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Molecular Cloning and Mrna Expression of Apn and Pxbre Genes in Diamondback Moth, Plutella Xylostella(L.)

Author: BaoHongWei
Tutor: YangYiZuo
School: Nanjing Agricultural College
Course: Agricultural Entomology and Pest Control
Keywords: Plutella xylostella Cry1Ac APN Glycolipid Glycosyltransferase mRNA expression
CLC: S433.4
Type: Master's thesis
Year: 2009
Downloads: 3
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Abstract


The diamondback moth, Plutella xylostella L., has short reproductive cycle, strong reproductive capacity and wide adaptability. These characteristics make it to be the most widely and serious pest on crucifers. This pest has developed serious resistance to many kinds of insecticides. Bacillus thuringiensis (Bt) toxins is widely used against the diamondback moth for its high specificity and environmental safety. However, several field populations of P. xylostella showed resistance to Bt toxins after a few years of extensive use. In order to expoit the environment-benign Bt toxins in the long term, it is necessary to understand Bt resistance mechanisms in P. xylostella. Although a lot of works have been published, the resistance mechanisms are still unclear. In this study, four Pxbre genes encoding glycosyltransferase genes which is associated with the elongration of carbohydrate structures on glycosylphingolipid, the possible candidate Bt receptor, were cloned and sequenced in P. xylostella, and mRNA expression levels of 9 APNs,1 ALP and 5 Pxbre genes were compared between the susceptible and Cry1Ac-resistant strains of P. xylostella.1. Relative mRNA expression levels of nine APNs and one ALP in fourth-instar larva midgut of Plutella xylostellaReduced expression levels of aminopeptidases (APNs) and alkaline phosphatases (ALPs) may be related to Bt resistance in insects. Relative mRNA expression levels of nine APNs and one ALP in fourth-instar larva midguts were compared between the susceptible and Cry-resistant strain (SZBT) of P. xylostella with semi-quantitative PCR, but no difference was observed. It suggests that these Bt receptor genes are not involved in Cry1Ac resistance in the SZBT strain through modification of expression levels.2. Cloning, sequence analysis, and mRNA expression patterns of Pxbre genes from Plutella xylostella Glycosylphingolipid has been identified as a receptor for Bacillus thuringiensis crystal toxin Cry5B in the nematode Caenorhabditis elegans. Four glycosyltransferase genes (bre) were sequentially involved in the elongration of carbohydrate structures on glycosylphingolipid. Loss of function of any one of these genes resulted in resistance to Cry5B. Full-length cDNAs of four Pxbre genes of P. xylostella were cloned with RACE technique. The four Pxbre genes (designated as Pxbre-1, Pxbre-3, Pxbre-4, and Pxbre-5) shared high similarity with their homologous partners in nematodes. It is suggested that the four Pxbre genes from P. xylostella may have similar functions as in nematodes.There are several amimo acid polymorphisms for each of the four Pxbre genes between the susceptible and resistant SZBT strains. No specific amino acid substitution was found in the SZBT strain when compared with the susceptible strain. In one clone of Pxbre-3, an insertion mutation was detected. By comparing between the cDNAs and gDNAs, we found that the insertion in Pxbre-3 was produced by an unspliced intron. This insertion introduced a premature stop codon and the putative protein was truncated. The relevance of this mutation in Pxbre-3 with Bt resistance is unclear.The mRNA expression of Pxbre-1 to Pxbre-5 in developmental stages (larva, pupa, and adult) was compared in the susceptible strain with Real-time PCR, and all the five genes showed highest expression in the adult stage. Pxbre-1, Pxbre-4 and Pxbre-5 had higher expression in the Cry1Ac-resistant SZBT strain than in the susceptible strain, whereas Pxbre-2 (bre2-like) and Pxbre-3 had higher expression in the susceptible strain than in the resistant SZBT strain.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant pest and its control > Lepidoptera pests
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