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Metallothionein(MT) are low-molecular-weight proteins widely distributed in the biosphere,which rich in Cys and can combine heavy metals.MT play an important role in improving heavy metal resistance in plants and lifting toxic heavy metals such as Cu,Zn,Pb,Ag,Hg,cd.The majority of MT in the C-and N-side has two Cys-rich domains.One of the mercapto-can chelate heavy metal ions and the formation of the complex non-toxic or low toxicity,also with heavy metals, such as stress-induced·OH generated by redox reactions occur,thereby reducing oxidative damage. A large number of studies shown that the Metallothionein can plant through its large number of Cys residues chelation to remove heavy metals and reactive oxygen species,so that plants from oxidative damage,many of metallothionein encoding genes have cloned from plants in recent years,, and in the study of gene expression patterns,tissue specificity and genetic structure,such as promoter,intron at chromosome positioning have made some progress,but the study of its function is still in its early stages.From the laboratory to build the wax plum cDNA library using EST technology from cloned Chimonanthus metallothionein gene.Means of biological information through the study of gene sequence analysis and function prediction.In order to further verify the possible functions.We construct a plant expression vector,and get same transgenic plants by the transformation of petunia.We did a preliminary analycis to these transgenenic plants.The main results were as follows:1 Analysis of Metallothionein and its encoded protein bioinformaticsDNAStar,DNAMAN cloning is analysis showed that the length of the cDNA sequence 1083bp, one of ORF240bp,encoding 80 amino acids,the theoretical molecular weight protein 7.76kDa, isoelectric point of 4.67.BLASTP at NCBI than up to a total of 21 have a higher amino acid sequence and its homology,ranked first in the Danshen putative metallothionin 2a protein,the similarity of 96.7%.2.Construction of the expression vector Metallothionein gene is located on Library pTriplEx2 cloning vector,activation of bacilli and SfiI restriction endonuclease digestion,at the same time added a SfiI restriction enzyme site of the ring pMD-18TM,and the vector fragment Recycling and connect,after digestion and PCR verification,cloning vector was successfully constructed;XbaI and SmaI,respectively,and then double-enzyme cloning vector and expression vector pC2301,Recycling and the expression vector fragment and connect by digestion and PCR to verify successful gene construct containing the expression vector.3.Nodule Agrobacterium-mediated genetic transformation and Km-resistant petunia plants was Nodule using Agrobacterium-mediated leaf disc transformation method,the exogenous gene into petunia,have been 28 Km~+ renewable plants.4.Transgenic petunia identification of molecular biologyFirst,screening of the resistant plants for GUS histochemical detection,initial screening and to identify transformed plants.And on the GUS staining positive PCR detection of plants again.The results showed that the GUS-positive lines showed a positive control with the same specific band, non the transformed plants did not get the specific band,which is further evidence of the foreign gene had been integrated into the petunia genome.5 Metal stress test in the transgenic plantsDoing heavy metal stress on a transgenic positive strains and control strains,Chosen as the stress of heavy metal copper,Plants were sprayed with 100mmol/l copper,after 10 days,the transgenic plants with the control strains have significant differences Preliminary descriptions of Metallothionein wax may have the function to against of heavy metals.
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