摘  要: 蓝藻是一类能进行光合放氧的并用于研究光合作用的原核生物，也是模式生物之一。集胞藻6803（Synechocystis sp.strain PCC 6803）是一种单细胞的蓝藻。研究发现蓝藻细胞的光合作用机制对CO2具有高亲和力，主要依赖于蓝藻体细胞内的CO2浓缩机制。本研究从集胞藻6803（Synechocystis sp.strain PCC 6803）中提取与CO2浓缩机制相关的基因组DNA，根据Gateway技术的要求，设计特殊引物P1／P2，利用高保真的KAPA HIFI酶PCR克隆获得带有attB位点的slr0012目的基因。将含有attB接头的slr0012目的基因通过BP 反应克隆到含有attP接头的Donor207载体上以产生入门载体pENTY-slr0012，再通过LR反应将入门载体pENTY-slr0012的slr0012基因克隆到35ss-3×HA载体上构建成植物双元表达载体35SS-slr0012-3×HA。经菌液PCR和测序鉴定后，将35SS-slr0012-3×HA重组表达质粒成功导入农杆菌GV3101中。通过生物信息学软件分析预测slr0012基因可能有核酮糖1,5 -二磷酸羧化/加氧酶的功能。该研究结果为进一步研究集胞藻6803 CO2浓缩机制相关基因在植物体内的功能奠定了基础。

关键词：集胞藻6803；slr0012基因；Gateway技术；植物双元表达载体

Cloning of 6803 Slr0012 of Synechocystis sp.strain PCC

and Construction Of their Plant binary expression vector

Abstract: Cyanobacteria are prokaryotes capable of oxygenic photosynthesis and used to study photosynthesis, are one of the most popular model organisms. Synechocystis sp.strain PCC6803 is single cell cyanobacteria. The research has shown that the photosynthesis of cyanobacteria has a high affinity for CO2, which is mainly dependent on the CO2 concentration mechanism in cyanobacteria. In this study the genome of DNA related CO2 concentrating mechanism was extracted from Synechocystis sp.strain PCC 6803, according to the requirements of Gateway Technology, design specific primers, using KAPA HIFI enzyme PCR to obtain the slr0012 target gene with attB sites.By the BP recombination reaction, the PCR product containing attB was transfered to an attP-containing Donor207 to create an entry clone,nanely pENTY-slr0012,again by the LR recombintion reaction, the pENTY-slr0012 of slr0012gene was cloned into 35ss-3xHA to create Plant binary expression vector, namely 35SS-slr0012-3×HA. After PCR and sequencing, the recombinant expression plasmid of 35SS-slr0012-3×HA was successfully introduced into Agrobacterium GV3101. By bioinformatics software, slr0012was predictioned the function of the ribulose 1,5-bisphosphate carboxylase. The results laid a foundation for further research on the function of the Synechocystis sp.strain PCC 6803 CO2 concentration mechanism related genes in the plant.

Key words: Synechocystis sp.strain PCC 6803; slr0012gene; Gateway technology; Plant binary expression vector

目    录

摘  要 - 1 -

引言 - 2 -

1.材料与方法 - 4 -

1.1实验材料 - 4 -

1.1.1感受态细胞 - 4 -

1.1.2基因组DNA和载体 - 4 -

1.1.3酶和试剂 - 5 -

1.1.3.1酶 - 5 -

1.1.3.2抗生素 - 5 -

1.1.3.3引物 - 5 -

1.1.3.4生化试剂及配方 - 5 -

1.2仪器设备 - 6 -

1.3实验方法 - 6 -

1.3.1生物信息学分析软件 - 6 -

1.3.2KAPA HIFI高保真酶PCR反应 - 7 -

1.3.3PCR产物纯化 - 7 -

1.3.4检测PCR反应 - 8 -

1.3.5Gateway技术 - 8 -

1.3.5.1BP反应 - 8 -

1.3.5.2LR反应 - 8 -

1.3.6大肠杆菌转化 - 9 -

1.3.7质粒提取 - 9 -

1.3.8农杆菌转化 - 10 -

2.结果与分析 - 10 -

2.1slr0012目的基因的序列分析 - 10 -

2.2集胞藻Synechocystis sp.PCC 6803 slr0012目的基因的扩增 - 11 -

2.3入门载体的构建 - 12 -

2.4 35SS-slr0012-3×HA植物双元表达载体的构建 - 15 -

2.5 35SS-slr0012-3×HA植物双元表达载体导入农杆菌 - 15 -

3.讨论 - 16 -

参考文献 - 18 -

致谢 - 20 -