Of Gus Gene In The Optimization Seaweed Expression System

The application of GUS gene to optimize the expression system of Laminaria japonica has been studied in this thesis. Firstly, the background values of GUS gene product were investigated in several types of kelp materials, then GUS gene was used as a reporter gene to compare the driving efficiency of four common used promoters from land plants or algae. A high efficient eukaryotic algae source promoter was selected to drive GUS gene to get stable expression. Both histochemical and qualitative flucrometric GUS assays have been carried out in female gametophytes, male gametophytes, young parthenogenetic sporophytes and diploid sporophytes of L. japonica. The results showed that no background was detected by using histochemical GUS assays in these kelp materials after cultured in sterilized seawater for 1 month. The background values detected by qualitative fluorometric GUS assays were 3.4監.3~ 5.2監.8~ 14.4?1.8~ 12.5 ?1.4 (pmol 4MU mg?protein min1) in these 4 types of materials, respectively. The background value was similar to that in high plants, which suggests that GUS gene can be used as a reporter gene in L. japonica. FCP promoter (from eukaryotic algae), AMT promoter (from algae virus), and two common promoters used in land plant genetic engineering, i.e. Ubi promoter and CaMV3SS promoter, were used to drive GUS gene. Young parthenogenetic sporophytes were used as gene recipients and biolistic bombardment was used as method. Result of fluorometric assay indicated that CaM V35S and FCP promoter were more efficient in kelp than the other two promoters. The FCP promoter-GUS gene cassette was introduced into female gametophytes using biolistic bombardment and young sporophytes were induced by inducing pathenogenesis. GUS activity was significantly detected using histochemical assay and the specific band of FCP promoter-GUS gene cassette was amplified by using PCR, which implicates that the foreign gene has been integrated and FCP promoter can drive stable expression in kelp. The sensitivity of parthenogenetic sporophytes to basta was studied in order to screen for higher efficient selective marker in kelp. LD50 of basta correlated to different algal length was calculated using statistic method. Result showed that within the length from 0.5 to 1.6 cm, LD5O of basta had no relationship with length. It was interesting to find that within a very narrow content range(2-5 ii g/ml), the toxic effect was even more apparent than much higher contents. Since Lam maria was more sensitive to basta than to chloramphenical and hygromycin, bar gene encoding resistance to basta has the potential to be more efficient selective marker.