Construction Of Suppression Subtractive CDNA Library Of Phelldendron Amurense Under Drought Stress And Metallothionein Ⅱ Gene Cloning

Phellodendron amurense is the level two protection plant in the country; their bark which is rich in alkaloids, such as berberine, has great medicinal value. P. amurense is an ancient tropical relict plant. Because it has gone through the heat from the tertiary to a series of cold climate change; it has higher adaptability under abiotic stress such as high temperature, drought, and cold.To study the gene expression of P. amurense under drought stress, in the present study, suppression subtractive hybridization (SSH) was employed to construct cDNA subtracted library using cDNA from P. amurense seedlings under drought stress as tester and cDNA from normal growth plant as driver. In the library, the rate of recombination was 95 %, the size of inserts was 300～800 bp. 265 new genes were obtained by DNA sequencing 816 positive clones picked randomly, and partitioned to 16 classes after homological analysis of Blast against NT, NR, SWISSPROT, KEGG database. 44 drought stress associated genes, such as heat shock protein cognate 70, dehydration responsive protein 22, universal stress protein, metallothioneinⅡ, late embryogenesis abundant protein, etc. were obtained, which makes 16.6% of the overall genes. These genes included osmotic regulator, signal component regulatory protein and anfioxidant enzyme. 160 sequences had been accepted by GenBank and EST database.Metallothionein gene (MY) was selected from the library of further studies. First, a reverse MT gene-specific primer was designed according to the known sequences, and 5' end sequences was achieved by 5' rapid amplification of cDNA ends (RLM-RACE) technology. After 5' RACE products been cloned into pGEM-T vector, transformed to Top 10 competent cells and sequenced, MT full-length sequence was obtained. PaMT2 mRNA was 852bp length, and ORF analysis showed that it has start codon ATG in 112bp, termination codon TGA in 315bp with 240bp open reading frame putatively encoding 79 amino acids. After Blast analysis, it was found that PaMT2 has 80% homology with citrus, genes in the DNA sequence, and homologous regional covers 100%; its amino acid sequence has 70% homology in the 55 amino acid sequences with Salix MT2A protein. The result shows that PaMT2 has significant homology with known MT protein, and could be predicted with similar function with known MT gene.In summary, we employed SSH technology to construct a subtractive cDNA library of P. amurense seedlings under drought stress,analyses the differentially expressed genes through bioinformatics analysis related to drought stress,cloned the full sequence Of metallothionein gene．It is helpful for moleCUlaur biology research of P．amurense．Meanwhile,a wealth Of drought-related genes will facilitate plant genetic engineering．...