| NAC transcription factor is peculiar kind of transcription factors in plants.Transcription factors involved in the expression of plant genes in different conditions,and they plays a key role in plant response to various biotic and abiotic stress factors. Chrysanthemum is traditional famous flower of China with high ornamental and practical value. But the abiotic stresses limit their growth and ornamental value, such as low temperature, drought, saline and alkali. So, it is an important significance to cultivate the Chrysanthemum cultivars of comprehensive resistance. Because of its complex genetic background, the progress of the salt-responsive molecular mechanism of chrysanthemum has been slowed. Chrysanthemum lavandulifolium is wild diploid species near the source of the cultivated-chrysanthemum, whose genetic background is simple, distribution is wide and salt tolerance is strong. In this study, the complete cDNA sequence of ClNAC9and CINAC39were cloned by3’RACE. Analysis of genes bioinformatics、expression pattern, and transformed into Arabidopsis thaliana in order to explore the expression pattern and function of NAC genes. Then,they transformed into Chrysanthemum xgrandiflora ’niu9717’and are expected to provide some basic theories for further research NAC function. The main results are as follows:1. Two homologous NAC genes were screened from the EST libraries in chrysanthemum transcriptome database and amplified by3’RACE to obtain the full-length cDNA of ClNAC9and ClNAC39. ClNAC9contains an651bp ORF encoding a217residue peptide. ClNAC39contains an849bp ORF encoding a283residue peptide.Searching conserved domain and comparison of deduced amino acid sequences showed that the two genes are members of NAC transcription factors and they both have conserved structure domain of NAM. Analysis of2genes use MEGA5software, we found that CINAC9is in branch of SENU5subfamily and ClNAC39is in branch of NAP subfamily.2.Quantitative real-time PCR (qRT-PCR) showed ClNAC9and ClNAC39were expressed under different abiotic stresses conditions. The expression of ClNAC9increased rapidly in response to drought, salinity, hot or ABA stress; while the expression of ClNAC39was induced to drought, salinity, hot, ABA or SA. But they were induced only under the low temperature condition.3. ClNAC9and ClNAC39were transformed into Arabidopsis thaliana (Col-0) with Agrobacterium GV3101containing the expression vectors pBI121-ClNAC9and pBI121-ClNAC39, respectively. The positive Arabidopsis transformants were selected using MS medium containing kanamycin resistance and RT-PCR. Under NaCl、NaHCO3and drought stresses, transgenic Arabidopsis with over-expression of ClNAC9and ClNAC39showed lower MDA contents and higher activity of SOD and POD than the wild type.these results indicated over expression of CINAC9and CINAC39in Arabidopsis could enhance the ability to endure stresses.4. After sterilized shoots were obtained from axillary bud, a series of factors that influenced Chrysanthemum×grandiflora ’niu9717’ tissue culture were studied. The tissue culture system was developed from two regeneration ways from either axillary bud way or callus way. Reproduction coefficient of axillary bud was3.2; plantlets can be regeneration, with the callus inducing rate over100%, the callus differentiation rate over92.3%, rooting rate over100%.5. Transgenic plants were obtained by Agrobacterium-mediated transfer system from Chrysanthemum xgrandiflora’niu9717’leaf. To select the resistant shoots, the concentration of kanamycin was10mg/L in callus inducing culture and8mg/L in rooting culture. Explants were pre-cultured for2-3days before infection. The pre-cultured explants were incubated for15min or10min in the bacterial suspension which concentration (OD600) was0.6or0.8. Co-cultivation period was2days, delay cultivation period was3days. The target gene integrated into the genomic DNA of Chrysanthemum xgrandiflora ’niu9717’ was detected and confirmed by PCR and RT-PCR analysis. |
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