Cloning and Functional Analysis of Calcineurin Subunits A and B in Development and Fecundity of Nilaparvata lugens (Stål)

doi:10.1016/j.rsci.2022.01.003

Abstract

Abstract:

Serine/threonine phosphatase calcineurin (CN) is a unique but confounding calcium/calmodulin- mediated enzyme, which is composed of a catalytic subunit A (CNA) and a regulatory subunit B (CNB). We cloned six transcripts for CNA named from NlCNA-X1 to NlCNA-X6, one CNB named NlCNB1 and one CNB homologous gene NlCNBH1 from Nilaparvata lugens. All of them are constitutively transcripted in various tissues and developmental stages. The primary structure of the six isoforms showed obvious differences in the length and composition of the amino acid sequence between the two binding domains of Ca²⁺/calmodulin (CaM) and CNB. Ca²⁺-binding EF-hand motifs were found in NlCNB1 and NlCNBH1. The specific gene silencing of NlCNA, NlCNB1 and NlCNBH1 respectively by RNAi resulted in drastical reduction in survival rate, female weight, eclosion rate and fecundity of N. lugens. These results showed that NlCNA, NlCNB1 and NlCNBH1 were required for N. lugens growth and reproduction. The negative effects of NlCNB1 silence on nymph mortality (97%), molting malformation (90%) and female sterile (50%) were more serious than those of NlCNA or NlCNBH1. qRT-PCR and enzyme-linked immunosorbent assay (ELISA) analyses indicated that the nymphs with silenced NlCNA, NlCNB1 or NlCNBH1 showed impaired hormone and energy metabolism. In nymphs, the contents of 20-hydroxyecdysone (20E) after NlCNB1 RNAi and phenoloxidase after NlCNA RNAi were particularly decreased. These results suggested that NlCNA is involved in immunity of N. lugens by regulation of phenoloxidase, while NlCNB1 may control the growth and development of N. lugens by 20E signaling pathway in addition to interact with CNA. Injection of 70 ng/μL dsNlCNB1 resulted in 77.0% down regulation of NlCNB1, and the nymph mortality was up to 57.9% at 10 d after injection. Therefore, NlCNB1 could be a potential candidate target used for strategy design in control of N. lugens. Our results revealed the importance of CN in the regulation of the growth and development of N. lugens, which provided a basis for further study of the molecular mechanism of CN.

Key words: Nilaparvata lugens, calcineurin, calcineurin catalytic subunit, calcineurin regulatory subunit, 20-hydroxyecdysone, phenoloxidase

Wang Weixia, Zhu Tingheng, Wan Pinjun, Wei Qi, He Jiachun, Lai Fengxiang, Fu Qiang. Cloning and Functional Analysis of Calcineurin Subunits A and B in Development and Fecundity of Nilaparvata lugens (Stål)[J]. Rice Science, 2022, 29(2): 143-154.

Figures/Tables 10

Table 1. Genes cloned from N. lugens and their sequence characteristics.

Gene ID	Blastn NCBI (Size)	Blast N. lugens genome	Accession No.	No. of exons	Full length (bp)	ORF length (bp)	Protein sequence length (aa)	MW (kDa)	pI
NlCNB1	XM_022349071.1 (1 396 bp)	Scaffold 5431	MT780262	/	720	75‒587	171	19.4	4.31
NlCNBH1	XM_022328413.1 (1 012 bp)	Scaffold 8	MT780263	4	994	31‒603	191	21.9	4.66
NlCNA-X1	XM_022350490.1 (2 008 bp)	Scaffold 262 Scaffold 262 Scaffold 262 Scaffold 262 Scaffold 262 Scaffold 262	MT780264	16	1 758	100‒1 707	536	59.8	6.65
NlCNA-X2	XM_022350490.1 (2 008 bp)		MT780265	15	1 743	100‒1 692	531	59.2	6.65
NlCNA-X3	XM_022350512.1 (1 960 bp)		MT780266	15	1 710	100‒1 659	520	58.0	6.52
NlCNA-X4	XM_022350540.1 (1 932 bp)		MT780267	14	1 686	100‒1 635	512	57.1	6.07
NlCNA-X5	XM_022350540.1 (1 932 bp)		MT780268	14	1 683	100‒1 632	511	57.0	6.18
NlCNA-X6	XM_022350548.1 (1 929 bp)		MT780269	14	1 680	100‒1 629	510	57.0	6.18

Fig. 1. Expression analyses of NlCNA, NlCNB1 and NlCNBH1 at various tissues (A?C) and developmental stages (D?F) in N. lugens. SG, Salivary gland; FB, Fat body; E-1, 0?48 h embryo; E-2, 72?96 h embryo; E-3, 120?144 h embryo; 1N-1d?5N-3d, From the first day of the 1st instar nymph to the third day of the 5th instar nymph; NF, Newly emerged female; NM, Newly emerged male; F-2d, Adult females at 2 d after eclosion; M-2d, Adult males at 2 d after eclosion. Three biological replications (Mean ± SE) were carried out for each gene based on independent samples, and relative expression levels of target genes were calculated by the equation Y = 10 (Ct internal ? Ct target) / 3 × 100%. Different lowercase letters above the bars indicate significant differences among different tissues by the Duncan's multiple range test at P < 0.05.

Fig. 2. Effects of dsRNA on survivorship of N. lugens (A?C) and mRNA levels of target genes (D?F). N. lugens (A?C) and mRNA levels of target genes (D?F). The 3rd instar nymphs were injected with specific dsRNA and observed for phenotypic variations at 24 h intervals. Individuals treated with dsGFP were used as a control. The survival rate was calculated from four biological replicates (Mean ± SE). For each treatment, 30 nymphs were used. mRNA levels of target genes from five nymphs at 4 and 10 d after injection in N. lugens were analyzed by qRT-PCR with the 2?ΔΔCT method from three biological replicates (Mean ± SE). Significant differences between the treatment and control are indicated with asterisks (*, P < 0.05).

Fig. 3. Effects of dsRNA injection on nymphal molting. A, Normal phenotype nymphs injected with dsGFP. B?F, Abnormal phenotype nymphs injected with dsNlCNB1. About 90% nymphs with silenced NlCNB1 failed to shed the exuviae completely and did not molt into next stage.

Fig. 4. Effects of dsRNAs on eclosion rate (A) of N. lugens and body weight of newly emerged females (B). The 4th instar nymphs were injected with specific dsRNA. Individuals treated with dsGFP were used as a control. The eclosion rate was calculated from three biological replicates (Mean ± SE). For each treatment, 30 nymphs were used. The newly emerged females were weighted with an electronic balance from three biological replicates (Mean ± SE). For each treatment, five nymphs were used. Different lowercase letters above the bars indicate significant differences among different treatments by the Duncan's multiple range tests at P < 0.05.

Table 2. Effects of NlCNA, NlCNB1 and NlCNBH1 on fecundity of N. lugens.

dsRNA	No. of eggs per female		No. of offspring		Hatchability (%)
dsRNA	Mean ± SE	No. of samples	Mean ± SE	No. of samples	Mean ± SE	No. of samples
dsGFP	504.5 ± 56.9 a	12	465.5 ± 50.3 a	12	94.0 ± 2.3 a	12
dsNlCNA	326.9 ± 54.6 b	13	311.0 ± 55.5 b	13	86.2 ± 7.7 a	13
dsNlCNBH1	258.0 ± 62.3 b	14	244.4 ± 57.6 b	14	96.9 ± 1.4 a	12
dsNlCNB1	75.0 ± 38.6 c	14	62.3 ± 39.3 c	14	59.9 ± 18.1 b	7

Fig. 5. Developmental status of N. lugens ovaries. A?D, Normal ovaries were dissected and photo- graphed under a dissection microscope from females injected with dsGFP at 2 and 4 d after eclosion. E?H, Ovaries from females injected with dsNlCNA at 2 and 4 d after eclosion. I?L, Abnormal ovaries from females injected with dsNlCNB1 at 2 and 4 d after eclosion. M?P, Ovaries from females injected with dsNlCNBH1 at 2 and 4 d after eclosion.

Fig. 6. Expression analysis of 8 among 10 selected genes in response to RNAi. The 4th instar nymphs were injected with specific dsRNA. Nymphs treated with dsGFP were used as a control. Five newly emerged females were collected for RNA extraction and mRNA levels of target genes were analyzed by qRT-PCR with the 2?ΔΔCT method from three biological replicates (Mean ± SE). Different lowercase letters above the bars indicate significant differences among different treatments by the Duncan's multiple range tests at P < 0.05.

Fig. 7. Relative physiological and biochemical changes affected by dsRNAs. Calcineurin content (A), calcineurin enzyme activity (B), phenoloxidase content (C), pyruvic acid content (D), 20-hydroxyacdysone (20E) content (E) and trehalose content (F) were analyzed between different treatments of newly emerged females and males. Three replicates were conducted (Mean ± SE). Different lowercase letters above the bars indicate significant differences among different treatments by the Duncan's multiple range test at P < 0.05.

Fig. 8. Survival rate (A) and NlCNB1 mRNA abundance (B) of N. lugens that treated with different concentrations of dsNlCNB1. dsNlCNB1-700 to dsNlCNB1-0.07 refer to the concentrations of dsNlCNB1 used for injection from 700 to 0.07 ng/μL. The survival rate was calculated from three biological replicates with 30 nymphs each (Mean ± SE). mRNA level of NlCNB1 at 4 d after injection were analyzed by qRT-PCR with the 2-ΔΔCT method from three biological replicates (Mean ± SE). *, P < 0.05; **, P < 0.01.

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