Cloning And Sequence Analysis Of Strictosidine Synthase CDNA And Its Promoter In Brassica Napus

A partial STR-like cDNA section sequence (about 300bp, named G21) in Brassia napus was discovered in a suppression subtractive library constructed with early flower buds (length less than 0.2mm) of an apetalous line and its wild type. According to the partially known sequence, the gene-specific primers were designed to amplify the 5' and 3' cDNA ends, respectively. The full-length cDNA of this STR-like gene, named BnSTR, was cloned through RACE approach. Sequence analysis showed that BnSTR was 1408 bp long and contained an ORF from the 20^th bp to the 1291^th bp. cDNA sequence comparison performing by Blast Search showed that BnSTR had high homology with one member of STR-like family, A. thaliana STR (88%), indicating that BnSTR belonged to STR-like family. BnSTR showed low homology to STRs from other plant, animal and bacterium species except AtSTR. Although multiple sequence alignment of BnSTR with some STR-family members from plants, animals and bacterium revealed low conservation, most of the protein sites existed in the conservational region.Semi-quantitative RT-PCR and Northern blot analyses showed that compared with that in wild type, the expression of BnSTR in the early flower buds of apetalous mutant Apet33-10 declined obviously, revealing that the function of BnSTR could be associated with petal primordial initiation and growth.Promoter was an important cis-acting element of gene expression regulation. In order to understand the regulative mechanism of transcription of STR, Primer8, Primer7 and Primer3 were selected from a set of 10-mer primers to perform three-round single primer PCR reaction by the template of B. napus genome DNA. Afterward, a 986 bp target fragment was cloned from the third round PCR products. And the 3' end of the cloned DNA fragment was the partial coding region of the corresponding BnSTR gene. This 691 bp 5' flanking region of BnSTR gene (named BnSTR-pro, GeneBank accession number: EF566876) had some cis-acting elements including two TATA-box, two CAAT-box, two ARR1AT-box, two CACTFTPPCA-box, three DOFCOREZM motifs, three EBOXBNNAPA/ MYCCONSENSUSAT motifs, one POLLEN1LELAT52 motif, one GAREAT motif, one GATA-box, one NODCON1GM/OSE1ROOTNODULE motif, one ACGTATERD1motif, one REALPHALGLHCB21 motif, one GTGANTG10motif, one ROOTMOTIFTAPOX1 motif, one SEF4MOTIFGM7S motif, one SURECOREATSULTR motif, one ACGTT-box, one MYB1AT-box, one GT1CONSENSUS motif. All of these cis-acting elements had relation to activating gene transcription of plant, response to dehydration, phytochrome regulation, regulator in infected cells, GA induction. Some of them are organ-specific elements. BnSTR-pro was 62.87% homologous with the promoter of STR from A. thaliana.