Abstract
Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g−1 in transgenic plants. The M. oryzae population was constant at 31, 48, and 72 h after inoculation in transgenic plants, while it was increased in the inoculated control plants. This study successfully clarified that over-expression of the Pikh gene in transgenic plants can improve their blast resistance against the M. oryzae pathotype P7.2.
Highlights
Over the past few decades, using resistance genes in resistant rice cultivars has been more desirable among different blast disease control strategies (Ashkani et al, 2015)
An analysis of the Pikh gene cloned in a pDrive cloning vector indicates that it contains a 1206 bp coding region that encodes a 401 amino acid protein using homology searches run with the Basic Local Alignment Search Tool (BLAST)
Due to differences in the leucine quantity among the transgenic MR219 plants compared with the control plants, we conclude that this increasing quantity cannot only be due to the protein encoded by the transformed Pikh gene
Summary
Over the past few decades, using resistance genes in resistant rice cultivars has been more desirable among different blast disease control strategies (Ashkani et al, 2015). ∼100 blast resistance genes have been identified and mapped in various rice genotypes (4% are from wild species, 45% are from Japonica, and 51% are from Indica cultivars), while only 19 of these genes have been cloned and characterized (Sharma et al, 2012) Most of these blast resistance genes belong to the nucleotide-binding site-leucine rich repeat (NBSLRR) class of genes (Sharma et al, 2012). These genes can be applied with breeding and genetic engineering programs for introgressing a high degree of tolerance into well-performing commercial cultivars with susceptibility to blast disease (Zhang, 2007). Agrobacterium-mediated transformation is a desired method for a wide range of plant modifications (Tzfira and Citovsky, 2006)
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