서강대학교

초록/요약

Abiotic stresses, such as high temperature, drought, salinity, cold, flooding, heavy metal etc., cause adverse effects on the growth and the productivity of crop plants. The burden of high temperature stress on crop plants is likely to increase because of the climate changes associated with global warming. Thus there is an urgent need to identify high temperature stress inducible genes for redesigning crops for tolerance at high temperature. To investigate plant response mechanism in genetic level under high temperature, seven cDNA clones inducible to high temperature were isolated from a cDNA library of hotpepper treated at 42oC. These cDNAs were sequenced and analyzed. Some of them showed high homology with the similar proteins of other species. Out of these seven cDNA clones, two were further functionally analyzed. One of the cDNA clones is Capsicum annuum Bax Inhibitor-1 (CaBI-1) encoding a Bax inhibitor-1 protein and the other is Capsicum annuum Protein Phosphatase 2C (CaPP2C) encoding a Protein phosphatase 2C protein. CaBI-1 contains an open reading frame (ORF) of 248 amino acids and CaPP2C contains 215 amino acids. Both of them showed significant amino acid sequence homology with the similar proteins of other species. Genomic gel blot analysis disclosed that CaBI-1 and CaPP2C might have single or two homologues in the pepper genome. CaBI-1 transcripts were preferentially induced in response to high or low temperatures, drought, high salinity, heavy metal, flooding and ABA. CaPP2C is also a multiple stress-inducible gene. Its transcript level was markedly increased by high temperature, flooding, heavy metal stress and ABA. These two genes were introduced into the Wisconsin 38 cultivar of tobacco (Nicotiana tabacum) by Agrobacterium mediated transformation under the control of the CaMV 35S promoter. Expression of the transgene in the transformed tobacco plants was demonstrated by RNA blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T1 lines showed that the transgene segregated in a Mendelian fashion. Transgenic tobacco lines that expressed the CaBI-1 and CaPP2C were compared with several different empty vector lines and they exhibited no phenotypic differences. In order to compare the thermotolerance of P35:CaBI-1 and P35S:CaPP2C plants with the untransformed control plants and the transformed control plants without an insert and with an insert in antisense orientation were exposed to 45oC. The transgenic tobacco lines having CaBI-1 and CaPP2C showed significantly better growth performance in the recovery phase following the stress. CaBI-1 and CaPP2C expression protects chlorophyll breakdown and protein degradation of heat stressed plants. In addition, the P35S:CaBI-1 and P35S:CaPP2C transgenic plants displayed markedly improved tolerance to severe water deficit, and to high salinity in comparison to the control plants. Microarray analysis revealed the up-regulation of some stress related genes in the transgenic plants which were validated by specific oligomer RNA blot analyses. The inductions of the transcript level of the CaBI-1 and CaPP2C target genes, as well as the CaBI-1 and CaPP2C transcripts showed positive correlation respectively. Thus, CaBI-1 and CaPP2C seems to be important determinant of stress response in plants. Moreover, CaBI-1 and CaPP2C, functions as transcription factors and signal transduction pathway component genes those activated and up-regulated many stress related downstream genes and thereby enhancing tolerance to different environmental stresses. The investigation for interaction of proteins in enhanced tolerance to stresses is required. Keywords: Abiotic stress; Capsicum annuum; Nicotiana tabacum; Bax inhibitor-1 protein; Protein phosphatase 2C; Thermotolerance; Drought tolerance; Salt tolerance; Over-expression; Up-regulation; Transgenic plant