Molecular cloning of the complete 11S seed storage protein gene of Coffea arabica and promoter analysis in transgenic tobacco plants
✍ Scribed by Pierre Marraccini; Alain Deshayes; Vincent Pétiard; William John Rogers
- Publisher
- Elsevier Science
- Year
- 1999
- Tongue
- English
- Weight
- 414 KB
- Volume
- 37
- Category
- Article
- ISSN
- 0981-9428
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✦ Synopsis
In this paper, we present the complete nucleotide sequence of the csp1 gene from Coffea arabica coding for the 11S-globulin seed storage protein. To investigate the sequences responsible for the regulated expression of this seed-specific coffee storage protein gene, about 1 kb of the 5'-upstream region from the csp1 gene was isolated using inverse polymerase chain reaction (IPCR) and then sequenced. Several DNA boxes were found in this coffee sequence that had similarity to those previously identified as being essential for grain (endosperm) specific expression in other plants. To study the ability of this sequence to direct grain-specific expression, the whole fragment, as well as a series of 5' deletions, was fused to the reporter gene β-glucuronidase (uidA) and analysed in transgenic Nicotiana tabacum plants. GUS measurements showed that all the deletions of the csp1 promoter directed the expression of the reporter gene in tobacco grain but not in the other tissues examined. GUS activities also revealed that the csp1 promoter constructs function as very strong promoters by comparison to the strength of the cauliflower mosaic virus (CaMV) 35S promoter. Therefore, this 11S promoter could represent a useful tool to change the expression of targeted genes in the grain of transgenic coffee plants. © Elsevier, Paris 11S storage protein / Coffea arabica / endosperm-specific promoter / coffee genetic engineering CaMV, cauliflower mosaic virus / csp1, coffee storage protein gene / CSPD, disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 37 ]decan}-4-yl) phenyl phosphate / GUS, β-glucuronidase / IPCR, inverse polymerase chain reaction / Ta, annealing temperature / TMAC, tetramethyl ammonium chloride / UTR, untranslated region / WAF, weeks after flowering