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Cloning and Functional Characterization of a Stress-Related Transcription Factor BnNAC5-3in Brassica Napus

Author: WangHongJuan
Tutor: YangGuangSheng; LiuPingWu
School: Huazhong Agricultural University
Course: Crop Genetics and Breeding
Keywords: Brassica napus drought stress salinity stress NAC
CLC: S565.4
Type: Master's thesis
Year: 2013
Downloads: 7
Quote: 0
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Abstract


The drought, salinity stresses negatively impact agricultural yield and quality. Improvement of plant stress tolerance is one of the most important goals of the agricultural breeders. More and more genetically modified crops that can tolerate plant stress were developed. But through transgenic technology to improve the stress resistance of Brassica napus was reported less. NAC transcription factor is key regulator in abiotic stress response. BnNAC5-3that belongs to the NAC family was cloned by homologous cloning and overexpressed in Brassica napus, which showed that BnNAC5-3gene could enhance Brassica napus tolerance to drought and salinity. The main results are as follows:1. The primer was designed according to BnNAC5-7full-length cDNA sequence, and with which BnNAC5-3were cloned. BnNAC5-3is899bp in full-length, encodes280amino acids and contains a conserved NAC domain. RT-PCR analysis showed that drought stress induced BnNAC5-3expression and the highest level of expression was at0.5hours after treatment of20%PEG (w/v). BnNAC5-3gene CDS was inserted into the PGBKT7vector, then transformed into yeast strain AH109. BnNAC5-3could activate reporter genes, which suggested that BnNAC5-3protein has transcriptional activation function.2. By agrobacterium-mediated method,390Brassica napus P35S::BnNAC5-3transgenic plantlets were obtained. Thirty five positive plants were identified by PCR, and the positive rate was about9%. The germination rate of transgenic TO generation seeds was significantly lower than the control in the10mg/L ABA aqueous solution, and the growth of transgenic seedlings was inhibited more in100mg/L ABA nutrient solution. These results suggested that overexpression BnNAC5-3enhanced plant ABA sensitivity. In5%PEG (w/v) nutrient solution culture, the control died earlier than Tl plants. Under15%PEG (w/v) or150mmol/L NaCl stress, the transgenic plants grew better. In greenhouse, T1transgenic lines of overexpression BnNAC5-3has better water retention and oxidation resistance than the control under normal conditions. Real-time PCR analysis showed that BnNAC5-3increased expressions of NCED3, key gene for the ABA synthesis, and ERD11, early response gene to dehydration in the overexpression lines.

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CLC: > Agricultural Sciences > Crop > Economic crops > Oil crops > Rapeseed ( Brassica )
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