| Nuclear proteins locate in the nucleus and play important role in many processes such as plant development, environmental stress and signal transduction. In this study, the screening of soybean cDNA library (Variety Tiefeng 8 treated under drought stress for 5 h) was completed using NTT system established previously, and some prositive clones were abtained. One of clones had RING finger domain and named as GmRZFP1. The homology, subcellular location, expression pattern and gene function were analyazed and results are as follows:(1) Totall 160 yeast clones were abtained from the screening of soybean cDNA library. The sequencing analysis showed that 22% genes involeved in singnal transdcution; 22% genes involved in stress response; 9% genes were related with transportion; 27% genes involeved in energy and primary metabolism; 20% proteins were unknown function. Among those prositive clones,57.4% proteins were anticipated as uncleus locational protein. (2) Bioinformatics analysis showed that the GmRZFP1 gene contains 927 bp, coding 308 amino acids, and has a C3HC4-type RING finger domain, Phylogenetic analysis showed that GmRZFPl belongs to C3HC4-type Zinc-finger subfamily. (3) Subcellular localization assay indicated that the 163GFP-GmRZFP1 fusion protein was located in the nucleus of plant. (4) Real-time PCR results showed that the expression of GmRZFPl was induced by drought, high salt, high temperature, low temperature, ethylene and ABA stresses, suggesting that GmRZFPl might involve in some stresses related signal pathways.(5) GmRZFPl gene was transformed into tobacco by Agrobacterium approach. The results of functional analysis showed that the over-expression of GmRZFP1 gene significantly improved resistance to drought and high-salt stress, and the size of parts above-ground and under ground of the transgenic tobacco were longer than the corresponding parts of wild-type after stresses treatment. The Chlorophyll contents in transgenic lines were higher than in wild-type.The DREB transcription factors play an important regulating role in plant stress response and over-expression of DREB gene significantly improved stress resistance of the transgenic plant. Proviously GmDREB1 and GmDREB3 had been transformed into Chinese wheat varieties Jimai 20 and shi 4185, respectively. In this reseasch, T4 generation of GmDREBl transgenic wheat and T3 generation of GmDREB3 transgenic wheat were identificated using PCR and functional analysis were compeled. The results are as follows:(1) Prositive rate of T4 generation of GmDREB1 transgenic wheat and T3 generation of GmDREB3 transgenic wheat were 45% approximately, which suggesting that GmDREBl gene and GmDREB3 genes have been integrated into wheat genome and had been inherited to next generation.(2) Under drought condition, agricultural properties of transgenic wheat such as spike numbers per plant, number of grains per plant, grain weight per plant and grains weight per thousand seeds were better than that in wild type. The drought-resistant parameter of transgenic wheat calculated as plants high, spike numbers per plant, number of grains per plant, grain weight per plant and grains weight per thousand seeds were medium strong level.In short, a novel C3HC4 type RING finger protein, GmRZFPl was isolated from soybean cDNA library using NTT system. Some characteristic of GmRZFP1 such as subcellular localization and expression pattern were compeled. Functional analysis of GmRZFP1 showed that overexpression of GmRZFPl can improve stresses tolerance of transgenic plants. Morever, some T3 or T4 generation DREB transgenic wheat were identificated using PCR and functional analysis proven that stress tolerance of transgenic wheat were improved comparing with wild type wheat plants. It is important for improving stress tolerance and yield of Chinese wheat varieties that further screening of genetic stable and reliable stress tolerant transgenic lines in the further.
|
PDF Full Text Request