| Chinese cabbage(B.rapa ssp.pekinensis),non-heading Chinese cabbage(B.rapa ssp.chinensis)are important vegetable crops,which have a long history of cultivation and emerge rich germplasm resources after the long-term artificial domestication.Currently,the development of omics technology such as genomics,transcriptomics and proteomics has provided us a great opportunity to systematically study the various plant responses to external signals.In the model plant Arabidopsis thaliana,it has been a mature system to study genes’ function and signal transduction network.However,studies on B.rapa are still limited so far.Therefore,genome-wide analysis of transcription factor families and identification of their functions in B.rapa will provide important theoretical guide for cultivation and breeding.In this study,we identified stress-and light-related transcription factor families(ZF-HD genes,Trihelix genes and PIFs)in B.rapa,and revealed their expansion,retention patterns,evolutionary rules,replication differentiation and mining key genes for stress responses by comparative genomics and gene expression analysis.At the same time,BcZF-HD14,BcTH28 and BcPIF3 were cloned from non-heading Chinese cabbage and functionally identified to reveal their synergistic effect in stress adaptation.The results are as follows:1.Identification and stress induced expression patterns of ZF-HD transcription factors in Chinese cabbageTotally 31 ZF-HD genes were identified in Chinese cabbage.We analyzed ZF-HD genes from multiple species and found that the BraZF-HD genes could be categorized into ZHD and MIF subfamilies.ZHD genes are plant-specific,nearly all intronless,and MIF genes possess only the zinc finger.Phylogenetic analysis suggested that ZHDs have expanded considerably during angiosperm evolution.In addition,the ZHD group has 24 members,which is twice as much as the Arabidopsis ZHD group,indicating that the Chinese cabbage ZHD genes have been retained more frequently than other group genes.In addition,most BraZF-HD genes are preferentially expressed in flower and significantly induced under photoperiod or vernalization conditions,as well as abiotic stresses.Thereby implying that they may play important roles in these processes,especially reproductive stage.2.Comparative genomic analysis of Trihelix family in Chinese cabbage reveals its evolutionary patterns and stress responsesTrihelix gene family is widely involved in plants’ stress and light responses.Here,a total of 52 Trihelix genes were identified in B.rapa,named as BraTH01 to BraTH52.All the Trihelix proteins are highly conserved and contain light responsive elements.By statistics of the number of Trihelix genes in different species,we found the expansion of Trihelix gene family started with angiosperm evolution.Trihelix family has five groups,SIP1 was more preferentially retained than other subgroups(GT-1,GT2,GTy,SH4),consistent with the gene dosage hypothesis.Then we investigated the evolutionary patterns,footprints and conservation of Trihelix genes in selected plants.Furthermore,among six abiotic stresses(Cold,Heat,PEG,NaCl,ABA and GA),most Trihelix genes were activated by salt and ABA treatment.3.BcZF-HD14 and BcTH28 synergistically promote proline accumulation and stress adaptation in non-heading Chinese cabbageBcZF-HD14 and BcTH28 are important genes from ZF-HD and Thrihelix transcription factor families respectively.In this study,BcZF-HD14 and BcTH28 are cloned from non-heading Chinese cabbage(Brassica rapa ssp.chinensis).overexpression of BcZF-HD14 and BcTH28 in Arabidopsis could promote accumulation of proline and seedlings adaptation under osmotic stress.RT-PCR result indicated that proline synthesis gene P5CS and circadian genes PIF4/7 were significantly induced in transgenic lines.Furthermore,yeast two hybrid result show that BcZF-HD14 and BcTH28 proteins could interact with each other.These results indicate that these two transcription factors may regulate proline accumulation by forming complex in non-heading Chinese cabbage.4.PIF-related genes are involved in plant hormones response in non-heading Chinese cabbagePhytochrome interacting factors(PIFs)are a group of transcription factors that negatively regulate photomorphogenesis,belonging to the fifteenth subfamily of bHLH transcription family in Arabidopsis.In Brassica rapa,PIFs belong to Ⅶ(a+b)subfamily of BrabHLH,including 18 members,in non-heading Chinese cabbage,19 PIF-related genes are identified,named BcbHLH01 to BcbHLH19.PIF-related genes are nonrandomly distributed on ten chromosomes.After the WGT in B.rapa and nonheading Chinese cabbage,PIF-related genes underwent a loss.RT-qPCR result show BcPIFs can be significantly induced by various plant hormones.These results will lay basis for future studies on PIF-related genes’ functions in B.rapa.5.BcPIF3 promotes plant growth and development through vernalization and photoperiod pathway in non-heading Chinese cabbagePIF3 is an important regulator gene in plant light-signaling pathway.We cloned BcPIF3 from non-heading Chinese cabbage(Brassica rapa ssp.chinensis).RT-PCR result indicated that BcPIF3 was more induced under long-day condition.Under vernalization,BcPIF3 raised at first,then decreased,and the peak appeared at 7 weeks.Yeast two hybrid assay showed that two proteins existing in light signaling pathway interact with BcPIF3,they are BcCAB1(CHLOROPHYLL A/B BINDING PROTEIN 1)and BcLHBIB1(Photosystem Ⅱ light harvesting complex gene 1.4).Furthermore,phenotypic analysis found that overexpression of BcPIF3 in Arabidopsis could promote root development and hypocotyl elongation in Arabidopsis seedlings.These results indicate that BcPIF3 is responsive to both photoperiod and vernalization and through associating with light related proteins,BcPIF3 promotes the growth of non-heading Chinese cabbage under long-day condition. |
