Differential Expression Screening And Functional Analysis Of Candidate Genes Involved In Cold Response In Non-Heading Chinese Cabbage

Temperature fluctuations and extreme freezing temperature at crown level, occurring during winter and early spring, cause recurrent losses of vegetables production. Cold stress severely affect the growth and development of plants through the imposition of osmotic stress, and secondary stress such as oxidative stress leading to membrane disorganization, metabolic toxicity, and inhibition of photosynthesis. Many plants increase in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. Freezing tolerance of such plants increases substantially after a period of exposure to low but non-freezing temperature. Cold acclimation of plant is a highly active process resulting from gene expression involved in physiological and metabolic adaptations to low temperature. Cold acclimation involves the remodeling of cell and tissue structures, the reprogramming of metabolism and gene expression, which encoding molecular chaperones, lipid, and sugar metabolism are suggested to be involved in cold adaptation. Studies on acquired freezing tolerance in Arabidopsis have contributed substantially towards the understanding of cold acclimation mechanisms. But the molecular basis of this acquired chilling tolerance or chilling acclimation is poorly understood in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). To date, non-heading Chinese cabbage has not been exploited to understand the molecular basis of its unusually high level of cold tolerance. Here cDNA-AFLP was used to identify transcripts that are strongly accumulated and induced in response to cold stress.1. Differential expression analysis of candidate genes involved in cold response in non-heading Chinese cabbage using cDNA-AFLPThe objective of this study was to determine temporal expression profiles of transcripts during cold acclimation of non-heading Chinese cabbage cold-tolerant inbred line043by using cDNA-AFLP.256primer combinations were used to investigate9960cDNA fragments. A total of77differentially expressed cDNA fragments were detected,57of which were up-regulated and20were down-regulated under cold treatment (4℃). These TDFs were classified into several functional groups with Blast in GenBank. Except for18function-unknown TDFs, the remained up-or down-regulated TDFs conferred functions of metabolism, photosynthesis and energy, transport, signal transduction, secondary metabolism, defense response, transcription, cytoskeleton, function unknown and hypothetical protein. Meanwhile, the expression patterns of three TDFs, TDF16#, TDF21#, and TDF57#showed a significant increase of differential expression after cold stress was analysed by qRT-PCR. These three TDFs might play important roles in promoting adaptation to cold stress. The result showed that all of the three TDFs were induced by cold stress, which suggest that play important roles in promoting adaptation to cold stress. This study provides important clues to understanding cold acclimation and low-temperature regulation mechanisms in non-heading Chinese cabbage and the three TDFs involved in cold responses need further research to determine their usefulness in breeding new resistance cultivars.2. Molecular cloning, characterization and function analysis of BcWRKY46from non-heading Chinese cabbageWRKY transcription factors belong to one of the largest families of transcriptional regulators in plants and form integral parts of signaling webs that modulate many vital processes during plant growth and development. BcWRKY46, a cDNA clone encoding a polypeptide of284amino acids and exhibited the structural features of group III of WRKY protein family, was isolated from the cold-treated leaves of non-heading Chinese cabbage using the cDNA-AFLP technique. Expression of this gene was induced quickly and strongly in response to various environmental stresses, including low temperatures, ABA, salt and dehydration. Constitutive expression of BcWRKY46in tobacco under the control of the CaMV35S promoter reduced the susceptibility of transgenic tobacco to freezing, ABA, salt and dehydration stresses. Our studies suggest that BcWRKY46plays an important role in responding to ABA and abiotic stress.3. Molecular cloning, characterization and function analysis of BcMCSU from non-heading Chinese cabbageUsing cDNA-AFLP techniques, a cold induced TDF16#homologous to AtMCSU was isolated from the leaves of non-heading Chinese cabbage which was named as BcMCSU. The full-length BcMCSU cDNA which consisted of a single open reading frame encoded a putative polypeptide of307amino acids. According to the functional domain analysis of the predicted amino acid sequence, the BcMCSU protein contains a Nifs domain at its N-terminus and a MOSC domain at the C-terminus. An efficiently overexpression vector of pEG103-BcMCSU was constructed and transformed into Arabidopsis thaliana by Agrobacterium mediated depressor permeating method. Transgenic Arabidopsis thaliana plants were obtained by resistance screening and PCR identification.4. Molecular cloning, characterization and function analysis of BcVIN3from non-heading Chinese cabbageUsing cDNA-AFLP techniques, a cold induced TDF57#homologous to AtVIN3was isolated from the leaves of non-heading Chinese cabbage which was named as BcVIN3. The full-length cDNA sequence of BcVIN3was1814bp, and encoding a putative polypeptide of582amino acids. According to the functional domain analysis of the predicted amino acid sequence, the BcVIN3protein contains a PHD-finger domain. An efficiently overexpression vector of pEG103-BcVIN3was constructed and transformed into non-heading Chinese cabbage’Suzhouqing’ by the pollen tube pathway method. Transgenic plants were obtained by resistance screening and PCR identification.