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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (4): 30-37    DOI: 10.13523/j.cb.20180405
研究报告     
转基因聚球藻7942中vp28基因表达效率及其光合特性分析
庄旻敏1,贾晓会1,2,施定基3(),朱嘉诚1,冯思豫1,何培民1,贾睿1()
1 上海海洋大学海洋生态与环境学院 上海 201306
2 北京师范大学生命科学学院 北京 100875
3 中国科学院植物研究所 北京 100093
vp28 Gene Expression and Photosynthetic Characteristics of Transgenic Synechococcus sp. PCC 7942
Min-min ZHUANG1,Xiao-hui JIA1,2,Ding-ji SHI3(),Jia-cheng ZHU1,Si-yu FENG1,Pei-min HE1,Rui JIA1()
1 School of Ocean Ecology and Environment Shanghai Ocean University, Shanghai 201306 China
2 School of Biosciences,Beijing Normal University,Beijing 100875, China
3 Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
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摘要： 背景

对虾白斑综合征病毒(white spot syndrome virus,WSSV)是对虾养殖业中危害最严重的病毒之一,至今尚无规模应用的有效药物防治方法。但近年来在WSSV免疫防治上进展较大。Vp28蛋白是WSSV囊膜上的主要结构蛋白,2004年以来其编码基因已在8种宿主中表达成功,在实验室试验中对WSSV的防治疗效显著,但目前尚未见到其在对虾产业中的应用。

 目的

利用对虾的天然饵料聚球藻表达Vp28重组蛋白,这种药食同源可简化操作,降低成本,有助其在生产中应用。

 方法

用荧光定量PCR方法检测转vp28基因聚球藻7942中vp28基因的表达效率。通过氧电极的方法测得转vp28基因型聚球藻在不同温度、光照、pH和盐度下的光合活性变化,找到它的最适生长条件。

 结果

检测了vp28基因表达效率为9.52%,是在鱼腥藻7120表达效率的3倍。最适采收时间是对数生长后期(15d左右)。转基因型蓝藻7 942的最适生长条件是:温度为40℃,盐度为0~0.1mol/L NaCl,pH为7.5,光强为450μmol/(m 2·s)。

结论

确定了vp28基因在聚球藻中的表达效率及该转基因藻的最适培养条件,这些研究结果为用转vp28基因型聚球藻7942规模制备药食同源的口服剂提供了依据。
关键词： 转vp28基因聚球藻;  光合活性;  实时荧光定量PCR;  vp28基因表达效率    
Abstract:

White spot syndrome virus (WSSV) is one of the most harmful viruses in the shrimp industry, and there has been no effective drug on mass scale up to now. In recent years, the immunological prevention of WSSV from shrimp has been progressed hopefully.Vp28 protein is the major structural component on the envelope of WSSV. Since 2004, its coding genes have been expressed in 8 species, and the control of WSSV has been proved remarkably in the laboratory. However, it has not been applied in shrimp industry yet. The shrimp bait, Synechococcus sp.PCC7942 was used as the acceptor to express vp28 gene, and this homology of medicine and food may be helpful for its application in the shrimp industry. The expression efficiency of vp28 in transgenic Synechococcus sp.PCC7942 has been detected by RT-qPCR method. And photosynthetic characteristics of transgenic Synechococcus sp.PCC7942 at different temperature, illumination, pH and salinity have been measured by the method of oxygen electrode. The expression efficiency of vp28 gene was 9.52% which was three times higher than that of Anabaena sp. PCC 7120. The most suitable harvest time was in late logarithm growth (the 15 th d). The optimum growth conditions of transgenic Synechococcus sp.PCC7942 were as follows: the temperature 40℃, the salinity 0~0.1mol/L NaCl, pH 7.5, light intensity 450μmol/(m 2·s). These data may be useful for scale preparation of oral drug made from transgenic Synechococcus sp.PCC7942.

Key words: Transgene Synechococcus with vp28 gene    Photosynthetic activity    RT-PCR    Expression of vp28 gene
收稿日期: 2017-11-21 出版日期: 2018-05-08
ZTFLH:  Q789  
基金资助: 国家863计划(2014AA093506);上海市科技兴农推广项目(沪农科推字(2017)第1-13号);上海市科委项目资助项目(16391903500)
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庄旻敏
贾晓会
施定基
朱嘉诚
冯思豫
何培民
贾睿

引用本文:

庄旻敏,贾晓会,施定基,朱嘉诚,冯思豫,何培民,贾睿. 转基因聚球藻7942中vp28基因表达效率及其光合特性分析[J]. 中国生物工程杂志, 2018, 38(4): 30-37.

Min-min ZHUANG,Xiao-hui JIA,Ding-ji SHI,Jia-cheng ZHU,Si-yu FENG,Pei-min HE,Rui JIA. vp28 Gene Expression and Photosynthetic Characteristics of Transgenic Synechococcus sp. PCC 7942. China Biotechnology, 2018, 38(4): 30-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180405    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I4/30

Strain Antibiotic Features Source
Anabaena sp. PCC 7120 wild type Institute of Botany, Chinese Academy of Sciences
Empty vector type 7120 Neomycin transferred pRL-489
vp28 type 7120 Neomycin Harboring vp28 gene
Synechococcus sp. PCC 7942 wild type Institute of Botany, Chinese Academy of Sciences,
Tianjin University of Science &Technology
Empty vector type 7942 Kanamycin transferred pRL-489
vp28 type 7942 Kanamycin Harboring vp28 gene
表1  本研究用的蓝藻品系
Primer name Primer sequence (5'-3') Size (bp)
vp28 forward AAGGATCCGGAGAGCGTCATGGATCTTTCTTTCAC 35
vp28 reverse CCCCCCGAATTCCACGATTTATTTACTCGGTCTC 34
  
Stage Cycle Temperature(℃) Time Content Fluorescence signal acquisition
Initial 1倍 95 15min Initial No
PCR reaction 40倍 95 10s Denaturation No
50~60 20s Annealing No
72 30s Extend Yes
表3  实时荧光定量PCR反应条件
图1  聚球藻7942野生型、转空载体型及转vp28基因型中Vp28蛋白的蛋白质印迹
图2  vp28基因的PCR产物用3%的琼脂糖凝胶电泳检测及纯化后vp28基因的熔解曲线
图3  回收的纯vp28片段标准曲线图
Growth times 3 6 9 12 15 18
Average Ct value 21.03 21.92 19.17 19.99 17.84 20.62
Average copy number(copies/ml) 38 642 19 810 156 136 84 362 423 779 52 570
表4  不同生育期转vp28基因型聚球藻7942的Ct值及拷贝数
Growth times 3 6 9 12 15 18
Average Ct 21.65 22.88 20.32 19.28 20.62 20.87
Average copy number(copies/ml) 24 261 9 635 65 850 143 760 52 570 43 574
表5  不同生育期转vp28基因型鱼腥藻7120的Ct值及拷贝数
图4  不同生长时期转基因型聚球藻7942 vp28基因的表达效率
  Fig.5 vp28 gene expression rates of transgenic Anabaena sp.7120 during different growth stages
图6  聚球藻7942野生型与空载体型、转vp28型的光合对环境因子响应曲线
图7  聚球藻7942野生型、空载体型和转vp28基因型的生长曲线(用分光光度计在OD750检测)
图8  聚球藻7942野生型、空载体型和转vp28基因型的生长曲线(用细胞计数板在光学显微镜下检测)
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