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Part:BBa_K5044044

esigned by: Chuming Chen Group: iGEM24_HUBU-China (2024-10-01)

Introduction

1.1 Background

Plastid transformation, or the introduction of foreign DNA into the chloroplast genome, has emerged as a powerful tool in plant biotechnology. This technique offers several advantages over nuclear transformation, including high-level transgene expression, the absence of gene silencing, and reduced risk of transgene escape via pollen. Kiwifruit (Actinidia chinensis), a woody vine with significant economic value, represents an attractive target for plastid engineering due to its potential for molecular farming and the production of valuable compounds.

The development of a stable plastid transformation system in kiwifruit is a significant step forward, as it extends the range of species that can be engineered using this technology. Prior to our work, successful plastid transformation had been limited to a few model plants and crops, such as tobacco, tomato, and poplar. The ability to transform the plastid genome of kiwifruit opens new avenues for the production of pharmaceuticals, vaccines, and other high-value products.

1.2 Objective

The primary objective of this project is to develop and characterize a stable plastid transformation system for kiwifruit (Actinidia chinensis). Specifically, we aim to:

Construct a Plastid Transformation Vector: Develop a vector, pQQC7, that carries a spectinomycin-resistance gene (aadA) and a green fluorescent protein (GFP) reporter gene, flanked by homologous sequences from the kiwifruit plastid genome to ensure targeted integration.
Establish a Reliable Transformation Protocol: Optimize the particle bombardment method for delivering the pQQC7 vector into kiwifruit leaf explants and establish a selection and regeneration protocol to obtain spectinomycin-resistant, transplastomic plants.
Verify Transgene Integration and Expression: Use molecular techniques, including PCR, Southern blot, Northern blot, and Western blot, to confirm the presence and expression of the transgenes in the transformed kiwifruit plants.
Evaluate Phenotypic Stability and Growth: Assess the growth and development of the transplastomic kiwifruit plants to ensure they exhibit normal phenotypes and are suitable for further biotechnological applications.

Materials and Methods

2.1 Construction Process

2.1.1 pYY34 Vector Construction

The pYY34 vector was constructed by ligating four digested DNA fragments using T4 DNA ligase. These fragments included:

The backbone obtained by digesting pBluescript II KS(+) with SacI and KpnI.
The GFP and aadA expression cassettes excised from pYY11 with SalI and SpeI.
The left homologous recombination region (LHRR, 1,092 bp) digested with KpnI and SalI.
The right homologous recombination region (RHRR, 1,185 bp) digested with BlnI and SacI.

The corresponding flanking sequences for homologous recombination were obtained by PCR amplification from the kiwifruit chloroplast genome (NCBI access number: NC_026690.1) using the following primer pairs:

(KpnI)AcLHRR-F/(SalI)AcLHRR-R for LHRR.
(BlnI)AcRHRR-F/(SacI)AcRHRR-R for RHRR.

2.1.2 pQQC7 Vector Construction

To construct the pQQC7 vector, the CrPpsbA and aadA fragments were PCR amplified using the following primer pairs:

(ApaI)CrPpsbA-F/(g10)CrPpsbA-R for CrPpsbA.
(g10)aadA-F/(SphI)aadA for aadA.

These fragments were then used in an overlap-extension PCR to generate a CrPpsbA-aadA fragment separated by the 5′ UTR from gene10 of bacteriophage T7 (T7g10). Finally, both the CrPpsbA-aadA fragment and pYY34 were excised with ApaI/SphI and ligated to generate the pQQC7 vector (NCBI access number: PP816932; plasmid number: 221601, Addgene).

2.2 Leaf Preparation and Transformation

Fresh young leaves of kiwifruit seedlings were placed abaxial side up on AcOsM (agar-solid MS medium, 0.1 M sorbitol, 0.1 M mannitol, 3% sucrose) overnight in the dark. Gold particles (0.6 μm diameter) coated with plasmid DNA pQQC7 were introduced into the plant cells using a biolistic gun (PDS-1000/He, BioRad, USA). After bombardment, the leaf samples were diced into 5 × 5 mm pieces and placed on AcReMs (agar-solid MS medium, 3% sucrose, and combinations of different hormones) containing spectinomycin.

2.3 Selection and Regeneration

Primary spectinomycin-resistant green calli began to appear after three months of incubation of bombarded leaf explants on AcReM3, which included 300 mg L−1 spectinomycin. After six months of selection, six green calli were obtained from 12 plates. These calli were able to grow into shoots and underwent additional rounds of regeneration to achieve homoplasmy. Young leaves of these transplastomic lines were subjected to further regeneration to ensure all plastids contained the transgene. After induction of rooting, the transplastomic lines were transferred to soil and did not exhibit any discernible phenotypic differences compared to wild-type plants.

2.4 Homoplasmy Verification

To confirm the presence of the transgene in the shoots, PCR was performed using specific primers (GFP-F/GFP-R) designed for the GFP gene, resulting in the amplification of a 720-bp PCR product. Additionally, a primer pair (psaB-aadA-F/psaB-aadA-R) was used to target the psaB region of the native chloroplast genome and the aadA marker, yielding a 2.9 kb product. Two PCR-positive lines (Ac-pQQC7#1, #6) were confirmed to have reached homoplasmy through Southern blot analysis. In the wild-type plants (Ac-WT), a 2.9 kb fragment was detected, while in the Ac-pQQC7, a 5.7 kb fragment corresponding to the integration of the transgene was observed.

Results

3.1 PCR Amplification Verification

Figure 1: Generation of Plastid-Transformed Kiwifruit

(A) Physical maps of the targeting region in the kiwifruit plastid genome and the pQQC7 vector.
(B) Preparation of kiwifruit leaves for particle bombardment.
(C) Selection of bombarded leaf explants on AcReM3 containing spectinomycin.
(D) Appearance of spectinomycin-resistant calli after three months of selection.
(E, F) Growth of shoots from the resistant calli.
(G, H) Additional rounds of regeneration to achieve homoplasmy.
(I, J) Progression of shoot growth and root induction.
(K) Growth comparison between transplastomic and wild-type plants.
(L, M) PCR amplification confirming the presence of the transgene.
(N) Southern blot analysis verifying homoplasmy.

To confirm the presence of the transgene in the transformed kiwifruit plants, we performed PCR using specific primers designed for the GFP gene and the aadA marker.

GFP Gene Amplification:Primers: GFP-F and GFP-RExpected Product Size: 720 bpResults: PCR amplification with these primers yielded a 720-bp product, confirming the presence of the GFP gene in the transplastomic lines (Fig. 1L).
aadA Marker Amplification:Primers: psaB-aadA-F and psaB-aadA-RExpected Product Size: 2.9 kbResults: PCR amplification with these primers resulted in a 2.9 kb product, indicating the successful integration of the aadA marker (Fig. 1M).

These results confirmed that the pQQC7 vector had been successfully integrated into the kiwifruit plastid genome.

3.2 Southern Blot Analysis

Southern blot analysis was conducted to verify the homoplasmy of the transplastomic lines.

Probes Used:A probe specific to the psaB region of the native chloroplast genome.A probe specific to the aadA marker.
Results:Wild-Type Plants (Ac-WT): A 2.9 kb fragment was detected, corresponding to the untransformed plastid genome.Transplastomic Lines (Ac-pQQC7#1, #6): A 5.7 kb fragment was observed, indicating the successful integration of the transgene and confirming homoplasmy (Fig. 1N).

This analysis provided strong evidence that the transplastomic lines had achieved complete replacement of the wild-type plastid genome with the modified one containing the pQQC7 vector.

3.3 GFP Expression Level Determination

To evaluate the expression level of the GFP gene in the transplastomic kiwifruit plants, we conducted several analyses, including Northern blot, Western blot, semi-quantitative analysis, and confocal microscopy.

3.3.1 Northern Blot Analysis

Purpose: To detect the presence and size of GFP mRNA transcripts.
Results:
Two transcripts were observed, with the smaller and more abundant transcript representing the expected full-length GFP mRNA (Fig. 2A).
The larger bands are likely due to read-through transcripts, a common phenomenon in plastids due to inefficient transcription termination (Lu et al., 2017; Zhou et al., 2007).

3.3.2 Western Blot Analysis

Purpose: To confirm the accumulation of the GFP protein.
Antibody Used: Anti-GFP antibody.
Reference Standard: Recombinant GFP (rGFP) as a reference.
Results:
A 27 kDa GFP peptide was detected, confirming the production of GFP in the transplastomic lines (Fig. 2B).

3.3.3 Semi-Quantitative Analysis

Purpose: To estimate the amount of GFP protein in the total soluble protein (TSP).
Method: Using a dilution series of recombinant GFP (rGFP) as a reference.
Results:
GFP accumulation reached approximately 2.5% of the TSP (Fig. 2C).

3.3.4 Confocal Microscopy

Purpose: To visualize the localization of GFP in the chloroplasts.
Results:
Confocal laser-scanning microscopy confirmed that GFP fluorescence was specifically localized in the chloroplasts of the Ac-pQQC7 lines (Fig. 2D).

Discussion

4.1 Significance of Successful Transformation

The successful development of a stable plastid transformation system for kiwifruit (Actinidia chinensis) using the pQQC7 vector is a significant milestone in plant synthetic biology. This achievement extends the range of woody species that can be genetically modified via plastid transformation, which was previously limited to poplar. The ability to introduce and express foreign genes in the chloroplasts of kiwifruit opens up new possibilities for the production of high-value compounds, such as pharmaceuticals, vaccines, and antibodies, with the potential for high-level accumulation (up to 75% of total soluble protein, TSP). Additionally, the use of plastid transformation reduces the risk of transgene escape through pollen, making it a more environmentally friendly approach compared to nuclear transformation.

4.2 Application Prospects

The establishment of a reliable plastid transformation protocol in kiwifruit provides an attractive biosynthetic chassis for molecular farming. Potential applications include:

Production of Edible Vaccines and Biopharmaceuticals: Kiwifruit could serve as a vehicle for the production of edible vaccines and biopharmaceuticals, offering a cost-effective and scalable method for delivering these products.
Enhanced Nutritional Value: By introducing genes that enhance the nutritional profile, such as those involved in the synthesis of vitamins, minerals, and other beneficial metabolites, the nutritional value of kiwifruit can be significantly improved.
Abiotic and Biotic Stress Tolerance: Genes conferring resistance to environmental stresses, such as drought, salinity, and pathogens, can be introduced to improve the resilience of kiwifruit crops, thereby increasing yield and sustainability.
Synthetic Biology Applications: The plastid genome's capacity for accommodating multiple transgenes and the absence of gene silencing make it an ideal platform for complex metabolic engineering and the construction of synthetic pathways.

4.3 Future Research Directions

While the current study has successfully established a plastid transformation system in kiwifruit, several areas warrant further investigation:

Optimization of Expression Levels: Further research is needed to optimize the expression levels of transgenes in the plastids, potentially by exploring different promoters, regulatory elements, and codon optimization.
Stability and Inheritance Patterns: Investigating the long-term stability of the transplastomic lines and the inheritance patterns of the modified plastid genomes will be crucial for ensuring the reliability of the system.
Scalability and Field Testing: Scaling up the transformation process and conducting field trials to assess the performance of transplastomic kiwifruit under real-world conditions will be essential for practical applications.
Safety and Regulatory Compliance: Addressing safety concerns and ensuring compliance with regulatory standards for genetically modified organisms (GMOs) will be important for the commercialization of transplastomic kiwifruit.
Expanding the Range of Transgenic Traits: Exploring the introduction of a broader range of transgenic traits, including those related to disease resistance, enhanced flavor, and extended shelf life, will further enhance the utility of this technology.

Appendix

6.1 Primer List

Below is a list of the primers used in the construction and verification of the pQQC7 vector and the analysis of transplastomic kiwifruit plants. All primers are listed with their respective sequences, restriction enzyme sites (where applicable), and purposes.

Primer Name	Sequence (5' to 3')	Restriction Enzyme Site	Purpose
(KpnI)AcLHRR-F	`GGTACC[sequence]`	KpnI	Amplification of LHRR for pYY34 construction
(SalI)AcLHRR-R	`GTCGAC[sequence]`	SalI	Amplification of LHRR for pYY34 construction
(BlnI)AcRHRR-F	`AGATCT[sequence]`	BlnI	Amplification of RHRR for pYY34 construction
(SacI)AcRHRR-R	`GAGCTC[sequence]`	SacI	Amplification of RHRR for pYY34 construction
(ApaI)CrPpsbA-F	`GGGCCCG[sequence]`	ApaI	Amplification of CrPpsbA for pQQC7 construction
(g10)CrPpsbA-R	`[sequence]`		Amplification of CrPpsbA for pQQC7 construction
(g10)aadA-F	`[sequence]`		Amplification of aadA for pQQC7 construction
(SphI)aadA	`GCATGC[sequence]`	SphI	Amplification of aadA for pQQC7 construction
GFP-F	`[sequence]`		PCR amplification for GFP gene verification
GFP-R	`[sequence]`		PCR amplification for GFP gene verification
psaB-aadA-F	`[sequence]`		PCR amplification for aadA marker verification
psaB-aadA-R	`[sequence]`		PCR amplification for aadA marker verification

Note: The specific sequences for each primer are not provided here and should be obtained from the original research or designed based on the target regions.

6.2 Media Formulations

AcOsM (Agar-Solid MS Medium with Osmoticum):Composition:Murashige and Skoog (MS) salts0.1 M sorbitol0.1 M mannitol3% sucrose0.8% agarpH: Adjusted to 5.8 before autoclaving

AcOsM (Agar-Solid MS Medium with Osmoticum):

Composition:
Murashige and Skoog (MS) salts
0.1 M sorbitol
0.1 M mannitol
3% sucrose
0.8% agar
pH: Adjusted to 5.8 before autoclaving

AcReMs (Agar-Solid MS Medium with Different Hormones and Spectinomycin):

Composition:
Murashige and Skoog (MS) salts
3% sucrose
Combinations of different hormones (e.g., BAP, NAA)
300 mg L−1 spectinomycin
0.8% aga
pH: Adjusted to 5.8 before autoclaving

AcSmM (Shoot Multiplication Medium):

Composition:
Murashige and Skoog (MS) salts
3% sucrose
300 mg L−1 spectinomycin
0.8% agar
pH: Adjusted to 5.8 before autoclaving

6.3 Detailed Experimental Procedures Vector Construction:

pYY34 Vector:

Digest pBluescript II KS(+) with SacI and KpnI.
Excise the GFP and aadA expression cassettes from pYY11 with SalI and SpeI.
Amplify the LHRR and RHRR by PCR using the specified primers.
Ligase the digested fragments to construct the pYY34 vector.

pQQC7 Vector:

PCR amplify the CrPpsbA and aadA fragments using the specified primers.
Perform overlap-extension PCR to generate the CrPpsbA-aadA fragment.
Excise the CrPpsbA-aadA fragment and pYY34 with ApaI/SphI.
Ligase the fragments to construct the pQQC7 vector.

Leaf Preparation and Transformation:

Place fresh young leaves abaxial side up on AcOsM overnight in the dark.
Coat gold particles (0.6 μm diameter) with plasmid DNA pQQC7.
Bombard the leaf explants using a biolistic gun (PDS-1000/He, BioRad, USA) at 1100 psi and a target distance of 9 cm.
Dice the bombarded leaf samples into 5 × 5 mm pieces and place them on AcReMs containing 300 mg L−1 spectinomycin.

Selection and Regeneration:

Incubate the bombarded leaf explants on AcReMs for three months to select for spectinomycin-resistant calli.
Transfer the resistant calli to AcSmM for shoot elongation and multiplication.
Subject the young leaves of these lines to additional rounds of regeneration to achieve homoplasmy.
Induce rooting and transfer the transplastomic lines to soil.

Verification:

PCR Amplification:
Use specific primers (GFP-F/GFP-R, psaB-aadA-F/psaB-aadA-R) to confirm the presence of the transgene.
Southern Blot Analysis:
Hybridize with probes specific to the psaB region and the aadA marker to verify homoplasmy.
Northern Blot Analysis:
Detect GFP mRNA transcripts to confirm transcription.
Western Blot Analysis:
Use an anti-GFP antibody and recombinant GFP as a reference to confirm protein accumulation.
Semi-Quantitative Analysis:
Estimate GFP accumulation using a dilution series of recombinant GFP.
Confocal Microscopy:
Visualize GFP fluorescence in the chloroplasts to confirm plastid localization.

6.4 Data and Figures

Figure 2: Analysis of GFP Expression in Transplastomic Kiwifruit Plants

(A) Northern blot analysis of GFP transcripts.
(B) Western blot analysis confirming GFP protein accumulation.
(C) Semi-quantitative analysis of GFP accumulation.
(D) Confocal microscopy showing GFP fluorescence in chloroplasts.

Acknowledgments

We would like to express our sincere gratitude to all the individuals and organizations that have contributed to the success of this project. Special thanks go to our advisors, mentors, and collaborators for their invaluable guidance, support, and expertise. We also thank the members of our laboratory and the broader scientific community for their constructive feedback and assistance. Additionally, we acknowledge the financial support from Hubei University,School of Life Science,Lab for Plastid Engineering,,which made this research possible.

8.1 Author Contact Information

Chuming Chren
Affiliation: Student leader of HUBU-China team,Lab for Plastid EngineeringSchool of Life Science,Hubei University, Wuhan 430074, China
Email: plastid@foxmail.com / Phone:+86 18071397098

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Basic Parts

Vector name	Code	Name	Function&Description	Type	Length(bp)
pMJ5	BBa_K5044000	T7 terminator	transcription terminator for bacteriophage T7 RNA polymerase	terminator	48bp
BBa_K5044007	lacI	The lac repressor binds to the lac operator to inhibit transcription in E.coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG)	CDS	1083bp
BBa_K5044002	Tc-ba-miR-CHS1-1		misc-feature	138bp
BBa_K5044003	pac	stem loop that binds the bacteriophage MS2 coat protein	misc_RNA	19bp
BBa_K5044005	T7 promoter	promoter for bacteriophage T7 RNA polymerase	promoter	19bp
pMJ6	BBa_K5044012	RBS	efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)	RBS	23bp
BBa_K5044017	TAT	HIV-1 TAT (48-60) is a cell-penetrating peptide derived from the human immunodeficient virus (HIV)-1 Tat protein residue 48-60. It has been used to deliver exogenous macromolecules into cells in a non-disruptive way.	misc_feature	33bp
BBa_K5044011	cp1	binds to a specific stem-loop structure in the viral RNA (Peabody, 1993)	CDS	387bp
pMJ11	BBa_K5044010	psaB	3' part psaB	misc_feature	1201bp
BBa_K5044018	rps14	Ribosomes, the organelles that catalyze protein synthesis, consist of a small 40S subunit and a large 60S subunit. Together these subunits are composed of 4 RNA species and approximately 80 structurally distinct proteins. This gene encodes a ribosomal protein that is a component of the 40S subunit. The protein belongs to the S11P family of ribosomal proteins. It is located in the cytoplasm. Transcript variants utilizing alternative transcription initiation sites have been described in the literature. As is typical for genes encoding ribosomal proteins, there are multiple processed pseudogenes of this gene dispersed through the genome. In Chinese hamster ovary cells, mutations in this gene can lead to resistance to emetine, a protein synthesis inhibitor. Multiple alternatively spliced transcript variants encoding the same protein have been found for this gene.	5'UTR	303bp
BBa_K5044019	pac	stem loop that binds the bacteriophage MS2 coat protein	CAAT_signal	19bp
BBa_K5044020	loxP	loxP is a 34bp DNA sequence located in the P1 bacteriophage, consisting of two 13bp inverted repeat sequences and an 8bp asymmetric spacer. This sequence is a specific recognition and binding site for the Cre recombinase enzyme, used to catalyze the DNA strand exchange process.	misc_feature	34bp
BBa_K5044022	aadA	The Aada gene, also known as the aminoglycosid-3 '-adenylate transferase gene, is a gene found in prokaryotes. The enzyme encoded by this gene has nucleotide transferase activity and is able to modify aminoglycoside antibiotics so that these antibiotics lose the ability to bind to the target and develop resistance.	misc_feature	792bp
BBa_K5044021	CrPpsbA	The selective marker gene aadA is driven by CrPpsbA.	misc_feature	273bp
BBa_K5044028	psbZ	Photosystem I exists in the body as trimer and monomer forms. Its structure has been determined to be the most complex membrane protein. The most notable feature of the PSI protein structure is that the auxiliary factor accounts for more than 30% of the total molecular weight of photosystem I. The auxiliary factor not only plays a decisive role in the function of the protein, but also plays an important role in the assembly and structural integrity of PSI. A monomer unit of photosystem I is composed of 127 cofactors and various different proteins (such as PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaG, and 16 other proteins) covalently bound together, and research has shown that the binding sites of most cofactors and proteins are specific and highly conserved.psbZ is a photosynthetic subunit.	misc_feature	131bp