Investigation Of Molecular Mechanism Of Cold Resistance Of Camellia Sinensis Based On Proteomics And Functional Identification Of CsCBF3

Tea(Camellia sinensis(L.)O.Kuntze),one of the important economic wooden plants in the world,are mainly grown in subtropical and tropical regions.With the development of tea industry,the tea planting area extends to 38 north latitude.Most tea plantations in China,especially the alpine tea field,often suffer from cold damage in winter and ’cold in late spring’in spring.The critical problems of breeding cold-resistant varieties is to improve the cold resistance of tea plant.It is of great important for the molecular breeding to study the cold resistance mechanism,analysis gene regulatory networks of responding to low temperature,explore and use the resistance gene pertinently.In our study,we used the cold-resistant Camellia sinensis var.’Shuchazao’(SCZ)and cold-susceptible Camellia sinensis var.assamica ’Yinghong 9’(YH9)as materials treated by cold acclimation.The changes of physiological and biochemical,and the expression level of key genes in respons to low temperature were compared and analyzed in SCZ and YH9.Proteomic study of tea response to cold acclimation was carried out.Moreover,the function of the key cold resistance gene CsCBF3 was identified.The main results are listed as follows:1.After cold acclimation,EL50 of SCZ and YH9 decreased significantly.The EL50 of SCZ decreased from 5.7℃ to-9.4℃ and YH9 decreased from-2.3℃ to-6℃.Significant chimeric and zipper structures were formed in the chloroplast of SCZ.After cold acclimation,the value of Fv/Fm was decreased,the activity of POD and CAT,the content of MDA,osmotic,soluble protein,soluble sugar and proline were increased in SCZ and YH9.While the Fv/Fm,POD,CAT activity,soluble sugar,especially sucrose content in YH9 were much lower than SCZ.2.After cold acclimation,the genes of ICE-CBF-COR pathway CsCBF1,CsCBF3 and CsDHNs were largely induced between the two varieries.And their expression level in SCZ higher than in YH9.The transcriptional levels of CsCBFl in SCZ and YH9 was increased by 282 and 29 times respectively,and CsDHN3 was increased by 68.7 and 9.2 times,respectively.After cold acclimation,the expression of key genes CsSPS,CsINV5 and CsRS2 in the glucose metabolism pathway are increased in SCZ.After cold acclimation,the transcription levels of synthetic genes CsOAT,CsP5CS and CsP5CR in the proline metabolic pathway was increased more in SCZ with than YH9,while the transcription levels of degradation gene CsProDH and CsP5CDH was decreased with lower than YH9.3.Using iTRAQ quantitative proteomics,380 differential abundance proteins(DAPs)were identified with 34 shared in SCZ and YH9.Their expression patterns were the same in SCZ and YH9 except for TEA009793.1(chlorophyll A-B binding protein of LHCII type 1).RT-qPCR was used to verify the transcriptional level of 25 DAPs.4.380 DAPs was enrichment in GO and KEGG.113 GOs terms were significantly enriched,which closely related to abiotic stresses,such as stomatal closure,response to cold,response to osmotic stress.Photosynthesis-antenna proteins,Glutathione metabolism,Anthocyanin biosynthesis,Zeatin biosynthesis,Synthesis and degradation of ketone bodies,Plant-pathogen interaction the 6 KEGGs pathways were significantly enriched.5.Based on the functional analysis of differential proteins,the molecular mechanism of SCZ cold resistance higher than YH9 has the following points.After cold acclimation,the expression of 14-3-3 protein decreased in SCZ is beneficial to the accumulation of LAE protein in tea plant;significant up-regulated ABCC1 and ABCB1 transporters facilitated stomatal closure and reduced transpiration;significantly decreased antenna protein(LHC)reduced the absorption of light energy by tea trees and alleviated photoinhibition caused by energy in plants;highly expressed SUS,gbE1,glgA,pgm,bglX,bglB are beneficial to the accumulation of glucose,fructose and starch;high expression of PAL,CHS,F3’5’H is beneficial to the accumulation of flavonoids and enhances the ability to scavenge free radicals.In addition,dehydration(TEA010673.1)was more abundant in SCZ than YH9,which was consistent with the results of Western bolt hybridization.6.Based on transcription and genome database,a new CBF/DREB transcription factor named CsCBF3 was cloned and identified,and GenBank accession number was MH017428.1.CsCBF3 contained 783 bp length of the OFR frame,which encode proteins consisted of 260 amino acids.The molecular weight of the protein is 28.06 kDa and isoelectric point is 4.84.The gene contains the typical AP2 domain of CBF transcription factor family with conserved characteristic sequences on both sides.There is not introns in CsCBF3.By analysising of CSCBF3 gene information of 10 tea varieties with different cold resistance,we found that CsCBF3 could be used as one of the indicators of cold resistance of tea plant.CsCBF3 can be expressed in all tissues and the highest expression in roots.The expression of CsCBF3 was induced by 4℃ at low temperature,and the transcription level at 12h was 300 times that of the control.In addition,NaCl treatment can induce the expression of CsCBF3,while SA treatment can inhibit the expression of CsCBF3.7.By DAPI staining and yeast single hybridization,CsCBF3 was localized in the nucleus and had transcriptional activation function.We analyzed the freezing CsCBF3 transgenic Arabidopsis thaliana,and the transcriptional expression levels of ABA-independent/dependent downstream COR gene The results showed that CsCBF3 can significantly improve cold tolerance of transgenic plants through ABA-independent pathways.