| Abstract: Tobacco is the important economic crops of leaf, as a model transgenic plant has played an important role in the development of biotechnology. In order to effectively start the foreign gene specifically expressed in tobacco leaf and improveme tobacco quality,we cloned a promoter rbcS from tabacco leaves,and transformed the SLC1-1 gene (encoding sn-2 Acyltransferase) to tobacco, analysis of transgenic tobacco crude fat content of the leaves. The results are as follows:1,According to published under the NCBI tobacco ribulose 1,5 - diphosphate oxygenase / carboxylase (X02353) primers were designed using RT-PCR from tobacco (Nicotiana tabacum) cultivar NC89 genome isolated rbcS promoter sequence, amplified fragment length 979bp. The sequence analysis showed that the promoter contains multiple conserved sequences by PlantCARE, and the more close to the start codon sequence of the more conservative regions, the promoter sequences contain not only the necessary promoter CAAT box, TATA box, but also with G-box, I -box, box I, box II, ATC-motif and other 12 kinds components of light response element, in addition, also contains an ABA homeopathic acting elements (ABRE), two MeJA responsive element, an ethylene response element and a tobacco-specific sequences of the 5'-tcttacacgtggcacctc-3'. The sequense blast with GenBank: X02353 showed that cloned rbcS promoter sequence from tobacco NC89 in the TATA-box, CAAT-box, G-box and other important functional areas found no change in base, but in other regions of the existence of seven bases mutation, including four base substitutions and 3 bp insertion, corresponding to 99.08% homology region.2,Constructed plant expression vector rbcS: pBI121, to 35S: pBI121 as a positive control transformed tobacco plants were GUS-positive staining obtained, for functional verification. The results showed that the tobacco rbcS promoter is a leaf tissue-specific promoter, and the leaves start gene expression than CaMV35S strong start to the foreign gene in transgenic tobacco leaves in the high expression provides a new tool for the follow-up gene can occur in tobacco leaf tissue-specific and light-induced expression of the foundation.3,We were constructed 35S: SLC1-1 and rbcS: SLC1-1 gene expression vector, transformed into tobacco, and oil of transgenic tobacco was tested. The results showed that the average oil content of transgenic tobacco increased 0.57 percentage, the highest increase compared with the control by 1.3 percentage, Note the increase oil content in tobacco is feasible by transform SLC1-1 gene.4,Detection of transgenic tobacco phenotype variation, by pollen germination tests, experiments and frozen sections of floral organs flanking sequence analysis, a preliminary analysis of transgenic tobacco caused by mutation causes organ. Mutant phenotype expression in the T0 generation of transgenic tobaccoes and found two leaves and floral organs of the mutant plants, leaves became small and thin, the main vein in the fork tip action, extends to both sides. An inflorescence did not open, inflorescence column shorter than usual, no flower formation; another small number of flowering plants, with flowers form only, without calyx, corolla missing the top part of the observed style and filaments found in some style and filaments growing together, there are only four filaments, there are one to several growth is not normal in 5 Filaments. Normal pollen germination and pollen viability test showed no difference,slices was observed when flower was small occurred withered. Flanking sequence analysis of the SLC-9 plants may be hexokinase gene or alata 48A gene; SLC-10 plant flanking sequences homologous to the gene sequences not found, insert it into the position that there may be non-coding region genome sequence. |
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