Cloning And Functional Activity Analysis Of The Promoter Of Metallothionein From Halostachys Caspica

Trace metal ions are essential,but excessive amounts can cause serious poisoning to organisms.Metallothionein(MT)is a small molecule substance expressed in various organisms,which can bind a large number of metal ions through abundant metal–thiolate cluster,participate in the transport of metal ions in vivo,regulate the balance of metal ions in the body,and relieve the toxicity of heavy metals and scavenge free radicals,ect.Meanwhile,the rich and varied metallothioneins in plants are mainly controlled by the regulatory elements on the promoter.This study was based on the mRNA sequence of the metallothionein gene already obtained in the previous period.Firstly,the full-length sequence of the metallothionein gene was cloned and the total length was 2334 bp.It consists of three exons and two introns.Respectively,the lengths of the three exons are 64 bp,79 bp,and 94 bp.The length of each intron is 836 bp and 1261 bp.Then three specific primers were designed based on the first intron sequence of 836 bp for cloning to obtain the metallothione promoter of Halostachys caspica by chromosome walking method.The length of the promoter fragment is 842 bp.On-line analysis of the promoter sequence reveal that the promoter contains the core sequences contained in the general promoters: TATA-box and CAAT-box,it also contains a variety of cis-regulatory elements: light-responsive elements,drought-responsive element,Methyl jasmonate-responsive element,salicylic acid responsive element,endosperm-specific expression regulatory elements,meristem-specific activation response element,etc.By comparing with the core sequence of reported metalloid response elements,two copper response elements(CURE)and four metalloid-responsive elements different from other plants were found on the promoter sequence,the core sequence respectively,5'-TGCAACG-3'(contains 2),5'-TGCAACT-3',5'-GAGAGAA-3'.It is speculated that the promoter is involved in the fine regulation of gene expression under various stress conditions.In order to verify the promoter activity and the key regions affecting promoter activity,the promoters of different lengths of HcMT were designed to replace the CaMV 35 S constitutive promoter in the plant expression vector pCAMBIA1304 before the reporter gene GUS,based on previous studies on the bioinformatics of HcMT promoter.GUS histochemical staining of N.benthamiana leaves with different lengths of promoters transiently showed a dark blue precipitate,indicating that promoters of different lengths of the HcMT gene have the activity of initiating downstream gene expression.Quantitative analysis of the enzyme activity of GUS produced by promoters of different lengths under non-stress showed that the GUS showed the highest enzyme activity under the effect of the CaMV 35 S promoter;other GUS enzymes under different lengths of HcMT promoter have a certain activity but no significant difference between each other(P>0.05).The P518 promoter had the highest GUS enzyme activity in all promoters of the metallothionein fragment,which was 28.27 pmol/h/g;while the GUS enzyme activity initiated by longer fragment promoter(P842 and P654)was only 45.10% and 34.31% of the P518 promoter.It shows that the length of the promoter is not related to the expression level of the gene,and it is presumed that there is a negative regulatory element between the-842bp~-518 bp fragment of the promoter,leading to the gene expression being blocked or inhibited.Treatment of transiently injected N.benthamiana leaves with metal ions and saline solution showed that the GUS enzyme activity under the P842 promoter was significantly higher(P<0.05)than other promoters under 100 ?M copper ion stress.The results showed that there was a copper response element between the promoters of metallothionein promoters from-842 to-654 bp.Under 300 ?M zinc ion stress,the promoter activities of P518 and P654 were significantly higher than those of P329 and P416 promoters.It was speculated that promoters may contain zinc ion regulatory elements between-654 to-518 bp.In addition,it has been found that the promoter can respond to the salt environment in a short region(P329).