氯霉素乙酰转移酶(CAT)基因在海带中的表达  

EXPRESSION OF CHLORAMPHENICOL ACETYLTRANSFERASE (CAT) GENE TRANSFERRED INTO LAMINRIA JAPONICA

作　　者：武建秋[1] 秦松[1] 邓田[1] 郭晓林[1] 曾呈奎[1] 

机构地区：[1]中国科学院海洋研究所,青岛266071

出　　处：《海洋与湖沼》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

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:

关 键 词：CAT基因 海带 选择标记 遗传转化 氯霉素 

分 类 号：Q949.288.4[生物学—植物学] Q785