Isolation And Analysis Of CPCysA Gene From Wintersweet(Chimonanthus Praecox) And Construction Of The Expression Vector

Utilization of sulfur in soil includes processes of sulfate taken up into roots, transportation, assimilation, distribution etc, These metabolic processes are concerned with and regulated by series of enzyme and protein. Among them,the last reaction of sulfur incorporated into cysteine is catalyzed by O-acetylserine (thiol) lyase (OASTL) from O-acetylserine (OAS) and sulphide. Recent research in plant shows that the sulfate assimilation is closely connected to the mechanism of response to stress of heavy metal such as cadmium(Cd), Research of OASTL which is the key enzyme of sulfate assimilation process in plant is important to fully understand all kinds of the regulating mechanism in the process of sulfur assimilation, furthermore, OASTL overexpression in plant is practically useful, since they can potentially be applied for the phytoremediation of soil and water or might have an environmental advantage in some conditions, for example, they can be more tolerant to stress such as heavy metals,H₂S and SO₂.In this paper, the full-length cDNA of CPCysA was isolated from Chimonanthus praecox which is rare and traditional native tree, adopting the method of randomly sequencing the ESTs of full-length cDNA library. Moreover, this paper analysed the sequence and expression in different tissues, and constructed a plant expression vector to verify the function of OASTL overexpression in plant, The main results are as follows:1.Based on the preliminary analysis of ESTs sequenced from flower organ cDNA library of Chimonanthus praecox , selected ESTs to sequence reversely, at last isolated a full-length cDNA of OASTL-A gene, nominated as CPCysA. (GeneBank accession number: DQ639764)2. The sequence analysis shows that the full-length cDNA of CPCysA is 1294bp,contains an 981bp open reading frame (ORF) from 103bp to 1084bp, which encodes a predicted protein of 326 amino acids with molecular weight of 34279Da, 103bp 5'non-coding region and 210bp 3' non-coding region including repeated polyadenylation signal sequence AATAA and polyA tail.3. BLAST analysis showed that the amino acid sequence of CPCysA gene was significantly...