Expression Analysis And Functional Characterization Of A Heat Responsive Gene CaHMA-like In Pepper

High temperature is one of the most important abiotic factors limiting plants growth,development and geographical distribution.Pepper(Capsicum annuum L.),originating from tropical and sub-tropical regions in mid-south America,is very sensitive to high temperature.The optimal temperature for pepper growth ranges from 21°C to 27°C.When the temperature is higher than 32°C,pepper plants will suffer from heat stress,resulting in poor pollination and fertilization,and even blossom and fruit drops,which seriously restricts its seasonal and regional production.The difference in thermotolerance among different pepper varieties were observed,however,its molecular mechanism remains to understand.Based on cDNA-AFLP technology,we got one gene fragment in our previous research work,which was up-regulated in the thermotolerant pepper line but was down-regulated in the thermosensitive pepper line.By homology comparison,the gene(Capana03g002300)was found containing a HMA domain(heavy metal-associated domain),so we named this gene CaHMA-like.In this study,the thermotolerant line R9 and the thermosensitive line B6 were used to explore the gene structure and expression characteristics of CaHMA-like,and its function in forming heat tolerance of plants was also studied.The main research results are as follows:(1)The CDS region of CaHMA-like gene contains 327 bp nucleotide,coding 108 amino acids.Homology comparison showed that the homologous genes of CaHMA-like also existed in other plant species,such as tomato,potato,rice,maize.The result of sequence alignment showed that the the amino acids sequence of this gene in different plant species is highly conserved.There were three heat shock elements(HSEs)in CaHMA-like gene promoter region.(2)The subcellular location of CaHMA-like gene was the plasma membrane.The transcripts of CaHMA-like gene were detected in pepper roots,leaves and seedlings by qRT-PCR technique.Under the 40? heat stress(HS),the expression of Ca HMA-like reached a peak in 2 h in the thermotolerant line R9,while in the thermosensitive line B6,the expression was suppressed.The gene was also expressed weakly under low temperature in B6.At the Meantime,CaHMA-like was significantly induced by heavy metal Cu and Cd.Inaddition,it was upregulated by ABA,MeJA,SA and Put in different degrees.(3)To elucidate the function of the CaHMA-like gene,we constructed a prokaryotic expression vector for CaHMA-like.The heterologous expression of CaHMA-like could enhance the viability of Escherichia coli(E.coli)under heat stress.A VIGS technique was used to silence the CaHMA-like gene in pepper plants.After heat and heavy metal stress treatment,the tolerance of Ca HMA-like silenced leaves against heat and heavy metal stress were inhibited,and the chlorophyll content decreased.Plant overexpression vector of pVBG2307-GFP and pVBG2307-GFP-CaHMA-like were constructed and transferred into Arabidopsis thaliana ecotype Col-0.Transgenic A.thaliana constitutively overexpressing CaHMA-like gene displayed enhanced thermotolerance and survival rate compared with wild-type plants and plants with empty vector.