Pectinases From Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): Putative Accessory Digestive Enzymes

Abstract

The cell wall in plants offers protection against invading organisms and is mainly composed of the polysaccharides pectin, cellulose, and hemicellulose, which can be degraded by plant cell wall degrading enzymes (PCWDEs). Such enzymes are often synthesized by free living microorganisms or endosymbionts that live in the gut of some animals, including certain phytophagous insects. Thus, the ability of an insect to degrade the cell wall was once thought to be related to endosymbiont enzyme activity. However, recent studies have revealed that some phytophagous insects are able to synthesize their own PCWDEs by endogenous genes, although questions regarding the origin of these genes remain unclear. This study describes two pectinases from the sugarcane weevil, Sphenophorus levis Vaurie, 1978 (Sl-pectinases), which is considered one of the most serious agricultural pests in Brazil. Two cDNA sequences identified in a cDNA library of the insect larvae coding for a pectin methylesterase (PME) and an endo-polygalacturonase (endo-PG)—denominated Sl-PME and Sl-endoPG, respectively—were isolated and characterized. The quantitative real-time reverse transcriptase polymerase chain reaction expression profile for both Sl-pectinases showed mRNA production mainly in the insect feeding stages and exclusively in midgut tissue of the larvae. This analysis, together Western blotting data, suggests that Sl-pectinases have a digestive role. Phylogenetic analyses indicate that Sl-PME and Sl-endoPG sequences are closely related to bacteria and fungi, respectively. Moreover, the partial genomic sequences of the pectinases were amplified from insect fat body DNA, which was certified to be free of endosymbiotic DNA. The analysis of genomic sequences revealed the existence of two small introns with 53 and 166 bp in Sl-endoPG, which is similar to the common pattern in fungal introns. In contrast, no intron was identified in the Sl-PME genomic sequence, as generally observed in bacteria. These data support the theory of horizontal gene transfer proposed for the origin of insect pectinases, reinforcing the acquisition of PME genes from bacteria and endo-PG genes from fungi.

Identification and expression profiling of novel plant cell wall degrading enzymes from a destructive pest of palm trees, Rhynchophorus ferrugineus

“…Within Curculionidae, apart from our results, GH28 has been reported only from S. oryzae . The B last 2GO annotation indicates that RferGH28u1 was identified through the degenerate PCR approach and shares 80.10% sequence similarity with a functionally characterized endo‐polygalacturonase (AHC08666) from S. levis (Table S2; Evangelista et al ., ). We also found that the CE8 class of enzymes was identified exclusively in the Curculionidae family, specifically from S. oryzae , S. levis and the RPW (Table ).…”

Estudos funcionais e estruturais de pectinases e xilanases com potencial para aplicações biotecnológicas

“…31 As pectinases de microorganismos estão relacionadas com processos de infecção, definindo seu potencial de patogenicidade, 32 e as de insetos estão provavelmente vínculadas com seu potencial de herbivoria. 33 As pectinases mais extensamente estudadas são as que agem sobre a região homogalacturonana; essas enzimas são subclassificadas de acordo com seu mecanismo de ataque a esse substrato. As protopectinases (PPase) convertem a protopectina em pectina solúvel, agindo diretamente (PPase-A) sobre a cadeia de ácido poligalacturônico (Poli-AGal)…”

“…28,33,35 Os plasmídios resultantes foram construídos para expressar a proteína-alvo fusionada, em seu C-terminal, a um c-myc epitope, usado em ensaios de imunodetecção, mais uma cauda de histidina (6xHis-tag), usada na purificação da proteína. 28,33,35 Os clones da levedura P. pastoris expressores das enzimas recombinantes Sl-PME e Sl-EndoPG foram gentilmente cedidos ao nosso grupo pelo Prof. Dr. Flavio Henrique Silva para realização dos estudos estruturais das Sl-Pectinases que serão descritos neste capítulo.…”

“…33,35 Em exemplo, o alinhamento da Sl-PME com a estrutura de S. Como mencionado anteriormente, evidencias sugerem que a origem das PMEs na família Curculionidae deva-se a um evento evolucionário conhecido como transferência horizontal, onde um fragmento de DNA de um organismo integra-se ao genoma de um outro organismo. Mais precisamente neste caso, acredita-se que esse evento ocorreu entre uma bactéria e o inseto ancestral comum aos coleópteros.…”

“…29,81,130 Dentre esses organismos, os micro-organismos representam a maior fonte de bioprospecção das EDPCV, isso porque eles possuem maior quantidade e diversidade de representantes. Entretanto, apenas 0,1-1% dos micro-organismos são atualmente cultiváveis em laboratório, o que representou por muitos anos a impossibilidade de estudar as enzimas produzidas pela fração majoritária e não-cultivável desses organismos.…”