| In the plant genetic engineering, high active constitute promoters have been used to make foreign gene express efficiently in many transgenic plants. However, the substance and energy in cell have been burdened by continual expression of foreign genes during all time and in all space, the development of transgenic plants would been influenced. By using tissue-specific promoters, the foreign gene expression can be effectively driven in specific tissues and organs, and the negative effects of the expression of foreign genes to transgenic plants can be avoided.In this paper, a series of 5'-flanking deletion of citrin gene promoter was fused to a B-glucuronidase gene, which was used as the reporter gene. By Agrobacterium tumefaciens- mediated, the fused genes were transformed into cells of Nicotiana tabacum L. Some transgenic tobacco plants demonstrated by molecular analysis were obtained and used to investigate the mode of reporter gene expression in transgenic plants. This work may not only lay a foundation for studying the relationship between the structure of citrin gene promoter and its function, but also provide a base for foreign gene orientational expression in transgenic plants by citrin gene promoter. The main studies and results were reported as follows:1. Construction of the plant expression vectorsA 1.9kb fragment of the 5'-upstream region of the salt-soluble globulin seed storage protein gene (citrin) was cloned by PCR from the citrus superior cultivar "Jincheng orange"(Citrus sinensis Osbeck). The results from sequenc analysis predicted that the 5'-upstream region of citrin may be a seed-specific expression promoter. Five plant expression vectors containing gus gene driven by fragments of citrin gene promoter at different length were constructed, which were pA-gus pB-gus pC-gus pD-gus, pN-gus.2. Obtainment of transgenic tobaccoThe leaves of in vitro seedlings of Nicotiana tabacum L. cv. Wisconsin 38 were used as the test material. The protocol for in vitro genetic transformation in tobacco was usedto transfer different fused gene into tobacco. Some transgenic plants demonstrated by molecular analysis were obtained.3. Analysis of the characters of transgenic tobacco plantsThe pptr plantlets were classified into different clones, Clone A, Clone B, Clone C Clone D and Clone N, according to vectors containing different fused gene. The regeneration plantlets were transferred into soil. Clone A Clone B Clone C and Clone D grew normally, but Clone N was short and more ramifying, its leaves were narrow, and its vegetation period was longer than the Control. The results indicated that it was caused by introduction and insert site of foreign gene.4. Analysis of the expression position and level of reporter gene in transgenic tobacco plantsGUS activity of histochemical staining in different organs, such as root, stem, leaf, seed and ovule, had been tested The result showed that: in Clone A, the GUS activity could be tested in ovules and seeds , and slight in root of single; in Clone B and C, GUS activity was tested in leaves and seeds; in Clone Dand N, there was no GUS activity being tested in any organs.Therefore, it was confered that citrin gene promoter could effectively drive foreign gene specific expression in seed and ovule, and it was seed-specific expression promoter.The results of GUS fluorescence analysis showed that there was different in organs in the GUS activity. The activity in root and stem was very low. That in leaf introduced pB-gus and pC-gus promoter was significantly high, which were 2.7 and 3.6 times as high as that of 35S promoter. In seed, the activity of transgenic plantlets containing pA-gus, pB-gus and pC-gus promoter was 4.5, 4.3 and 3.5 times as high as that of 35S promoter. The above data suggested that the citrin gene promoter was a high active seed-specific promoter.5. Bioinformatics analysis of citrin gene promoterAccording to bioinformatics theory, citrin gene promoter sequence was compared and analyzed with internet database. It not only had some typical e...
|
PDF Full Text Request