| The Brassica rapa of Cruciferae family,composed of three subspecies: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,also known as small cabbage,green cabbage,etc.,originated in the middle and lower reaches of the Yangtze River in China,is one of the popular vegetables.With a long cultivation history,it has abundant germplasm resources and extensive planting areas.However,non-heading Chinese cabbage often suffers from various stresses from the external environment during its life cycle(growth and development),such as biotic stress and abiotic stress,which will seriously affect its normal growth and development.Among many abiotic stresses,cold stress has caused more serious damage to the normal growth and development of non-heading Chinese cabbage.Therefore,it is of great theoretical significance and practical application value to study the molecular regulation mechanism of non-heading Chinese cabbage cold stress-related genes and signal transduction.Both non-heading Chinese cabbage and Arabidopsis belong to the Cruciferae family.Compared to the model plant Arabidopsis,studies on cold stress-related genes and response regulation mechanisms in non-heading Chinese cabbage are relatively few.In this study,RNA-Seq and miRNA-Seq were used to mine cold-related genes and miRNAs.In addition,four cold stress-related genes BcCOR15B,BcCOR15A,BcHHP3 and BcHHP5 were identified in non-heading Chinese cabbage,and their physical and chemical properties,hydrophilicity,transmembrane region,subcellular localization,protein structure,expression pattern and regulation characteristics under the cold stress and other treatments were analyzed,non-heading Chinese cabbage gene silencing plants were used for functional research.The main results are as follows:1.Using RNA-Seq and miRNA-Seq to analyze cold stress-related genes and miRNAs.According to the transcriptome sequencing results,1,860 new genes were discovered,of which 1,613 were functionally annotated.Among them,13 common differentially expressed genes were detected in all materials,including 7 up-regulated and 6 down-regulated.At the same time,we used qRT-PCR to confirm the changes of these gene expression levels.In addition,we sequenced miRNA of the same material.Our findings revealed a total of 34,182,333 miRNA reads,88,604,604 kinds of miRNA,among which the most common size was 24 nt.In all materials,the number of common differential miRNAs is 8.According to the corresponding relationship between miRNA and its target genes,we carried out GO and KEGG analysis on the set of target genes that each group of differentially expressed miRNAs.Through the analysis,it is found that the distribution of candidate target genes in different materials is different.2.Two cold-related genes,BcCOR15B and BcCOR15A,were isolated from non-heading Chinese cabbage.Their open reading frames are 429 and 390 base pairs in length,encoding 142 and 129 amino acids,respectively.The predicted amino acid sequences of BcCOR15B and BcCOR15A share the respective 99.30%and 95.56%highest homology with the amino acid sequences of the corresponding BrCOR15B and BrCOR15A in Chinese cabbage.Sequence analysis and phylogenetic analysis showed that the relationship between BcCOR15B/A and BrCOR15B/A was relatively close.Structural analysis showed that the structure of BcCOR15B/A proteins were both relatively stable and conservative.Subcellular localization indicated that BcCOR15B and BcCOR15A proteins were both localized on the chloroplast.qRT-PCR analysis showed that BcCOR15B/A were both induced to express by cold stress and other different treatment.Prokaryotic expression and western blot analysis revealed that the two proteins were both hydrophilic.The identification and analysis of cold-related genes BcCOR15B/A in non-heading Chinese cabbage might provide a reference for further study on agronomic traits of vegetable and crop plants in cold environments.3.BcHHP3 was isolated from non-heading Chinese cabbage,it has an open-reading frame of 1044 base pairs,encoding 347 amino acids,a molecular weight of 39.35 kDa,isoelectric point of 9.08,an instability index of 48.35,and grand average of hydropathicity of 0.382.Multi-alignment and phylogenetic analysis showed that BcHHP3 bears a high similarity to AtHHP2.As predicted by SOMPA and SWISS-MODEL online tools,the structure of the BcHHP3 protein is relatively stable and highly conservative.Quantitative rverse transcript polymerase chain reaction analysis showed that BcHHP3 was induced to co-express under cold stress and ABA treatment.The BcHHP3-GFP fusion protein was localized on the cell membrane and nuclear membrane.TYMV-VIGS test shows that BcHHP3 is related to cold stress regulation.4.BcHHP5 was isolated from non-heading Chinese cabbage.Sequence analysis and phylogenetic analysis indicated that BcHHP5 in non-heading Chinese cabbage is similar to AtHHP5 in Arabidopsis thaliana.Structure analysis showed that the structure of the BcHHP5 protein is relatively stable and highly conservative.Subcellular localization indicated that BcHHP5 was localized on the cell membrane and nuclear membrane.Furthermore,qRT-PCR analysis showed that BcHHP5 was induced to express by cold stress and other different treatment.In non-heading Chinese cabbage,BcHHP5-silencing assay,inhibiting the action of endogenous BcHHP5,indicated that BcHHP5-silencing might have a negative effect on cold tolerance,which was further confirmed.All of these results indicate that BcHHP5 might play a role in abiotic response.This work can serve as a reference for the functional analysis of other cold-related proteins from non-heading Chinese cabbage in the future. |
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