Cloning And Characterization Of The Gene Encoding Metallothionein In Celery

The normal growth and development of plants depend on the suitable external conditions,which seriously affect the yield and quality of crops.On the other hand,with the development of China’s industry and the accelerated process of urbanization,industrial wastewater,vehicle emissions,urban waste discharged into rivers,air,soil,finally accumulated a large number of heavy metals.The presence of heavy metals in air,soil and water has a serious impact on biological organisms,and its biological enrichment in the food chain is very dangerous for human being.Celery（Apium graveolens L.）belongs to the Apium of Apiaceae.It is one of the most important vegetables which are widely cultivated in China and even worldwide.The abiotic stresses and heavy metal pollution cause great harm to the growth of celery.In our study,celery was taken as the experimental material.We cloned the Agmt2 gene,which the encoded AgMT2 protein is thought to improve the anti adversity ability（especially resistant to heavy metal stress）.Then the expression profile was performed under different stresses for preliminary verification of function of the gene.We also purified AgMT2 protein,and obtained the transgenetic T3 plant of Arabidopsis thaliana,focusing on the response mechanism of heavy metal stress.The main results are as follows:1.The Agmt2 genes were cloned from two celery cultivars ’Liuhe Huangxinqin’ from Nanjing and ’Ventura’ from USA,respectively.The Agmt2 gene showed a length of 231 bp open reading frame（ORF）and encoded with 76 amino acid residues,with two introns of 385 bp.Sequence analysis indicated that AgMT2 belonged to the plant Metallothio 2 superfamily.The predicted molecular weight and pI value of the AgMT2 proteinwere 7.5 kDa and 4.95,respectively.There was a 99.56 similarity in nucleotide acid site in ’Liuhe Huangxinqin’ compared to ’Ventura’.The amino acid sequences of AgMT2 in two cultivars were completely identical.Multiple sequence alignment showed that domain of MT2-like proteins was highly conserved.AgMT2 further demonstrated a very close evolutionary relationship with MT2 from Nicotiana tomentosiformis,N.sylvestris and N.Plumbaginifolia in Solanaceae.QuantitativeRT-PCR（qRT-PCR）revealed that Agmt2 gene expression profiles exhibited tissue specificity in celery.Agmt2 gene up-regulated more significantly in ’Ventura’ than in ’Liuhe Huangxinqin’ under eight metal ions(Co²⁺,Ni²⁺,Fe²⁺,Mg²⁺,Mn²⁺,Ca²⁺,Cu²⁺,Zn²⁺)and four abiotic stress treatments（heat,cold,salt and drought）.2.pET-30a（+）-AgMT2 expression protein vector was successfully constructed and a large number of cultivation of Escherichia coli cells was induced in our study.The results showed that the AgMT2 protein concentration was 0.42 mg/mL,and SDS-PAGE showed the molecular weight was about 20.1 kDa.In addition,the cells carrying AgMT2 expression and the blank cells were treated with heavy metal concentrations in the culture medium to make the identification of AgMT2 metal tolerance.The results showed that under the stress of Cd²⁺,Cu²⁺ and Zn²⁺,the growing trend of AgMT2-expressed E.coli cells was stronger than the control and the ability of carrying Zn²⁺ and Cd²⁺ was stronger than that of Cu²⁺.It suggested that AgMT2 is a functional fusion protein which can be effectively expressed in E.coli with specific binding of metal iron.3.Agmt2-transgenic T3 A.thaliana was obtained using floral dip transformation.Identification of transgenic A.thaliana was performed by different methods,such as culture medium selection,GUS staining,PCR verification,phenotypic observation and other methods.In addition,in order to confirm the two functions（heavy metal detoxification and free radical scavenging abilities）of AgMT2,A.thaliana was treated with heavy metal irons,irrigated with 100 μM of Cd²⁺,Cu²⁺,Zn²⁺ and distilled water for 15 days,respectively.The results showed that the GSH content,total chlorophyll content,SOD and POD activity in transgenic A.thaliana plants were significantly higher than those in control plants under heavy metal stress.It indicated that after heavy metal stress,Agmt2 transgenic plants showed more significant heavy metal detoxification and free radical scavenging abilitied than the control plants.In conclusion,this study provides important information for the study of genes related to adversity in important vegetables of Apiaceae,laying the basis for the new varieties’breeding of celery.