| 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（CAT gene，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。 |
| 关键词: CAT基因 海带 选择标记 SV40启动子 遗传转化 |
| DOI： |
| 分类号: |
| 基金项目:国家攀登计划B资助项目，PDB－6－4－1;国家自然科学基金资助项目，
39400076,39670367号 |
附件 |
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| EXPRESSION OF CHLORAMPHENICOL ACETYLTRANSFERASE (CAT) GENE TRANSFERRED INTO LAMINARIA JAPONICA |
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WU Jian-qiu, QIN Song, DENG Tian, GUO Xiao-lin, ZENG Cheng-kui
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Institute of Oceanology,the Chinese Academy of Sciences,Qingdao,266071
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| Abstract: |
| In order to study the expression of foreign genes in Laminaria japonica and to determine suitable markers, experiments were undertaken from December, 1994 to April, 1995. The materials used were young sporophytes of L. japonica. They were provided by Rongcheng Seeding Farm in December, 1994. The methods are as follows:
1. Sterilization and culture of materials. Dipping materials in autoclaved seawater (20–30min), then transfer to 1.5% KI (W/V, 20–30min), and at last autoclaved water (20–30min). Finally materials were recovered in autoclaved seawater for 20min. the culture condition was 10h/14h (ratio of light period to darkness period), 50μE/(m2.s) and (10.0±0.5)°C.
2. Introduction of CAT gene (cat), pCAT-control plasmid (SV40 promoter-cat- SV40 enhancer, 4780bp, by a Biolistic PDS-1000/He Particle Delivery System. 5 sporophytes were used as one sample and were bombarded twice. The bombardment parameters were: vacuum 6.1×103 Pa, rupture disk 1100 psi and the distance between samples and macrocarrier holder 6cm.
3. Transient expression of cat. 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 Kit (Boehringer Mannkeim GmbH Germany) after 48h culture in darkness. The amount of CAT was counted by comparing OD at 405nm with CAT standard sample.
4. Selection and stable expression of cat. After bombardment, sporophytes underwent a week of recovery period in non-selective medium and then selected in liquid medium containing 200mg/L chloramphenicol. The medium was renewed every 7 days. 75 transgenic sporophytes, 20 negative controls and blank controls were used. When all controls died the survived bombarded sporophytes were transferred to non-selective medium. CAT was detected by the same method as mentioned above.
Results are as follows:
1. Cat transient expression. The results are shown in Fig.1. the amount of CAT is 952.0 pg/mg protein. It is almost twice of that in control (negative control: 561.0 pg/mg protein; blank control: 549.8 pg/mg protein).
2. Selecting and stable expression of cat. All control died after 14 days of culture but 11 of 75 bombarded sporophytes were alive. High CAT content was detected in two of them, sample 4 was 848.7 pg/mg protein (Fig.2). It is about three times of that for the control samples (309.7 pg/mg protein), which indicates that cat had stably expressed in L. japonica. Another sample has a CAT amount of 345.6 pg/mg protein.
The results of this paper suggest that cat can be a suitable selectable marker for genetic transformation of L. japonica, and that SV40 promoter can work in kelp. |
| Key words: CAT gene (cat), Laminaria japonica, Selectable marker, SV40 promoter, Genetic transformation |
