Feng-Xiang Lai

Event-specific qualitative and quantitative detection of transgenic rice Kefeng-6 by characterization of the transgene flanking sequence

The transgenic rice line Kefeng-8 harboring insect-resistant genes Cry1Ac and CpTI showed ideal field performances characterized by high resistance to rice stem borer (Scirpophaga incertulas) and leaf roller (Cnaphalocrocis medinalis). This GM rice line is likely to be approved in China in the near future. In this study, we estimated the insert number of foreign genes, analyzed the flanking sequences of T-DNA in rice genome, and presented an event-specific detection method for this line. The results show that the foreign gene inserted one copy between position 11,774 and 11,805 of chromosome 11 (AC120536.5) in Kefeng-8 genome. Based on these inserts and border sequences, the event-specific qualitative and quantitative PCR system was established for Kefeng-8. The qualitative detection limit was assessed to be 0.1%, and the limit of quantitative method was assessed to be 100 initial template copies. Two mixed rice sample with known Kengfeng-8 contents were used to verify the quantitative method, from which the expected results were observed. This study provides a reliable method and information for detection, identification, and quantification of the presence of GM rice Kefeng-8.

Identification and Function Analysis of enolase Gene NlEno1 from Nilaparvata lugens (Stål) (Hemiptera:Delphacidae).

The enolase [EC 4.2.1.11] is an essential enzyme in the glycolytic pathway catalyzing the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP). In this study, a full-length cDNA encoding α-enolase was cloned from rice brown planthopper (Nilaparvata lugens) and is provisionally designated as NlEno1. The cDNA sequence of NlEno1 was 1,851 bp with an open reading frame (ORF) of 1,305 bp and encoding 434 amino acids. The deduced protein shares high identity of 80–87% with ENO1-like protein from Hemiptera, Diptera, and Lepidoptera speices. The NlEno1 showed the highest mRNA expression level in hemolymph, followed by fat body, salivary gland, ovaries and egg, and showed trace mRNA levels in testis. The mRNA of NlEno1 showed up-regulated level in virulent N. lugens population Mudgo, IR56 and IR42 when compared with TN1 population. Injection of double-stranded RNA (dsRNA) of NlEno1 into the adults significantly down-regulated the NlEno1 mRNA level along with decreased eggs and offspring. Moreover, injection of NlEno1-dsRNA decreased mRNA level of Vitellogenin (Vg) gene. These results showed that the NlEno1, as a key glycolytic enzyme, may play roles in regulation of fecundity and adaptation of N. lugens to resistant rice varieties.

Reference genes for quantitative real-time PCR analysis in symbiont Entomomyces delphacidicola of Nilaparvata lugens (Stål)

Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a major rice pest that harbors an endosymbiont ascomycete fungus, Entomomyces delphacidicola str. NLU (also known as yeast-like symbiont, YLS). Driving by demand of novel population management tactics (e.g. RNAi), the importance of YLS has been studied and revealed, which greatly boosts the interest of molecular level studies related to YLS. The current study focuses on reference genes for RT-qPCR studies related to YLS. Eight previously unreported YLS genes were cloned, and their expressions were evaluated for N. lugens samples of different developmental stages and sexes, and under different nutritional conditions and temperatures. Expression stabilities were analyzed by BestKeeper, geNorm, NormFinder, ΔCt method and RefFinder. Furthermore, the selected reference genes for RT-qPCR of YLS genes were validated using targeted YLS genes that respond to different nutritional conditions (amino acid deprivation) and RNAi. The results suggest that ylsRPS15p/ylsACT are the most suitable reference genes for temporal gene expression profiling, while ylsTUB/ylsACT and ylsRPS15e/ylsGADPH are the most suitable reference gene choices for evaluating nutrition and temperature effects. Validation studies demonstrated the advantage of using endogenous YLS reference genes for YLS studies.

A Genome-Wide Identification and Analysis of the Basic Helix-Loop-Helix Transcription Factors in Brown Planthopper, Nilaparvata lugens

The basic helix-loop-helix (bHLH) transcription factors in insects play essential roles in multiple developmental processes including neurogenesis, sterol metabolism, circadian rhythms, organogenesis and formation of olfactory sensory neurons. The identification and function analysis of bHLH family members of the most destructive insect pest of rice, Nilaparvata lugens, may provide novel tools for pest management. Here, a genome-wide survey for bHLH sequences identified 60 bHLH sequences (NlbHLHs) encoded in the draft genome of N. lugens. Phylogenetic analysis of the bHLH domains successfully classified these genes into 40 bHLH families in group A (25), B (14), C (10), D (1), E (8) and F (2). The number of NlbHLHs with introns is higher than many other insect species, and the average intron length is shorter than those of Acyrthosiphon pisum. High number of ortholog families of NlbHLHs was found suggesting functional conversation for these proteins. Compared to other insect species studied, N. lugens has the highest number of bHLH members. Furthermore, gene duplication events of SREBP, Kn(col), Tap, Delilah, Sim, Ato and Crp were found in N. lugens. In addition, a putative full set of NlbHLH genes is defined and compared with another insect species. Thus, our classification of these NlbHLH members provides a platform for further investigations of bHLH protein functions in the regulation of N. lugens, and of insects in general.

Ran Involved in the Development and Reproduction Is a Potential Target for RNA-Interference-Based Pest Management in Nilaparvata lugens.

Ran (RanGTPase) in insects participates in the 20-hydroxyecdysone signal transduction pathway in which downstream genes, FTZ-F1, Krüppel-homolog 1 (Kr-h1) and vitellogenin, are involved. A putative Ran gene (NlRan) was cloned from Nilaparvata lugens, a destructive phloem-feeding pest of rice. NlRan has the typical Ran primary structure features that are conserved in insects. NlRan showed higher mRNA abundance immediately after molting and peaked in newly emerged female adults. Among the examined tissues ovary had the highest transcript level, followed by fat body, midgut and integument, and legs. Three days after dsNlRan injection the NlRan mRNA abundance in the third-, fourth-, and fifth-instar nymphs was decreased by 94.3%, 98.4% and 97.0%, respectively. NlFTZ-F1 expression levels in treated third- and fourth-instar nymphs were reduced by 89.3% and 23.8%, respectively. In contrast, NlKr-h1 mRNA levels were up-regulated by 67.5 and 1.5 folds, respectively. NlRan knockdown significantly decreased the body weights, delayed development, and killed >85% of the nymphs at day seven. Two apparent phenotypic defects were observed: (1) Extended body form, and failed to molt; (2) The cuticle at the notum was split open but cannot completely shed off. The newly emerged female adults from dsNlRan injected fifth-instar nymphs showed lower levels of NlRan and vitellogenin, lower weight gain and honeydew excretion comparing with the blank control, and no offspring. Those results suggest that NlRan encodes a functional protein that was involved in development and reproduction. The study established proof of concept that NlRan could serve as a target for dsRNA-based pesticides for N. lugens control.

Knockdown of a putative argininosuccinate lyase gene reduces arginine content and impairs nymphal development in Nilaparvata lugens

Nilaparvata lugens is a typical phloem feeder. Rice phloem is high in simple sugars and very low in essential amino acids. Nilaparvata lugens harbors an ascomycete Entomomyces delphacidicola that hypothetically biosynthesizes several amino acids to meet the nutrition requirement of the planthopper. Among these amino acids, here, we focused on arginine biosynthesis. A complete cDNA of an E. delphacidicola gene, arginine-succinate lyase, EdArg4, the last step in arginine biosynthesis, was obtained. RNAi-mediated suppression of EdArg4 reduced arginine content in the hemolymph, and decreased the expression of several arginine biosynthesis genes. Silencing of EdArg4 delayed nymphal development and led to nymphal lethality. About 20% of the EdArg4 RNAi surviving adults were deformed. The most obvious defect was wider and larger abdomen. The EdArg4 RNAi-treated planthoppers had thickened wings and enlarged antennae, legs, and anal tubes and a few adults did not normally emerge. Arginine deficiency in the EdArg4 RNAi planthoppers repressed nitric oxide signaling, determined at the transcriptional level. We infer that E. delphacidicola biosynthesizes essential arginine to compensate for nutrition deficiency in N. lugens.

Ras-like family small GTPases genes in Nilaparvata lugens: Identification, phylogenetic analysis, gene expression and function in nymphal development

Twenty-nine cDNAs encoding Ras-like family small GTPases (RSGs) were cloned and sequenced from Nilaparvata lugens. Twenty-eight proteins are described here: 3 from Rho, 2 from Ras, 9 from Arf and 14 from Rabs. These RSGs from N.lugens have five conserved G-loop motifs and displayed a higher degree of sequence conservation with orthologues from insects. RT-qPCR analysis revealed NlRSGs expressed at all life stages and the highest expression was observed in hemolymph, gut or wing for most of NlRSGs. RNAi demonstrated that eighteen NlRSGs play a crucial role in nymphal development. Nymphs with silenced NlRSGs failed to molt, eclosion or development arrest. The qRT-PCR analysis verified the correlation between mortality and the down-regulation of the target genes. The expression level of nuclear receptors, Kr-h1, Hr3, FTZ-F1 and E93 involved in 20E and JH signal pathway was impacted in nymphs with silenced twelve NlRSGs individually. The expression of two halloween genes, Cyp314a1 and Cyp315a1 involved in ecdysone synthesis, decreased in nymphs with silenced NlSar1 or NlArf1. Cyp307a1 increased in nymphs with silenced NlArf6. In N.lugens with silenced NlSRβ, NlSar1 and NlRab2 at 9th day individually, 0.0% eclosion rate and almost 100.0% mortality was demonstrated. Further analysis showed NlSRβ could be served as a candidate target for dsRNA-based pesticides for N.lugens control.