Compositions and methods of using rna interference for control of nematodes
A nematode, Caenorhabditis elegans technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of worm-resistant transgenic plant regulation and other issues.
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Embodiment 1
[0105] Example 1: Identification and isolation of soybean cyst nematode RNAi target genes
[0106] Using total RNA isolated from the J2 stage of SCN, a cDNA fragment of about 400-500 bp in length was isolated using RT-PCR for construction of the binary vector discussed in Example 2. The PCR product was cloned into TOPO pCR2.1 vector (Invitrogen, Carlsbad, CA) and the insert was verified by sequencing. Gene fragments for all eight target genes were isolated using this method.
[0107] To obtain full-length cDNAs of soybean cyst nematode target genes, a RT-PCR method based on a highly conserved splicing leader sequence (SL1) present in various nematode species was used. Reactions were performed using the Superscript One-Step Kit (Invitrogen, Carlsbad, Calif., Cat. No. 10928-034) and primer sets. The forward primer consisted of a 22-mer SL1 sequence, and the reverse primer was gene-specific and located in the previously cloned cDNA region. PCR products were cloned into pCR4-...
Embodiment 2
[0110] Example 2: Binary vector construction for soybean transformation
[0111] To assess whether SCN targeting is effective in vivo, binary vectors were made using cDNA fragments for eight SCN target genes. The vector consists of an antisense fragment of the target (eg, soybean cyst nematode tcp-1), a spacer fragment, a sense fragment of the target (eg, soybean cyst nematode tcp-1), and a vector backbone. In this vector, the dsRNA of the target gene is expressed under the constitutive Super promoter (see US 5955,646, incorporated herein by reference). The selectable marker used for transformation was a mutated acetohydroxyacid synthase (AHAS) gene from Arabidopsis conferring resistance to the herbicide ARSENAL (Imazapyr, BASF Corporation, Florham Park, NJ). Expression of the mutant AHAS is driven by the ubiquitin promoter.
[0112] The gene fragment corresponding to SEQ ID NO:3 was used to construct the binary vector RTP1030. The gene fragment corresponding to SEQ ID NO...
Embodiment 3
[0113] Example 3: dsRNA bioassay targeting soybean cyst nematode target genes
[0114] dsRNA expression and acquired nematode resistance were confirmed using rooted explant assays. Details of this assay can be found in co-pending application USSN 12 / 001,234, the contents of which are incorporated herein by reference. The binary vectors RTP1030, RCB987, RSA131, RTP1095, RSA123, RSA012, RTP1169, RTP1269 described in Example 2 were transfected into disarmed Agrobacterium rhizogenes strain K599 using Soybean cotyledons were used as explants for transformation. Two to three weeks after inoculation and root induction according to the method of USSN 12 / 001,234, transformed roots formed at the severed ends of the explants. Soybean roots were excised from rooted explants, subcultured, and 1-5 days after subculturing, roots were inoculated with surface-sterilized SCN J2 juveniles in multiwell plates for target gene construct assays. As controls, soybean cv. Williams 82 control vect...
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