Part:BBa_K5044044
Plastid transformation vector for kiwifruit by HUBU-China
pQQC7 is a specialized plastid transformation vector designed for the stable integration of foreign genes into the chloroplast genome of kiwifruit (Actinidia chinensis). This vector is a powerful tool for plant synthetic biology, enabling the expression of genes of interest in the chloroplast, which can be particularly useful for the production of high-value compounds, such as pharmaceuticals, vaccines, and biopharmaceuticals. The pQQC7 vector includes several key components to ensure efficient and stable transformation:
Key Components:
Selectable Marker: Gene: aadA (aminoglycoside 3′-adenylyltransferase) Function: Confers resistance to spectinomycin, allowing for the selection of transformed plants. Promoter: psbA promoter from Chlamydomonas reinhardtii, ensuring strong and constitutive expression. Reporter Gene: Gene: GFP (Green Fluorescent Protein) Function: Serves as a visual marker to confirm successful transformation and to monitor gene expression. Promoter: Tobacco plastid 16S rRNA promoter (Prrn), known for its strong and constitutive activity in the chloroplast. 5' UTR: 5′ untranslated region from gene10 of bacteriophage T7 (T7g10), enhancing translation efficiency. Homologous Recombination Sequences: Left Homologous Recombination Region (LHRR): 1,092 bp, amplified from the kiwifruit chloroplast genome. Right Homologous Recombination Region (RHRR): 1,185 bp, amplified from the kiwifruit chloroplast genome. Target Site: These regions ensure precise integration of the vector between the trnfM and trnG genes in the kiwifruit plastid genome, facilitating homoplasmy (uniformity of the plastid genome). Plasmid Backbone: Origin: Derived from pBluescript II KS(+) and pYY11. Restriction Sites: SacI, KpnI, SalI, SpeI, BlnI, ApaI, SphI, providing flexibility for cloning and modification. Function:
Stable Integration: The pQQC7 vector is designed to integrate stably into the kiwifruit chloroplast genome, ensuring that the introduced genes are passed on to subsequent generations without the risk of transgene escape through pollen. High-Level Expression: The strong promoters and 5' UTRs ensure high-level expression of the genes of interest, making it suitable for the production of recombinant proteins and other valuable compounds. Visual Confirmation: The GFP reporter allows for easy visualization of transformed cells and tissues, facilitating the screening and selection of successfully transformed plants. Usage in Projects:
Cloning: Clone your gene of interest into the pQQC7 vector using appropriate restriction enzyme sites or Gibson assembly. Ensure that the gene is positioned downstream of the PrrnG10L regulatory element for optimal expression. Transformation: Use a biolistic (particle bombardment) method to introduce the pQQC7 vector into young kiwifruit leaves. Follow the detailed protocol for leaf preparation, particle coating, and bombardment. Select for transformed plants by culturing the bombarded leaf explants on medium containing spectinomycin. Transfer the resistant calli to shoot multiplication medium for further growth and regeneration. Verification: Confirm the presence and correct integration of the construct using PCR and Southern blot analysis. Assess the expression level of the target gene using Northern blot, Western blot, and confocal microscopy to visualize GFP fluorescence. Applications: Pharmaceuticals and Vaccines: Use the pQQC7 vector to produce therapeutic proteins, vaccines, and antibodies in kiwifruit. Enhanced Nutritional Value: Introduce genes that enhance the nutritional profile, such as those involved in the synthesis of vitamins, minerals, and other beneficial metabolites. Stress Tolerance: Engineer kiwifruit to be more resilient to environmental stresses, such as drought, salinity, and pathogens. Synthetic Biology: Utilize the chloroplast's capacity for multiple transgenes and the absence of gene silencing to construct complex metabolic pathways. Design Considerations:
Promoter Strength: The choice of the Prrn and psbA promoters ensures robust and constitutive expression. Translation Efficiency: The T7g10 5' UTR enhances translation efficiency, leading to higher protein yields. Stability and Homogeneity: The use of homologous recombination sequences ensures precise and stable integration, reducing the likelihood of random insertions. Regulatory Compliance: The design complies with regulatory standards for genetically modified organisms (GMOs), ensuring safety and suitability for both research and potential commercial applications. This comprehensive description provides users with a detailed understanding of the pQQC7 vector, its function, and how to use it effectively in their projects. If you have any additional questions or need further information, please feel free to contact us.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 129
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 129
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 118
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 129
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 129
Illegal NgoMIV site found at 1846 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2091
Illegal BsaI.rc site found at 818
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