Agrobacterium-mediated transformation of indica rice with chitinase gene for enhanced sheath blight resistance

Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to 5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was correlated with sheath blight resistance.     

Enhanced resistance to stripe rust disease in transgenic wheat expressing the rice chitinase gene RC24

Stripe rust is a devastating fungal disease of wheat worldwide which is primarily caused by Puccinia striiformis f. sp tritici. Transgenic wheat (Triticum aestivum L.) expressing rice class chitinase gene RC24 were developed by particle bombardment of immature embryos and tested for resistance to Puccinia striiformis f.sp tritici. under greenhouse and field conditions. Putative transformants were selected on kanamycin-containing media. Polymease chain reaction indicated that RC24 was transferred into 17 transformants obtained from bombardment of 1,684 immature embryos. Integration of RC24 was confirmed by Southern blot with a RC24-labeled probe and expression of RC24 was verified by RT-PCR. Nine transgenic T₁ lines exhibited enhanced resistance to stripe rust infection with lines XN8 and BF4 showing the highest level of resistance. Southern blot hybridization confirmed the stable inheritance of RC24 in transgenic T₁ plants. Resistance to stripe rust in transgenic T₂ and T₃ XN8 and BF4 plants was confirmed over two consecutive years in the field. Increased yield (27–36 %) was recorded for transgenic T₂ and T₃ XN8 and BF4 plants compared to controls. These results suggest that rice class I chitinase RC24 can be used to engineer stripe rust resistance in wheat.     

Development of transgenic finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) resistant to leaf blast disease

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R₀ and R₁ progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R₁ lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.     

水稻金属耐受蛋白基因OsMTP2生物信息学及表达分析

为了解水稻(Oryza sativa)响应重金属污染及对重金属积累的机制,利用生物信息学手段对水稻金属耐受蛋白(metal tolerance protein, MTP) OsMTP2的结构特征进行预测,并分析OsMTP2基因的表达特征。结果表明,该蛋白由415个氨基酸组成,具有很强的疏水性。mRNA原位杂交结果表明,OsMTP2在叶片输导组织中有较强的表达,但在根、花药中没有检测到明显的杂交信号。这说明水稻金属耐受蛋白基因OsMTP2主要在输导组织中表达,并在金属离子运输中发挥重要作用。     

Chloramphenicol Acetyl Transferase (CAT) Protein Is Expressed in Transgenic Tobacco in Field Tests following Attack by Insects

The expression of chloramphenical acetyl transferase (CAT) protein driven by the wound-inducible promoter from the proteinase inhibitor II K (pin2) gene was examined in whole tobacco (Nicotiana tabacum L.) plants under field conditions. Mechanical wounding of the field-grown leaves caused an accumulation of CAT protein in these leaves which begins several hours after wounding and continues to accumulate for about 36 hours. When sections of leaves were assayed for accumulation of CAT protein following wounding, the CAT protein was found to accumulate in the apical portions of the leaves. When endogenous insects attacked the leaves of transgenic plants grown in the field, the plants responded by inducing CAT protein. The mesophyll cells of the leaf were the site of expression of the CAT protein rather than the mid-vein or major veins within the leaf blade, indicating that the wound-inducible pin2 promoter specifically directs the synthesis of novel genes in tissues preferentially consumed by larval insects.     

Enhanced fungal resistance in Arabidopsis expressing wild rice PR-3 (OgChitIVa) encoding chitinase class IV

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants. J.-H. Pak and E.-S. Chung contributed equally to this work.