectric points are 5.36 and 5.32, and the protein lengths are 308 and 272 aa, respectively. On the multistage structure, McAQP is arranged by six long alpha helices via counterclockwise rotation to form a barrel channel, whereas four monomers form a tetramer to exercise function. The relative expression of McAQPs quantified by qRT-PCR showed that both McAQP X1 and McAQP X2 were up-regulated with increasing relative humidity, which was generally consistent with the transcriptome sequencing results. Meanwhile, under long-term stable high humidity stress, both Vg and VgR were up-regulated under RH 75% and down-regulated under RH 90%. Under 24 h emergency high humidity stress, Vg expression level was down-regulated with increasing humidity, while VgR was up-regulated under RH 90%.【Conclusion】High humidity environment affects the reproduction, water content and body color of M. crassicauda. The aquaporin sequence structure in subfamily II is relatively conservative, without strong species-specific differentiation. NPA site is important for the function of aquaporin, and the asparagine residue plays a structural role for water molecules through the central channel. It is speculated that the expression change of AQPs may be an important means for M. crassicauda to cope with humidity changes.

Key words: Megoura crassicauda, high relative humidity stress, reproduction, aquaporin (AQP), protein structure, bioinformatics

Table 1

Primers used for qRT-PCR"

基因名称<BOLD>G</BOLD>ene name 正向引物Forward primer (5′-3′) 反向引物Reverse primer (5′-3′)
McAQP X1 TGTGTTTGACGCACCAATGTC AACGCAGTGCCAAACAGTTC
McAQP X2 AACCGGGACAAGGATTCGTC TAGATGGGCGGCAGCAATAG
McVg GCATTAGCCACTATGTTTCA CGTATTGCTCCATTGTTGT
McVgR AGATTGATTGCCACGACGGT TTCCGTCACATCTCCACGTC
McActin GATCATTGCCCCACCAGAAC TTTACGGTGGACAATGCCTG

Fig. 1

Growth and physiological development of M. crassicauda Data were presented as mean±SE. Different lowercases on the columns indicated significant differences (P<0.05). The same as below"

Fig. 2

Phenotype and water content of M. crassicauda under each humidity"

Table 2

The expression of aquaporin’ transcripts in M. crassicauda"

基因
Gene		 基因ID
Gene ID		 RH 60% vs RH 75% RH 60% vs RH 90% RH 75% vs RH 90%
log2FC P值P value log2FC P值P value log2FC P值P value
McAQP X1 PB.6653.1 0.4487 5.62E-07 0.1090 0.2594 -0.3282 6.01E-05
McAQP X2 PB.17934.1 0.4067 0.5397 0.5026 0.4215 0.1111 0.8436
PB.13731.1 -0.3547 0.1984 0.5392 0.0317 0.9044 6.57E-05
PB.26879.1 NA NA 5.3254 0.0122 5.2597 0.0170
PB.11356.1 -0.1972 0.4002 -0.5149 0.0297 -0.3053 0.1182

Table 3

Basic physicochemical properties of McAQP sequences"

基因
Gene		 登录号
Accession number		 分子量
Molecular weight (kDa)		 理论等电点
Theoretical pI		 长度
Length (aa)		 原子总数
Total number of atoms		 脂肪系数
Aliphatic index		 不稳定系数
Instability index
McAQP X1 PP908450 33.89 5.36 308 4806 108.93 39.27
McAQP X2 PP908451 28.94 5.32 272 4078 107.21 22.79

Fig. 3

Phylogenetic tree of McAQPs and aquaporins from other insect species based on amino acid sequence"

Fig. 4

Alignment of partial sequences from the three branches of the AQP multiple sequence evolution tree"

Fig. 5

The secondary structure prediction of McAQP X1 (A) and McAQP X2 (B) The blue represents α-helix and the red represents β-sheet"

Fig. 6

Three-dimensional structure prediction of McAQP X1 and McAQP X2"

Fig. 7

The relative expression level of McAQP X1, McAQP X2 under long-term stable high humidity and 24 h emergency stress"

Fig. 8

The relative expression level of Vg and VgR under long-term stable high humidity and 24 h emergency stress"

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