摘要
于1994年12月-1995年4月，在山东荣城海带育苗场采集海带幼孢子体，用基因枪法将氯霉素乙酰转移酶基因（CATgene，cat）导入海带幼池子体中，48h后检测到瞬间表达。转化后恢复培养一周，以致死剂量氯霉素筛选。两周后对照组全部死亡，转化组万株海带中有11株存活。转化三个月后经CAT酶联免疫（CATELISA分析，在11株存活海带中有2株检测到CAT基因的表达。结果初步证明了CAT基因是海带基因工程的有效选择标记；同时也证明SV40启动于是适用于大型褐藻的启动子元件。
In order to study the expression of foreign genes in Laminaria japonica and todetermine suitable markers, experiments were undertaken from December, l994 to April, l995. Thematerials used were young sporophytes of L. japonica. They were provided by Rongcheng SeedingFarm in December, 1994. The methods are as follows:1. Sterilization and culture of materials. Upping materials in autoclaved seawater (2O-3Omin),then transfer to 1.5% K1 (H;/ L, 20- 30min), and at last autoclaved water (2o-3omin). Finallymaterials were recovered in autoclaved seawater for 2omin. The culture condition was 10h / l4h(ratio of light period to darkness Period), 5OUE / (m2· s) and (l0.0 0.5)t.2. 1ntroduchon of CAT gene (cat), pCAT-control plasmid (SV4O promoterical-SV4O enhancer,4 75obP), by a Biolistic PDS-1000/ He Particle Delivery System. 5 sporophytes were used as onesample and were bombarded twice. The bombardment Parameters were' vacuum 6.l X 1O3Pa, rupturedisk 1100 psi and the distance between samples and macrocarrier holder 6cm.3. Transient expression of cal. One bombarded sample (5 sporophytes, bombarded with DNA),negative control (5 sporophytes, bombarded without DNA) and blank control (5 sporophytes,non-bombarded) were detected by using CAT ELISA Xit (Boehringer forkeim GmbH Dermany)after 48h culture in darkness. The amount of CAT was counted by comparing OD at 4o5nm withCAT standard sample.4. Selection and stable expression of cal. After bombardment sporophytes underwent a week ofrecovery period in non-selechve medium and then selected in liquld medium contrining 2Oomg / Lchloramphenicol. The medium was renewed every 7 days. 75 tusgenic sporophytes, 20 negativecontrols and blank controls were used. When all controls died the survived bombhoed sporophyteswere transferred to non-selechve mediurn. CAT was detected by the sam method as menhonedabove.Results are as follows:1. Cal transient expression. The results are shown in Hg. l. The amount of CAT is 952.O pg /mg protein. It is almost twice of that in control (negative control: 56l.O pg / mg Protein, blankcontrol f 549.8 pg / mg protein).2. Selecting and stable expression of cat. All control died after l4 days of culthe but 1l of 75bombarded sporophytes were alive. High CAT content was detected in two of them, samp1e 4 was848.7 pg / mg protein (Flg.2). It is about three times of the for the control samples (3o9.7 pg / mgProtein), which indicates that cat had stably expressed in L japonica. Another sample has a CATamount of 345.6 pg / mg protein.The results of this paper suggest that cal can be a suitable selectable marker for genetictransformation of L japonica, and that SV40 promoter can work in kelp.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
1999年第1期28-33,共6页
Oceanologia Et Limnologia Sinica
基金
国家攀登计划B资助!PDB-6-4-1
国家自然科学基金!39400076
39670367
关键词
CAT基因
海带
选择标记
遗传转化
氯霉素
CAT gene(cat) Laminaria japonica Selectable marker SV40 promoter Genetic transformation
