| Brassica rapa crops originated from China,have a long history of cultivation and variety richness.In botany classification,they belonging to Cruciferae Brassica plants,including Chinese cabbage(Brassica rapa ssp.pekinensis),non-heading Chinese cabbage(Brassica rapa ssp.Chinensis)and turnip(Brassica rapa ssp.rapa).Among them,non-heading Chinese cabbage is a popular vegetable and widely planted in the region along the Yangtze River.Like Arabidopsis thaliana,non-heading Chinese cabbage also can induce cold acclimation through a series of transcriptional regulation the mechanisms,thus improving its cold resistance,while CBF cold responsive pathway dominated by CBF transcription factor family genes is one of the most important these transcriptional regulation.Thus,system study of CBF cold responsive pathway is very important for better understanding cold and other stresses regulation mechanisms in non-heading Chinese cabbage.In this study,CBF genes were isolated and cloned from non-heading Chinese cabbage,and their stress analysis of expression pattern and regulation characteristics had been analyzed,furthermore,we studied on the relationship between the upstream regulation of BcCBF3;through small RNA sequencing,the role of CBF genes played in the relationship between cold resistance and pathogenic relevance were discussed;in addition,the whole-genome identification and analysis of MYB transcription factors were achieved in Chinese cabbage,which could act as reference for understanding the upstream regulation factor MYB of CBF genes in non-heading Chinese cabbage and even all Brassica rapa crops.In this paper,the main research results are as follows:1.In this study,eight CBF-like genes were isolated from non-heading Chinese cabbage(Brassica rapa ssp.chinensis).The deduced CBF proteins shared high similarity with their Arabidopsis orthologs and localized to the nucleus.Furthermore,quantitative real time PCR(qPCR)analysis showed that BcCBFl-3 were induced by cold(4?)but not drought or abscisic acid(ABA),indicating that they are involved in an ABA-independent pathway;however,BcCBF4-6 were regulated by both drought and ABA,suggesting that they were involved in an ABA-dependent pathway.Nevertheless,unlike Arabidopsis,BcCBF4?6 showed response to both cold and ABA,indicates ABA-independent and ABA-dependent parts of CBF pathway in NHCC might not be completely separate,and these genes may act as the connection points in the network.BcCBFs were also participating in the response to biotic stresses.2.Gateway technology was utilized to construct the over-expression vectors of BcCBFl,BcCBF2,BcCBF3 and BcCBF4,subsequently transformed into onion epidermal cells for subcellular localization,the results showed that all the four BcCBF proteins were located to the nucleus.Then,Agrobacterium with over-expression vector was transformed into Arabidopsis;Cblcv induced VIGS vector was obtained and transformed into non-heading Chinese cabbage.Conduction determination indicated that over-expressed BcCBF3 transgenic plant had higher cold tolerance.QPCR analysis further showed that there was no direct relation between the expression of BcCBF2 and BcCBF3,and the upstream regulator of BcCBF3,BcMYB15,showed feedback inhibition of BcCBF3,while the expression of downstream BcCOR15A was positively related with BcCBF3 and it could further amplified the expression change of BcCBF3.3.The BcCBF3 promoter sequence were analyzed and two WRKY binding cis-element W-boxs were identified.Expression and GUS analysis showed that two W-boxs both have a role in the binding with WRKY.We further studied the expression pattern of CBF related genes in BcWRKY33 over-expressed non-heading Chinese cabbage plant.The results indicated that constitutive expression of BcWRKY33 activated the transcription of BcCBF3,whereas their expression levels under cold were higher than in untreated plants.The expression pattern of BcCBF4 in the transgenic plants indicated that although it response to ABA like WRKY33,the ABA response mechanism of them were independent.In addition,BcWRKY33-GST recombinant protein has been obtained from prokaryotic expression,which could be foundation for experimental verification of protein-DNA interaction.4.In this study,two small RNA libraries were constructed based on non-heading Chinese cabbage leaves infected by TuMV and healthy leaves,and sequenced using the Illumina-Solexa high-throughput sequencing technology.A total of 86 conserved miRNAs belonging to 25 known miRNA families and 45 novel ones were identified.Differential expression analysis showed that 42 miRNAs were down-regulated and 27 miRNAs were up-regulated in response to TuMV stress.A total of 271 target genes were predicted using a bioinformatics approach,these genes are mainly involved in growth and resistance to various stresses.We further selected 13 miRNAs and their corresponding target genes to explore their expression pattern under TuMV and/or cold(4?)stresses,and the results indicated that some of the identified miRNAs could link TuMV response with cold response of non-heading Chinese cabbage.5.We firstly analyzed the cis-elements in the promoters of CBF genes from non-heading Chinese cabbage and cabbage,and found that all these CBFs contained MYB-binding domain in their promoters,indicating extensive roles that MYBs act in the upstream regulation of CBF genes.Then,we analyzed 256 R2R3-MYB genes from Chinese cabbage.The R2R3-,3R-and atypical MYB proteins in Chinese cabbage formed 45 subgroups based on domain similarity and phylogenetic topology.Moreover,RNA-seq data revealed tissue-specific and distinct R2R3-BrMYB expression profiles,and quantitative real-time PCR(qPCR)analysis in leaves showed stress and ABA signal responsive expression.Our results indicate that members of C11,C14,C31,C36 and C38 subgroups might participate in mutiple stress response in Chinese cabbage,while C42 members only showed cold response,indicating that they might be cold specific regulons.Members from these subgroups might exist candidate up-stream regulons of CBF genes. |
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