Function Characterization Of Dehydrin CaDHN1 And Expression Analysis Of Dehydrin Gene Family In Pepper(Capsicum Annuum L.)

Pepper(Capsicum annuum L.),which is widely cultivated in the wold,is an important flavoring that commands universal application as seasonings with vegetables.A range of abiotic stresses,such as low temperature,are the important factors limiting plant growth and geographical distribution.Pepper is very sensitive to low temperature.The optimal growth temperature for pepper ranges from 24°C to 28°C,the growth of plants is retarded below12°C and completely stop below 5 ?,so low temperature affect the yield and quality of pepper.Resistance breeding by molecular technique is an efficient approach to improve plant resistance to environmental stresses.In this study,cold-related genes CaDHN1 was cloned.Expression patterns of the CaDHN1 gene under abiotic stresses and signaling molecules were performed.Function of CaDHN1 gene was studied using virus-induced gene silencing(VIGS)and transgenic techniques with Arabidopsis plants(Arabidopsis thaliana).In addition,the genome sequence of pepper has been published,which is convenient for characterizing dehydrin gene family in pepper.Therefore,we analyzed CaDHN gene family using bioinformatics method and systematically investigated the expression patterns of DHN genes in pepper different tissues and under various treatment.The results provide a foundation for further functional study and will be valuable for improving the adaptability of pepper to unfavorable growing conditions.And the main results in all experiments were as follow:1.Based on the previously characterized cold-associated pepper seeding cDNA library,we obtained a 262 bp pepper cDNA,cloned the full-length and named CaDHN1.GenBank with accession No.JX402924.Pepper CaDHN1 contained an ORF of 660 bp and encoded a protein of 219 amino acids.2.Tissue expression profile analysis revealed CaDHN1 was expressed predominantly in fruits and flowers.The expression of CaDHN1 was markedly upregulated in response to cold,salt,osmotic stresses and salicylic acid(SA)treatment,but no significant change by abscisic acid(ABA)and heavy metals treatment.A VIGS technique was used to silence the CaDHN1 in pepper plants,Under cold stress,the pTRV2:CaDHN1 plants aggravated the visible symptoms of leaf damage in seedlings.The MDA and electrical conductivity measurements were both significantly increased,while chlorophyll levels were markedly decreased in CaDHN1-silenced plants than that ofpTRV2:00 plants.The leaf discs from the silenced pepper plants were exposed to NaCl or mannitol solutions.CaDHN1-silenced leaf discs were more yellow than those of control plants,and some were blackened after 3 days of salt treatment,and the same was true of mannitol-treated plants.Chlorophyll levels were also significantly reduced in CaDHN1-silenced plants compared with controls.The expression patterns of several stress-related genes(KIN1,Mn-SOD,POD)were monitored by qRT-PCR.Cold stress induced Mn-SOD,POD and KIN1 expression in both control and CaDHN1-silenced plants.However,after cold treatment,expression of these genes in CaDHN1-silenced plants was noticeably lower than in control plants.CaDHN1-silenced pepper plants showed obvious lower resisting abiotic stress.PVBG2307-CaMV35S-CaDHN1 overexpressed vector was constructed and transferred into Arabidopsis.The transgenic plants were treated with cold or salt stress.The results showed that after low temperature treatment,survival rate of CaDHN1 transgenic arabidopsis was significantly higher than that of wild type.Under salt stress,the wild-type plants grew significantly weaker than transgenic plants.After salt stress on the vertical medium,we found that the number of fibrous root and fresh weight of wild type plants was significantly lower than those of the transgenic plants.These results suggest that there is a positive correlation relationship between the expression of CaDHN1 gene and cold,salt resistances of plants.3.The conserved amino acid sequence of dehydrin(PF00257)was used to search in pepper genome database(PGP)(http://peppper genome.snu.ac.kr/)and protein database(http://peppersequence.genomics.cn/page/species/index.jsp)with the HMMER3.0 software.As a result,a total of 7 DHN candidate genes were identified in pepper,with four presented on chromosome 2;one on chromosome 4 and two on chromosome 8.Based on the number and order of Y-,S-and K-motifs,the seven DHNs were classified as YSK2-,Y3SK2-and SK3-type proteins.In order to analyze the phylogenetic relationships among pepper,Arabidopsis,rice,barley and tomato,we constructed an unrooted phylogenetic tree of dehydrin proteins.The DHNs could be divided into five groups: YnSKn-,SKn-,Kn-,YKand KS-type proteins?Interestingly,pepper only had YnSKn-and SKn-types.Tissue-specific expression results showed that the seven DHN genes were expressed in all tissues and might be involved in the growth and development of pepper.The gene expression profiles analysis suggested that most of the CaDHN genes were induced by various stresses(low temperature,salt and mannitol)and signaling molecules(ABA,SA and MeJA).