Cloning and Characterization of karmoisin Homologue Gene (Nlka) in Two Brown Planthopper Strains with Different Eye Colors

doi:doi.org/10.1016/j.rsci.2016.02.005

Abstract

Abstract:

The brown planthopper (BPH), Nilaparvata lugens, is a destructive insect pest of rice throughout Asia. Different from brown-eye color wild type, BPH also has red-eye color mutation phenotype. As a visible genetic marker, the red-eye mutant in BPH is a valuable material. To reveal the eye color mutation mechanism, a karmoisin homologue gene (named as Nlka) was cloned from BPH. And karmoisin is always deemd as a xanthommatin-related gene in other insects, encoding phenoxazinone synthetase (PHS). Nlka is consisted of 7 exons and encodes a protein with 502 amino acids (NlKA). NlKA showed high amino acid identities with its insect homologues (48.8%-51.8%). Nlka transcripts can be detected at all the developmental stages and in all tissues tested, including egg, nymph, adult, body wall, ovary, fat body, midgut and Malpighian tubule. However, no constant In/Del or non-synonymous mutation was observed between the mutant and the wild type strains. Quantitative real-time PCR experiment also showed that Nlka transcript level had no significant differences between them. These results indicated that Nlka is not the target gene causing the red-eye color mutation phenotype of BPH. Through the second structure and motif analysis, the present study also showed that all the proteins deduced from the karmoisin genes in insects may be members of monocarboxylate transporters (MCTs) rather than PHSs.

Key words: Nilaparvata lugens, red-eye mutant, karmoisin, monocarboxylate transporter, phenoxazinone synthetase, rice, gene clone

Shu-hua Liu, Jian Tang, Ju Luo, Bao-jun Yang, Ai-ying Wang, Jin-cai Wu. Cloning and Characterization of karmoisin Homologue Gene (Nlka) in Two Brown Planthopper Strains with Different Eye Colors[J]. Rice Science, 2016, 23(2): 104-110.

Figures/Tables 6

Table 1 Oligonucleotide primers used for reverse transcription polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE) and quantitative real-time PCR (qRT-PCR).

Primer	Sequence (5′-3′)	Description
ka-F1	ATGTCGGAAACCGAGAAAGTGC	RT-PCR
ka-R1	GAACAAACAGAGGCAGGGATGC
ka-F2	GTCCGATTGCGTTCGACTTGTG	3′-RACE
ka-R2	CTCCGCTGCAACTCCACATAGACC	5′-RACE
ka-F3	GGTCCGATTGCGTTCGACTTGTG	qRT-PCR
ka-R3	TGGATGAGTTGCGAGGTGGCTGT
β-actin F	ACGCCATCCTCCGTCTGGACTT
β-actin R	CAAAGTCCAGGGCAACGTAGCA

Fig. 1. cDNA sequences and deduced amino acid sequences of Nlka in N. lugens.The stop codon is indicated by an asterisk. The amino acids in boxes indicate the 11 predicted transmembrane domains.

Fig. 2. Genomic structure of the Nlka gene in N. lugens. Red boxes indicate the exons (1-7), and the spaces between two boxes indicate the introns (I-VI).

Fig. 3. Analysis of deduced amino acids of Nlka and karmoisin and monocarboxylate transporter (MCT) genes in other insects. A, Amino acid sequence alignments; B, Phylogenetic relationship analysis. Nlka, Nilaparvata lugens, KT304312; Dbka, Drosophila busckii, ALC46387.1; Dmka, Drosophila melanogaster, NP_001189213; Dpka, Danaus plexippus, EHJ77874.1; CbMCT, Cerapachys biroi, EZA53034; CfMCT, Camponotus floridanus, EFN65757; CqMCT, Culex quinquefasciatus, XP_001857555; HlMCT, Habropoda laboriosa, KOC69627; ZnMCT, Zootermopsis nevadensis, KDR23400.

Fig. 4. mRNA expression characters of Nlka in different tissues and development stages of brown planthopper. A, Reverse transcription PCR amplification of Nlka from total RNA in N. lugens; B, Relative mRNA expression levels at different developmental stages.Bw, Body wall; Ov, Ovary; Fb, Fat body; Mg, Midgut; Mt, Malpighian tubule; 1st, The first instar nymphs; 3rd, The third instar nymphs; 5th, The fifth instar nymphs; F, Female adults; M, Male adults.The relative expression level (fold) was expressed as mean ± SE (n = 3). Different lowercase letters above the columns indicate the significant differences at the 0.05 level.

Fig. 5. Relative expression levels of Nlka in red-eye mutant and brown-eye wild individuals at different developmental stages.1st, The first instar nymphs; 3rd, The third instar nymphs; 5th, The fifth instar nymphs; F, Female adults; M, Male adults; NIL-BB, Brown-eye wild type; NIL-rr, Red-eye mutant type.The relative expression level (fold) was expressed as mean ± SE (n = 4). Asterisk above the column indicates the significant difference at the 0.05 level.

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