| Rapeseed(Brassica napus L.) is one of the most important commercial crops in China. However, it is often subjected to various stresses which seriously affect the production of rapeseed in the actual production process.In the process of long-term evolution, plant formed a complex signal transduction and gene regulatory network system to defense against many kinds of stresses. Transcription factors play very important roles in this system, such as WRKY transcription factor superfamily, which defined by the highly conserved WRKYGQK amino acid sequence and a zinc-finger motif in the N-terminal. WRKY transcription factors can act as repressors as well as activators in regulating downstream gene expressions by specifically binding to the(T)(T)TGAC(C/T)(W-box) sequence in promoter regions. In recent years, studies have shown that WRKY transcription factors are widely involved in the transcriptional regulation of plants such as growth and development, metabolism and biotic and abiotic stress responses, etc. Some also displayed pleiotropic functions, playing different roles in different aspects of biological processes in plants at the same time, making it possible that plants can coordinate the balance of plant growth and defense.In this study, we cloned a c DNA sequence of Bn WRKY28 gene from the B.napus variety ning RS-1, which belongs to II-c subtribe of WRKY super family.And we studied its functions preliminarily by sequence analysis, inducing expression pattern analysis, subcellular localization analysis, overexpression phenotypic analysis, hormone treatment, q RT-PCR, stress resistance identification and detection of endogenous hormones, etc. The functions analysis about Bn WRKY28 transcription factor were described as follows: 1. The cloning of Bn WRKY28The Bn WRKY28 gene from B.napus was obtained by homology cloning.The c DAN sequence containing an open reading frame of 939 bp, encoding a polypeptide of 313 amino acids with a putative molecular weight of 34.973 k Da, and theory isoelectric point of 6.67. Utilizing Plant CARE and PLACE software to analysis the 1kb sequence upstream the Bn WRKY28 revealed many cis-elements related to the growth and development and stress responses, suggesting that Bn WRKY28 gene may involve in the regulation of these signaling pathways. 2. Subcellular localization and transcriptional activation analysisBn WRKY28::GFP fusion construct was transformed into the Arabidopsis protoplast, and Bn WRKY28 was shown to locate in the nucleus, complying with the general features of transcription factors. Transcription activation analysis in yeast showed that the Bn WRKY28 protein had the transcriptional activation activity. 3. Analysis of induced expression patterns of Bn WRKY28The expression pattern by q PCR analysis in Westar showed that the expression of Bn WRKY28 could be induced by signaling molecules such as salicylic acid(SA), Methyl jasmonic acid(Me JA) and necrotrophic fungi(S.sclerotiorum), which indicated that Bn WRKY28 may be involved in diverse signaling pathways. 4. Genetic transformation into B.napus and phenotype analysis of Bn WRKY28 overexpression plantsUsing agrobacterium-mediated genetic transformation, Bn WRKY28 overexpression transgenic plants were obtained by transforming the seedling hypocotyl and tissue culture. Compared with the controls, transgenic plants showed many different phenotypes in the growth and development, such as abnormal seed germination, shorter seedling hypocotyl, decreased lateral root development and increased main root growth, dwarf plant, changed leaf shape, inadequacy matured seeds and other related phenotypes. 5. Bn WRKY28 is involved in GA pathwayThe shorter hypocotyl phenotype can be partly restored by applying exogenous GA. Expression analysis of the genes involved in GA metabolism and transduction pathways showed that a critical for GA synthesis gene GA20ox1 was down-regulated significantly and GA catabolic genes GA2ox1/GA2ox2 were up-regulated in Bn WRKY28 overexpression plants. Moreover, the target genes GID1 a, GID1 b, SLY1 negatively regulated by GA showed a pattern of increased expression. 6. Bn WRKY28 is involved in biotic and abiotic stressDetermination of the endogenous SA and JA in seedlings showed that the JA level decreased significantly, while the SA level had no significant difference between the transgenic plants and control. Genes related to SA pathway appeared an up-regulated trend while genes involved in JA pathway showed a down-regulated trend. In addition, overexpress Bn WRKY28 seems to reduce the resistance to necrotrophic fungi, B. cinerea in B.napus, which indicate that Bn WRKY28 is likely to positively regulate SA-dependent resistance pathway and negatively regulate JA-dependent resistance pathway. Furthermore, when treated with PEG-6000 on seedling for simulated drought, the overexpression plants displayed increased tolerance to PEG-6000.To sum up, Bn WRKY28 plays an important role in regulating plant growth and development as well as in coping with external stress at the same time. |
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