GUS基因在优化海带表达系统中的应用

【作者】 吴韵；

【导师】 秦松；

【作者基本信息】 中国科学院海洋研究所 ， 海洋生物学， 2001， 硕士

【摘要】 本文研究了如何应用GUS基因优化海带表达系统。首先研究了GUS在各种海带材料中的本底值，而后以GUS基因作为报告基因比较了几种基因工程常用启动子在海带中启动瞬间表达的效率。选择表达效率较高的真核藻类启动子与GUS基因融合，在海带中获得了稳定表达的结果。 分别用组织化学染色法和荧光分析法对海带雌配子体、雄配子体、孤雌生殖海带幼孢子体和二倍体海带孢子体等四种不同的材料的GUS本底进行了检测。组织染色的结果表明，各种海带材料在无菌海水中培养一个月后都没能检测到GUS本底。荧光分析法所得数据显示，这四种材料的GUS活性分别为3.4±0.3、5.2±0.8、14.4±1.8、12.5±1.4(pmol MUmg^-1 protein min^-1)，与高等植物类似，说明GUS基因可作为报告基因用于海带基因工程研究。 选用真核藻启动子FCP启动子、藻类病毒的AMT启动子、高等植物基因工程中常用的高效Ubi启动子和CaMV35S启动子，与GUS基因融合构建四种质粒，通过基因枪的方法转化孤雌生殖海带幼孢子体，比较GUS基因在海带中的瞬间表达量。实验结果表明，与CaMV35S和FCP融合的GUS基因在海带中表达效率较高。 采用基因枪的方法用FCP启动子-GUS基因转化海带雌配子体，通过诱导孤雌生殖获得孤雌生殖海带幼孢子体，用组织染色法检测到了GUS活性，并从海带的总DNA中扩增到了FCP启动子-GUS基因的特征条带，从而提示了FCP启动子能引导外源基因在海带中稳定表达。 为了找寻高效筛选元件进行了孤雌生殖海带对草丁膦的敏感性实验，用统计学的方法得出了草丁膦对不同长度孤雌海带的半致死剂量。研究发现海带全长0.5—1.6cm范围内，草丁膦的LD₅₀与海带长度不相关，同时发现浓度在2—5μg/ml狭小范围内的草丁膦对孤雌海带比高浓度具有更为明显的毒性反应。本文结果提示草丁膦抗性基因—bar基因有可能成为海带基因工程更为理想的选择标记，因为海带对草丁膦比对氯霉素和潮霉素更敏感，而后两者是目前采用的筛选压力。 本文结果为海带表达系统的优化，提供了有效的报告基因、启动子和选择标记元件。更多还原

【Abstract】 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.更多还原