| Promoter is a special DNA sequence in the 5’end of gene, which can bind with RNA polymerase and initiate the transcription process.According to their action style and function, promoters applied in plants can be divided into three categories:constitutive promoter, tissue-specific promoter, and inducible promoter. Tissue-specific promoter can make foreign gene highly express in specific tissues or organs. Fruit specific promoter is an important tissue-specific promoter, which can control the foreign gene expression in transgenic plants. Cloning and study of fruit-specific promoter can lay the foundation for the improvement of fruit quality.In our previous research related to postharvest banana fruit ripening, we have isolated two fruit specificly and highly expressed genes:granule-bound starch synthase gene (GBSS) and banana lectin gene (BanLec). A length of 670bp banana lectin gene promoter had been isolated and proved to be fruit-specific, highly expressed and ABA induced. In order to further study its function, we obtained a 344bp promoter sequence by using Genome Walker method according to BanLec promoter sequence (670bp). Altogether we obtained 1014bp and named BanLec-1. The result of bioinformatics analysis showed that there were three basic promoter regions in the 423-473,556-606 and 834-884. The transcriptional start site (+1) was base A and located at 111bp of 5’-upstream the initial codon ATG. BanLec-1 contained several ABA response elements. Compared with BanLec gene (670bp), BanLec-1 (1014bp) had more tissue-specific expression controlling elements (CAATBOX1 at -648,-548), cold regulatory elements (MYCCONSENSUSAT at -690,-684), injury regulatory elements (WBOXNTERF3 at -871,-743), and ABA and other hormone-related response elements (DPBFCOREDCDC3 at -838, ABRE at -818). We used BanLec-1 promoter to replace the CaMV 35S promoter in pCAMBIA1304 vector to construct vector 1304-L and then transformed it to banana root, leaf and fruit by paricle bombardment. The result of detection of GFP and GUS activity showed that it was also a fruit specific promoter, and had higher activity than BanLec promoter and CaMV 35S.Granule bound starch synthase gene was obtained through the fruits of SSH library by DNA microarray analysis. Northern blot analysis showed that the Granule bound starch synthase gene was fruit specific and highly expressed. So we believed the promoter of this gene should be fruit specific and high expressed.According to starch synthase gene sequence, we obtained a length of 606bp promoter sequence by using TAIL-PCR method, named BR47-1. The result of bioinformatics analysis showed that there were two core promoter regions from 93 to 138 and from 182 to 227. The transcriptional start site (+1) of the BanLec-1 gene promoter was base C and located at 420bp of 5’-upstream the initial codon ATG The sequence with the basic transcriptional promoter elements:20 TATA-boxes,10 CAAT-boxes,3 G-Boxes. TATA-boxes located at -159,-523,-546 and -570, etc. CAAT-boxes located at -93,-128,-224,-284 and -532, etc. G-Boxes located in the -336,-541,-603. This promoter also contained CCGTCC-box, SORLIP5AT and other tissue-specific elements, which were located at -283,-46, respectively. Three ABA cis-acting elements ABRE are located at -257,-338 and -541. Two jasmonic acid responsed cis-regulatory elements: CGTCA-motif located at -412 and TGACG-motif located at -656. Two gibberellin responsed elements:P-box located at -84 and TATC-box located at -193. A saluylic acid responsed cis-regulatory element named TCA-element was located at the -549. Other two sugar-related components CGACGOSAM Y3 located at -283, WBOXHVISO1 located at -636 and -491.We used BR47-1 promoter to replace the CaMV 35S promoter in pCAMBIA1304 vector to construct vector p1304BR47 and then transformed it to root, leaf and fruit by paricle bombardment. The result of detection of GFP and GUS activity showed that this promoter was a fruit specific promoter and had higher activity than CaMV 35S. |
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