Functional characterization of ZmSSI2/FAB2 in maize mutant as a possible critical factor in fatty acid desaturation
PROJECT Proposal
Applicant’s Names: Marika Pavasini, Angela Gazzoli, Ukoji Stepheine Onyinyechi, Tommaso Foglia
Applicant’s IDs: 28701A, 24115A, 36939A, 24213A Date: 29th January 2024
Plant Molecular Biology
index
Introduction
Work Package 1
Work Package 2
Work Package 3
Future perspectives
This research project aims to
Specific
General
- Our gene could have an important activity for both the biosynthesis pathway of cuticle and stress responses, to drought, high temperature and biotic stresses.
- It might be responsible not only for the fatty acid desaturation phase in maize but could also play an important role in enhancing SA-defence genes.
- Functionally characterize our gene of interest (the Zm00001d004019 gene).
- Understand its role during plant development.
- Where and when is our gene expressed in the plant? Where does our gene product work in the cell? What is the role of our gene?
World production of top cereals
introduction
Relevance of maize
Maize is one of the oldest cultivated crops. Maize showed the highest production (1.2 billion tons in 2021) and fastest growth over the period (+104 percent since 2000) compared to the other major grains, as it has wider uses in the biofuel and animal feed sectors.
Source: FAO. 2022
introduction
Various mechanisms to resist drought -> tolerance at the cellular level is essential since it allows tolerant plants to maintain cellular homeostasis.
Maize tolerance to drought
Due to the steady changeset of the environment e.g. the decrease of the rainfall and high temperature, studying maize tolerance to drought becomes very important to face the possible decrease of yields.
In contrast, sensitive plants suffer rapid irreversible cell damage essentially due to the degradation of their membranes.
The study of plants’ cuticles could be very important in facing climate change.
introduction
The cuticle has an important role, giving the plants an advantage in reducing the loss of water from leaves surfaces. The maize cuticle is a protective and waxy layer that covers the outer surface of the aerial parts of maize plants, including leaves, stems, seeds and reproductive structures.
our gene - Zm00001d004019
In the fdl1-1 mutant is down-regulated and it is identified to be putatively involved in fatty acids biosynthesis, it is known as SSI2/FAB2. The putative gene product is known as Acyl-[acyl-carrier-protein] desaturase or stearyl acyl carrier protein desaturase (SACD). It is probably involved in the desaturation of the Stearoyl-ACP that led to the production of Oleoyl-ACP.
+ INFO
the putative expression pattern of our gene of interest
It is likely expressed in the plant inflorescence, the pericarp of the embryo and on immature seeds between 2-14 days after pollination.
Similarity with arabidopsis
Our gene has three Arabidopsis homologues, others have been identified in monocots such as rice (OS04G0379900) and sorghum (SORB1_3006G048700).
- To check the possible function of our gene we searched the literature for its homologue in Arabidopsis thaliana, the most similar to our gene is AT2G43710.
- This gene has an average 80% sequence similarity with our gene. Based on this similarity we can hypothesize that the two proteins have similar functions.
- The gene AT2G43710 is also known as SSI2/FAB2 and it is expressed in different parts of the Arabidopsis leaves, shoots, inflorescence and seeds. It belongs to the S-ACP-DES family. It is involved in lots of biological processes including fatty acid biosynthesis and plant immune defense.
- A research conducted by Kachroo et al., 2007 showed that the change in the levels of oleic acid (18:1) in Arabidopsis SSI2/ FAB2 mutant resulted in an alteration in salicylic and jasmonic acid-mediated immune response.
01
Work Package one
'Developing the lines to study our gene expression and gene function'
WP1:objectives
objectives
- Develop a Loss of function (LOF) mutant that will be useful in the characterization of our gene action in the fatty acid biosynthesis pathway.
- Develop overexpression lines to compare with the wild type and LOF mutants.
TASKs: Developing mutant lines using CRISPR-Cas9 gene editing method
CRISPR/Cas9 for targeted mutagenesis
Task 1.1: Design and construction of plasmid vector for loss of function mutant.
- Construct delivery in immature embryos using Agrobacterium mediated technique
Task 1.2: Screening LOF mutants
TASK 1.3: Developing overexpression lines
- Overexpression vector construction
- Build a construct which would include our gene placed under rice ubiquitin promoter
- Transformation in Agrobacterium and delivery of construct to maize embryo.
- The plants are screened and transformed lines are regenerated and will be used in further research
Material and Method of WP1
Figure 1: pRGEB32-BAR Vector for the gene editing in maize using CRISPR-Cas9 (Hunter, 2021).
Task 1.1: Generating LOF mutants
- CRISPR-P (http://crispr.hzau.edu.cn/CRISPR2/) was used to design specific guideRNA for the gene editing.
- Guide143v with the sequence ACTTCAGTGCAGTAGCACAGAGG was chosen (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/SCOR
- pRGEB32-BAR vector was used for driving the gene editing for the generation of LOF mutants.
Material and Method of WP1
Task 1.2: Screening LOF mutants
- Initial allele identification is done by direct sequencing of PCR products.
- T0 plants carrying at least two mutated alleles are backcrossed to the wildtype and F1 plants that are used for the subsequent 2 generations for allele isolation.
A: Typical workflow of genotyping strategy to achieve homozygous mutant lines for studying gene functions. B: determining sequences of bi-allelic PCR products directly from Sanger sequencing chromatograms (Hunter, 2021).
Material and Method of WP1
Task 1.3: developing and screening of overexpression lines.
- A plasmid containing our gene sequence driven by the rice ubiquitin promoter in PCAMBIA 1300 vector will be used to transfer Agrobacterium.
- Agrobacterium-mediated transformation of maize embryo.
- To confirm our overexpression line, we would perform qPCR
- To quantify our overexpression line at the RNA level, we perform the RT-qPCR on the mRNA
expected results
- The lines (LOF and Overexpression) generated at this WP will be used in comparing the fatty acid profile in all lines and the wild type in subsequent work packages.
- Morphological phenotypic analysis of the plant's traits during growth will be conducted.
- Phenotypic characterization will be helpful to determine usefulness of mutant in agronomy.
02
Work package two
aim of the work package
Characterize the role of our gene in the fatty acid biosynthesis pathway.
Our objectives include:
• Identifying the product of our gene. •Examining whether there is differential gene expression associated with specific tissues and distinct developmental stages.
aim of the work package
Guidelines
Question one
Question three
Question two
Where and when is our gene expressed in the plant ?
What is the role of our gene?
Where does our gene product work in the cell?
task 2.1: Real-time q-PCR of different tissues in different developmental stages.
Where and when is our gene expressed in the plant?
Sampling WT
Expected results
MasterMIx
Amplification
Extraction
Epidermids (V1-V7) Pericarp (2-20 DAP)
Real-time qPCR
Primers-TaqMan-Actin-Buffer
cDNA (RT from purified mRNA)
2-ΔΔCt
Task 2.2: Subcellular localization using GFP-chimera protein.
Built a fusion protein
Where does our gene product work in the cell?
Stable transfromation method
Recombinant Ti plasmid reinserted into Agrobacterium
Aim of the Task 2.2
We want to carry out a tissue specificity analysis by using the fusion protein under the control of the Zm00001d004019 promoter in maize WT. The experiment will be done on different tissues (epidermis and pericarp) and developmental stages.
Constructs will be then delivered into WT maize immature embryos
Co-coultivation
Observation under a confocal laser scanning microscope.
Microscope
Task 2.3: Heterologous complementation experiment in yeast.
What is the role of our gene?
This approach will help us to understand:
- The gene product by analysing the fatty acid produced by the mutant yeast
- Substrate specificity, thanks to the possibilities of adding to the medium different substrates.
- Better positioning our gene inside the fatty acid desaturation pathway.
Task 2.4: Labeling experiment + gC-MS.
What is the role of our gene?
This analysis needs to be performed on the first leaves at the emergence, and on leaves at different developmental stages, to assert whether it is possible to detect a specific expression pattern. On LOF, WT and OE.
- Analysis of the flow of carbon through the various leaf glycerolipids
- Analysis of the desaturation state of the fatty acids esterified to these glycerolipids
expected results
- Task 2.1: Will be hypothesized through the use of the 2-ΔΔCt method which will allow us to calculate the relative transcript level.
- Task 2.2: Expect to see the fluorescence (GFP) only in the organelles that express our gene’s construct.
- Task 2.3: We presume that our yeast-complemented cells will have a different ratio of unsaturated/saturated fatty acid compared to the wild-type (WT) strain.
- Task 2.4: Concerning the LOF plant and OE we will expect to detect a difference in the presence of labeled monounsaturated fatty acid compared to the WT. Moreover, if we analyze these labeled-isotope molecules over time in the wild type, we will detect possible differences in the labeled molecules between different developmental stages.
03
work package three
"COSTITUTIVE EXPRESSION OF SALICILIC ACID DEFENSE PATHWAY"
Check levels of SA/JA in LOF and WT
EXPERIMENT
- Extract the two phytohormones from frozen samples
- Perform quantification with high-performance liquid chromatography equipment coupled with a mass spectrometer.
80%
of purchases are emotional
Check the expression level of the defense genes related to SA
EXPERIMENT
- We decided to concentrate ourselves on the detection of the level of transcriptome associated with the SA defence pathway
- Analyse the expression level of the well known in corn SA defence gene ZmPAL and ZmPR1
- And perform a retro transcriptase qPCR to detect the expression levels
Expected results
- First of all we would like to detect a different concentration of these phytohormones between WT and LOFs
- Secondly it could be interest to investigate whether is possible to catch a correlation between the genotype (LOFS), the SA content and the phenotype
- From this task we would like to detect a difference in the level of expression of these two genes in the LOFs individuals
04
FUTURE PERSPECTIVE
FUTURE PERSPECTIVE
To perform in future other typology of experiment based on the idea that hight temperature could effectively improve the dwarf phenotype.
With temperature treatment the LOF mutant could be “WT-restored” only for the dwarf trait and not for susceptibility.
Be useful for the country with hot environment to reduce the use of pesticide, still having good yield.
THANK YOU FOR THE ATTENTION
Questions?
What is fdl1-1?
Cuticle -> vital and essential role in organ separation during embryo development. One of the possible genes responsible in maize is called Fused Leaves 1 (FDL1) which encodes for the transcription factor ZmFDL1/MYB94. A loss of function of a recessive mutant of this TF fdl1-1 resulted in the fusion of organs during embryo development post-pollination.
Example of chimera (fusion protein). The cDNA of our gene of interest fused with the marker GFP under the control of the promoter of our gene of interest.
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Stepheine Ukoji
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Transcript
Functional characterization of ZmSSI2/FAB2 in maize mutant as a possible critical factor in fatty acid desaturation
PROJECT Proposal
Applicant’s Names: Marika Pavasini, Angela Gazzoli, Ukoji Stepheine Onyinyechi, Tommaso Foglia Applicant’s IDs: 28701A, 24115A, 36939A, 24213A Date: 29th January 2024
Plant Molecular Biology
index
Introduction
Work Package 1
Work Package 2
Work Package 3
Future perspectives
This research project aims to
Specific
General
World production of top cereals
introduction
Relevance of maize
Maize is one of the oldest cultivated crops. Maize showed the highest production (1.2 billion tons in 2021) and fastest growth over the period (+104 percent since 2000) compared to the other major grains, as it has wider uses in the biofuel and animal feed sectors.
Source: FAO. 2022
introduction
Various mechanisms to resist drought -> tolerance at the cellular level is essential since it allows tolerant plants to maintain cellular homeostasis.
Maize tolerance to drought
Due to the steady changeset of the environment e.g. the decrease of the rainfall and high temperature, studying maize tolerance to drought becomes very important to face the possible decrease of yields.
In contrast, sensitive plants suffer rapid irreversible cell damage essentially due to the degradation of their membranes.
The study of plants’ cuticles could be very important in facing climate change.
introduction
The cuticle has an important role, giving the plants an advantage in reducing the loss of water from leaves surfaces. The maize cuticle is a protective and waxy layer that covers the outer surface of the aerial parts of maize plants, including leaves, stems, seeds and reproductive structures.
our gene - Zm00001d004019
In the fdl1-1 mutant is down-regulated and it is identified to be putatively involved in fatty acids biosynthesis, it is known as SSI2/FAB2. The putative gene product is known as Acyl-[acyl-carrier-protein] desaturase or stearyl acyl carrier protein desaturase (SACD). It is probably involved in the desaturation of the Stearoyl-ACP that led to the production of Oleoyl-ACP.
+ INFO
the putative expression pattern of our gene of interest
It is likely expressed in the plant inflorescence, the pericarp of the embryo and on immature seeds between 2-14 days after pollination.
Similarity with arabidopsis
Our gene has three Arabidopsis homologues, others have been identified in monocots such as rice (OS04G0379900) and sorghum (SORB1_3006G048700).
01
Work Package one
'Developing the lines to study our gene expression and gene function'
WP1:objectives
objectives
TASKs: Developing mutant lines using CRISPR-Cas9 gene editing method
CRISPR/Cas9 for targeted mutagenesis
Task 1.1: Design and construction of plasmid vector for loss of function mutant.
- Construct delivery in immature embryos using Agrobacterium mediated technique
Task 1.2: Screening LOF mutantsTASK 1.3: Developing overexpression lines
Material and Method of WP1
Figure 1: pRGEB32-BAR Vector for the gene editing in maize using CRISPR-Cas9 (Hunter, 2021).
Task 1.1: Generating LOF mutants
Material and Method of WP1
Task 1.2: Screening LOF mutants
A: Typical workflow of genotyping strategy to achieve homozygous mutant lines for studying gene functions. B: determining sequences of bi-allelic PCR products directly from Sanger sequencing chromatograms (Hunter, 2021).
Material and Method of WP1
Task 1.3: developing and screening of overexpression lines.
expected results
02
Work package two
aim of the work package
Characterize the role of our gene in the fatty acid biosynthesis pathway.
Our objectives include: • Identifying the product of our gene. •Examining whether there is differential gene expression associated with specific tissues and distinct developmental stages.
aim of the work package
Guidelines
Question one
Question three
Question two
Where and when is our gene expressed in the plant ?
What is the role of our gene?
Where does our gene product work in the cell?
task 2.1: Real-time q-PCR of different tissues in different developmental stages.
Where and when is our gene expressed in the plant?
Sampling WT
Expected results
MasterMIx
Amplification
Extraction
Epidermids (V1-V7) Pericarp (2-20 DAP)
Real-time qPCR
Primers-TaqMan-Actin-Buffer
cDNA (RT from purified mRNA)
2-ΔΔCt
Task 2.2: Subcellular localization using GFP-chimera protein.
Built a fusion protein
Where does our gene product work in the cell?
Stable transfromation method
Recombinant Ti plasmid reinserted into Agrobacterium
Aim of the Task 2.2
We want to carry out a tissue specificity analysis by using the fusion protein under the control of the Zm00001d004019 promoter in maize WT. The experiment will be done on different tissues (epidermis and pericarp) and developmental stages.
Constructs will be then delivered into WT maize immature embryos
Co-coultivation
Observation under a confocal laser scanning microscope.
Microscope
Task 2.3: Heterologous complementation experiment in yeast.
What is the role of our gene?
This approach will help us to understand:
Task 2.4: Labeling experiment + gC-MS.
What is the role of our gene?
This analysis needs to be performed on the first leaves at the emergence, and on leaves at different developmental stages, to assert whether it is possible to detect a specific expression pattern. On LOF, WT and OE.
expected results
03
work package three
"COSTITUTIVE EXPRESSION OF SALICILIC ACID DEFENSE PATHWAY"
Check levels of SA/JA in LOF and WT
EXPERIMENT
80%
of purchases are emotional
Check the expression level of the defense genes related to SA
EXPERIMENT
Expected results
04
FUTURE PERSPECTIVE
FUTURE PERSPECTIVE
To perform in future other typology of experiment based on the idea that hight temperature could effectively improve the dwarf phenotype.
With temperature treatment the LOF mutant could be “WT-restored” only for the dwarf trait and not for susceptibility.
Be useful for the country with hot environment to reduce the use of pesticide, still having good yield.
THANK YOU FOR THE ATTENTION
Questions?
What is fdl1-1?
Cuticle -> vital and essential role in organ separation during embryo development. One of the possible genes responsible in maize is called Fused Leaves 1 (FDL1) which encodes for the transcription factor ZmFDL1/MYB94. A loss of function of a recessive mutant of this TF fdl1-1 resulted in the fusion of organs during embryo development post-pollination.
Example of chimera (fusion protein). The cDNA of our gene of interest fused with the marker GFP under the control of the promoter of our gene of interest.