| CaMV35S promoter is widely used in plant genetic engineering. Due to its constitutive expression pattern, foreign gene driven by CaMV35S promoter can express throughout all tissues of plant at every developmental stage, at a high metabolic expense to host plant. Too many resources have been distributed for foreign products expression in untargeted tissues, which result in great waste of both materials and energy. Therefore, researches on specific promoters, ideal alternatives to constitutive promoters, have become great important contents in plant genetic engineering. Driven by specific promoters, foreign genes are directed to express at special times, in given places of plant or even in desired quantities. In this study, we have isolated and identified reproductive organ-specific promoter from cotton, which can provide potential controlling elements for efficient expression of foreign gene in cotton reproductive organ. Ultimately, by means of genetic engineering, our research will provide useful molecular tools for improving characters of reproductive organ in cotton.A novel technique, isocaudarners inverse PCR (II-PCR), has been established for chromosome walking. With availability of a fragment of target DNA sequence, flanking unknown DNA sequence was obtained rapidly by successive chromosome walk using Il-PCR method.An advanced method has also been developed for rapid isolation of transcription initiation side (RITIS) of target gene. Using RITIS, full-length cDNA sequence of target gene was obtained quickly. Upon obtainment of cDNA sequence, initiator was determined and accurate position of promoter was also localized.Using techniques above, two closely linked genes, nodulin-like and ADP-ribosylation factor 1 (arfl), their full-length cDNA sequences and promoters have been isolated from cotton. nodulin-like gene is 2353bp in size, including 5 introns and 6 exons. Its full-length cDNA is 1480bp, containing an 1125-bp ORF structure which encodes 375 amino acids. Northern blot analysis showed that nodulin-like gene could express preferentially in fibre and reproductive organs of cotton, such as bud, flower and boll. Arfl gene is 4360bp, including 6 introns and 7 exons. Northern blot result indicated that arfl gene could express predominantly in cotton reproductive organs. Promoter of arfl gene is 1629bp in size.Based on structural characters of promoters and distributions of controlling elements in promoter sequences of nodulin-like and arfl genes, three promoter truncations have been designed for each of them. They were fused to gus gene to produce 6 plant expression vectors, pBIN1GUS, PBIN2GUS, PBIN3GUS, pBIAlGUS, PBIA2GUS and pBIA3GUS, respectively. The 6 plant expression vectors have been introduced into Gossypium hirsutum Y18 via pollen tube method. Then, about 8.44kg seeds were collected from transformed plants of T0 generation. Screened by kanamycin and further identified by PCR amplification and GUS staining, 47 transgenic plants were obtained, among which 13 were transformed by pBINlGUS, 6 were from pBIN2GUS, 4 were produced by pBIN3GUS, 14 were generated from pBIAlGUS, 6 were obtained from pBIA2GUSand the rest 4 plants were selected from pBIA3GUS transformers.Further works on both GUS staining and fluorescent quantitative analysis have been performed with progenies of above transgenic cottons. Results showed that (1) core promoter of nodulin-like gene was localized between +109 and -221bp, which played crucial role in endowing the promoter with reproductive organ-specific character; (2) regulation elements which could enhance expressional strength of nodulin-like promoter in cotton reproductive organ were distributed between -221 and -677bp; (3) sequence of -677 to -1084bp could further enhance expression of gus gene. In promoter of arfl gene, elements in region of+133 to -277bp could direct specific expression of gus gene in anther, fibre and boll shell of cotton; elements in fragment of+133 to +748bp could determine expression of the promoter in certain tissues, such as ovary, Stigma, Fil... |
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