| 摘要: |
| 采用RT-PCR技术, 从雨生红球藻(Haematococcus pluvialis)中克隆出IPP异构酶基因(ipiHp1), 构建双元载体pCAMBIA3300-egfp-ipiHp1和基因枪转化载体pBlueScript SKⅡ-bar-egfp-ipiHp1, 利用根癌农杆菌侵染法和基因枪转化法, 将目的基因导入雨生红球藻中, 经过含50?g/mL草丁膦BBM培养基筛选得到阳性转化子。通过荧光观察报告基因egfp和PCR扩增分析, 证明ipiHp1基因整合到转化子的基因组中。生物量测定结果表明大部分转化子的生物量与野生型相似。虾青素含量测定发现, 农杆菌侵染法转化的转化子A3虾青素含量与野生型相比有显著变化, 平均值达到16.49mg/g, 与野生型相比提高了5.16%, 而基因枪转化法转化的转化子虾青素含量与野生型无显著性差异。 |
| 关键词: 虾青素 雨生红球藻 IPP异构酶 遗传转化 |
| DOI：10.11693/hyhz201304029029 |
| 分类号: |
| 基金项目:海洋公益性行业科研专项经费资助, 201305022号。 |
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| THE EFFECTS OF GENETIC TRANSFORMATION OF ISOPENTENYL DIPHOSPHATE ISOMERASE GENE ON THE ASTAXANTHIN CONTENT OF HAEMATOCOCCUS PLUVIALIS |
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WANG Na, LIN Xiang-Zhi, MA Rui-Juan, YAN Jin-Fei
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Engineering Research Center of Marine Biological Resource Comprehensive Utilization, Third Institute of Oceanography, State Oceanic Administration
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| Abstract: |
| Isopentenyl diphosphate isomerase gene (ipiHp1) is a key enzyme gene for biosynthesis of astaxanthin in Haematococcus pluvialis. In this study, the ipiHp1 gene fragment was amplified by RT-PCR. We constructed a binary vector pCAMBIA3300-egfp-ipiHp1 and an expression vector pBlueScript SKⅡ-bar-egfp-ipiHp1 for particle gun. The target gene was introduced into H. pluvialis by Agrobactrium tumefaciens-mediated transformation and particle. The transformants was selected and maintained in the BBM agar plates containing 50?g/mL Basta. The EGFP fluorescence assay and PCR analysis indicated that ipiHp1 gene was integrated into genomic DNA of the H. pluvialis. The majority of the transformants displayed similar biomass comparing with the wild type strain. Astaxanthin content detection found that the A. tumefaciens-mediated transformant strain A3 had significant difference comparing with the wild type strain (P<0.05). The average reached to 16.49mg/g, compared to wild type increased by 5.16%. The statistical analysis also showed that there was no significant difference (P>0.05) between particle gun transformants and wild-type strains in the variation of astaxanthin content. |
| Key words: astaxanthin Haematococcus pluvialis isopentenyl diphosphate isomerase (IPI) genetic transformation |
