Chorismate mutase gene from the potato cyst nematode Globodera rostochiensis
Inventors
Assignees
US Department of Agriculture USDA
Publication Number
US-8575427-B2
Publication Date
2013-11-05
Expiration Date
2029-07-24
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Abstract
The nucleotide sequence of a 992 bp region of cDNA and the nucleotide sequence of a 1973 bp (or a 1913 bp) of genomic DNA of the Gr-cm-1 gene were determined for G. rostochiensis. PCR primers and probes specific for G. rostochiensis and G. pallida were generated. PCR assays, including a real-time TaqMan PCR were used to identify G. rostochiensis and G. pallida and to differentiate G. rostochiensis from G. pallida. Transgenic hairy roots expressing Gr-cm-1 dsRNA were generated. There was a 52% reduction in the average number of females per root in the Gr-cm-1 dsRNA transgenic lines when compared with the infected control lines.
Core Innovation
The invention provides the nucleotide sequence of a novel chorismate mutase (cm) gene cloned from the potato cyst nematode Globodera rostochiensis, including cDNA and genomic DNA sequences. Species-specific PCR primers and probes were developed based on sequence polymorphisms between cm genes from G. rostochiensis and G. pallida. PCR assays, including real-time TaqMan PCR assays, were established for rapid, sensitive, and specific identification and differentiation of G. rostochiensis from G. pallida.
The infection and parasitism of host plants by G. rostochiensis, a devastating potato pest with eggs capable of lying dormant in soil for up to 30 years, are difficult to manage. Traditional bioassays for determining nematode pathotypes are time-consuming, taking almost two years, and current control strategies like pesticides, crop rotation, and resistant cultivars are not fully effective against certain virulent pathotypes such as Ro2. There is a critical need for rapid, specific diagnostic assays for quarantine and eradication purposes and novel strategies to develop nematode-resistant potato cultivars.
Claims Coverage
The patent contains nine claims with one clear independent claim related to an isolated polynucleotide. The claims focus on isolated nucleic acid sequences involved in nematode growth inhibition, expression vectors for plants, transformed cells, and methods for nematode population control using double stranded RNA.
Isolated polynucleotide for nematode growth inhibition
An isolated polynucleotide consisting of at least 50 contiguous nucleotides from a specific region of SEQ ID NO: 4, where uptake by G. rostochiensis of a dsRNA sequence comprising at least one strand complementary to this polynucleotide inhibits nematode growth.
Plant transformation vector comprising isolated polynucleotide
A plant transformation vector including the isolated polynucleotide and optionally a reverse fully complementary sequence, operably linked to a heterologous promoter functional in plant cells, which results in expression of doubled stranded RNA molecules that form dsRNA.
Transformed cell with isolated polynucleotide
A cell transformed with the isolated polynucleotide capable of expressing dsRNA targeting the nematode's cm gene.
Method for controlling G. rostochiensis population with dsRNA agent
Methods that use agents comprising double stranded RNA sequences, uptake by nematodes inhibits biological function, where the dsRNA corresponds to the isolated polynucleotide or at least 95% nucleotide identity thereto, to control nematode populations.
The claims collectively cover isolated polynucleotides targeting the cm gene region for nematode inhibition, vectors and transformed cells for plant expression of dsRNA, and methods using dsRNA agents to control G. rostochiensis parasitic nematodes by inhibiting their growth and infection capability.
Stated Advantages
The PCR and TaqMan assays provide a highly sensitive, rapid, and specific method for identifying and differentiating G. rostochiensis and G. pallida, eliminating lengthy traditional bioassays and reducing time from years to hours.
TaqMan assays allow processing of large numbers of samples efficiently without post-PCR gel analysis, enabling quarantine and eradication efforts with quicker and reliable data.
The RNA interference (RNAi) strategy using dsRNA expressing transgenic plants significantly reduces nematode infection and female development, offering a durable and environmentally safe control method for G. rostochiensis.
Documented Applications
Detection and identification of G. rostochiensis and G. pallida in biological and environmental samples using PCR and TaqMan PCR assays for quarantine and eradication protocols.
Generating transgenic tomato hairy roots expressing dsRNA targeting the G. rostochiensis cm gene to reduce nematode infection and reproduction.
Developing nematode-resistant agricultural plants, including potato, tomato, and eggplant, by expressing dsRNA targeting the nematode cm gene to control infection.
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