ABSTRACT
Background: selectable marker gene [SMG] systems are critical for generation of transgenic crops. Transgenic crop production without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and prepares gene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient and flexible strategy to regulate transgene expression
Objectives: this study aimed to construct a transformation vector based on Cre/loxP recombination system to enhance efficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenic plants by a pathogen inducible promoter
Materials and Methods: in pG-IPFFDD-cre[int]-gus[int] construct, cre recombinase and selectable marker gene [nptII] cassettes were placed between the two loxP recognition sites in direct orientation. Seed-specific Napin promoter was used for regulation of Cre expression in transgenic seeds. In the construct, loxP flanked sequence containing nptII and recombinase cassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and beta-glucuronidase coding region. The cunstuct was transformed into Nicotiana tabaccum via Agrobacterium-mediated transformation
Results: the results showed that both cre and nptII excision occurs in T1 progeny tobacco plants through seed-specific cre expression. The excisions were confirmed by methods activation of the gus gene, germination test on kanamycin-containing medium and molecular analysis. Inducibility of gus expression by FFDD-containing promoter in T1 leaf tissues was confirmed by histochemical Gus staining assay
Conclusions: the established system is not only an efficient tool for marker gene elimination but also provides possibility for inducible expression of the transgene
ABSTRACT
Plant diseases, caused by a wide range of phytopathogenic fungi, could be managed using of Trichoderma sp, as a biocontrol agent. Cell wall degrading enzymes like chitinase from T. harzianum are important means for fungal pathogen inhibition. Overexpression of these chitinase enzymes can improve the antagonistic potential of Trichoderma sp. strains. This study aimed to produce a new enhanced biocontrol system of Trichoderma harzianum, overexpressing chit42 gene. The improved T. harzianum could be an appropriate biocontrol agent for controlling the stem rot disease of canola caused by Sclerotinia sclerotiorum. T. harzianum protoplast cotransformation was carried out by pLMRS3-Chit42 and p3SR2 plasmids. The transformants were selected based on their growth on colloidal chitin containing medium. The improvement of transformants was investigated by quantification of mRNA using real-time quantitative polymerase chain reaction [RT-PCR] and measurement of chitinase activity in the medium containing colloidal chitin as the carbon source. Furthermore, the antagonistic activity of transformants against S. sclerotiorum was assessed by dual culture experiments. The overexpressing transformants of Chit42 displayed higher levels of chitinase activity to inhibit S. sclerotiorum growth compared with the wild type. The results indicated that the value of the chitinase activity [126.42 + 0.07 U/mL] of Chit42-9 increased 64.17 fold. Transcriptomic analysis demonstrated that Chit42-9 transformant expressed 5.2 fold of Chit42 transcript as compared with the parent strain. Biocontrol inhibition of this transformant was 4.98-fold more compared with the non-transformant type. The improved Chit42-9 transformant can be used for biocontrolling S. sclerotiorum, cause of stem rot disease in canola
ABSTRACT
Canola [Brassica napus L.] is an important oilseed crop. A serious problem in cultivation of this crop and yield loss, are due to fungal disease stem rot caused by Sclerotinia sclerotiorum. The pathogenesis-related [PR] proteins have the potential for enhancing resistance against fungal pathogen. Thaumatin-like proteins [TLPs] have been shown to have antifungal activity on various fungal pathogens. In this study, the tip gene isolated from cereal rye [Secale cereal L.] was introduced into canola plants. The amplified DMA fragment [about 500 bp] was analyzed and confirmed by restriction pattern and cloned into pUC19 and designated as pUCNG1. Comparison of the cloned fragment with the DNA sequence indicated that this gene contains no intron. The tip gene was predicted to encode a protein of 173 amino acids with an estimated molecular mass of 17.7 kDa. The deduced amino acid sequence of TIP showed a significant sequence identity with TLP from S.cereal and other plants. We used a transgenic over-expression approach in order to investigate anti-fungal activity of expressed TLP on Sclerotinia sclerotiorum. TLP was overexpressed under the CaMV35S constitutive promoter in [Brassica napus, R line Hyola 308]. Transformation of cotyledonary petioles was achieved via Agrobacterium tumefaciens LBA4404. The insertion of transgene was verified by the polymerase chain reaction [PCR] and genomic DMA dot blotting. Antifungal activity was detected in transgenic canola lines using detached leaf assay. The size of lesions induced by S. sclerotiorum in the leaves of transgenic canola was significantly retarded when compared to that detected in non-transgenic plants
Subject(s)
Brassica napus , Secale , Gene Expression , Plant Proteins , AscomycotaABSTRACT
Sclerotinia sclerotiorum is a phytopathogenic fungus which causes serious yield losses in canola. A pathogen inducible-promoter can facilitate the production of Sclerotinia-resistant transgenic canola plants. In this study, the "gain of function approach" was adopted for the construction of a pathogen-inducible promoter. The synthetic promoter technique was used, which involved the insertion of the dimerized form of the cisacting element [F] upstream of the minimal CaMV 35S promoter, which drives the expression of the beta-glucronidase [GUS] gene. The pGFF construct containing this synthetic promoter [SynP-FF] was used for stable transformation of the canola plant. Fluorometric GUS expression analysis indicated that the SynP-FF promoter is responsive to methyl jasmonate and S. sclerotiorum treatments. The results of histochemical GUS expression patterns showed strong reporter expression in leaf, flower and stem tissues of canola. Hence, the SynP-FF synthetic promoter, carrying fungal pathogen-inducible features, could be considered as a valuable tool for controlling the expression of transgenes to improve resistance against the same lifestyle pathogens
Subject(s)
Fungal Proteins , Gene Expression , Glucuronidase , Microscopy, Electron, Scanning Transmission , HistocytochemistryABSTRACT
The cDNA from the mycoparasitic fungus Trichoderma atroviride PTCC5220 encoding a 42 kDa chitinase [Chit42] was isolated. The nucleotide sequence of the cDNA fragment as having a 1263 bp open reading frame that encodes a 421 amino acid polypeptide, and a high homology was found with other reported Chit42 belonging to the Trichoderma sp. The 22 amino acid N-terminal sequence is a putative signal pep-tide for the possible secretion of the protein. The protein has been expressed and secreted as a mature form in Escherichia coll BL21[DE3] using the pelB leader sequence. The E. coll strain expressed Chit42 in an active form and secreted the protein into the medium. This recombinant chitinase has been shown to have inhibitory activity on mycelial growth and also, lytic activity on the cell wall of Rhizoctonia solani [AG2-2], causal agent of root rot in sugar beet in vitro. Expressed chitinase was optimally active at pH 5 and at 40°C. It is thermally stable at 60°C for more than 120 min at pH5
ABSTRACT
The genomic and cDNA clones encoding cellobiohydrolase II [cbhII] have been isolated and sequenced from a native Iranian isolate of Trichoderma parceramosum, a high cellulolytic enzymes producer isolate. This represents the first report of cbhII gene from this organism. Comparison of genomic and cDNA sequences indicates this gene contains three short introns and also an open reading frame coding for a protein of 470 amino acids. The deduced amino acid sequence includes a putative signal peptide, cellulose binding domain, linker region, and catalytic domain. Homology between this sequence and other reported Trichoderma CBHII proteins and also structural prediction of this enzyme are discussed. The coding sequence of cbhII gene was cloned in pPIC9 expression vector and expressed in Pichia pastoris GS115. The expression was confirmed by Northern dot blot, RT-PCR and enzyme activity staining