Name: In Planta Gene Expression and Measurement of Chlorophyll Fluorescence of Non-Photochemical Quenching Values in Bamboo Leaves
Uploaded: 2023-08-18T12:30:00
Description: Watch this Scientific Journal Video about In Planta Gene Expression and Measurement of Chlorophyll Fluorescence of Non-Photochemical Quenching Values in Bamboo Leaves at JoVE.com

in planta gene expression and measurement of chlorophyll fluorescence of non-photochemical quenching values in bamboo leaves

Udvikling af gentransformations- og redigeringsmetoder i bambus

The investigation of gene function in bamboo holds great promise for the advanced understanding of bamboo and unlocking its potential for genetic modification. An effective way of this can be achieved through the process of Agrobacterium-mediated infection in bamboo leaves, whereby the T-DNA fragment containing exogenous genes is introduced into the cells, subsequently leading to the expression of the genes within the leaf cells.
Bamboo is a valuable and renewable resource with a wide range of applications in manufacturing, art, and research. Bamboo possesses excellent wood properties such as high mechanical strength, toughness, moderate stiffness, and flexibility1, which is now widely used in a variety of household and industrial supplies, including toothbrushes, straws, buttons, disposable tableware, underground pipelines, and cooling tower fillers for thermal power generation. Therefore, bamboo breeding plays a crucial role in obtaining bamboo varieties with excellent wood properties for replacing plastics and reducing plastic usage, protecting the environment, and tackling climate change, as well as generating significant economic value.
However, traditional bamboo breeding faces challenges due to the lengthy vegetative growth stage and uncertain flowering period. Although molecular breeding techniques have been developed and applied to bamboo breeding, the process of bamboo gene transformation is time-consuming, labor-intensive, and complicated due to the callus induction and regeneration processes2,3,4,5. Stable genetic transformation often requires Agrobacterium-mediated methods, which involve tissue culture processes such as callus induction and regeneration. However, bamboo has a low ability for callus regeneration, greatly limiting the application of stable genetic transformation in bamboo. After Agrobacterium infects plant cells, the T-DNA fragment enters the plant cells, with the majority of T-DNA fragments remaining non-integrated in the cells, resulting in transient expression. Only a small portion of T-DNA fragments randomly integrate into its chromosome, leading to stable expression. The transient expression levels show an accumulation curve that can vary for each gene expressed from an Agrobacterium-delivered T-DNA. In most cases, the highest expression levels occur 3-4 days after infiltration and quickly decrease after 5-6 days6,7. Previous studies have shown that more than 1/3rd of mutations in gene-edited plants obtained without selection pressure for resistance come from the transient expression of CRISPR/Cas9, while the remaining less than 2/3rd come from stable expression after DNA integration into the genome8. This indicates that T-DNA integration into the plant genome is not necessary for gene editing. Moreover, selection pressure for resistance significantly inhibits the growth of non-transgenic cells, directly affecting the regeneration process of infected explants. Therefore, by using transient expression without selection pressure for resistance in bamboo, it is possible to achieve non-integrated expression of exogenous genes and study gene function directly in plant organs. Hence, an easy and time-saving method can be developed for exogenous gene expression and editing in bamboo9.
The developed exogenous gene expression and gene editing method is characterized by its simplicity, cost-effectiveness, and the absence of expensive equipment or complex procedures9. In this method, the bamboo endogenous violaxanthin de-epoxidase gene (PeVDE) was used as the reporter for exogenous gene expression without selection pressure. This is because the edited PeVDE in bamboo leaves reduces the photoprotection ability under high light and demonstrates a decrease in the non-photochemical quenching (NPQ) value, which can be detected through chlorophyll fluorescence imaging. To demonstrate the effectiveness of this method, another bamboo endogenous gene, the cinnamoyl-CoA reductase gene (PeCCR5)9, was knocked out using this system and successfully generated mutants of this gene. This technique can be used for the functional characterization of genes that have functions in bamboo leaves. By overexpressing these genes transiently in bamboo leaves, their expression levels can be enhanced, or by gene editing, their expression can be knocked down, allowing for the study of downstream gene expression levels, leaf phenotypes, and product contents. This provides a more efficient and feasible approach for gene function research in bamboo. This technique can be applied to the functional characterization of genes that function in bamboo leaves. By overexpressing these genes transiently in bamboo leaves, their expression levels can be enhanced, or by gene editing, their expression can be knocked down, allowing for the study of downstream gene expression levels, leaf phenotypes, and product contents. Additionally, it is important to note that, due to extensive polyploidization, the majority of commercially important genes in bamboo genomes are present in multiple copies, resulting in genetic redundancy. This poses a challenge for performing multiplex genome editing in bamboo. Prior to the application of stable genetic transformation or gene editing techniques, it is crucial to quickly validate gene functions. In addressing the issue of multiple gene copies, one approach is to analyze transcriptome expression profiles to identify genes that are actively expressed during specific stages. Furthermore, targeting the conserved functional domains of these gene copies allows for the design of common target sequences or the incorporation of multiple target sites into the same CRISPR/Cas9 vector, enabling the simultaneous knockout of these genes. This provides a more efficient and feasible approach for gene function research in bamboo.

Forfatter Spotlight: Fremme af genredigering i bambusblade for bæredygtige plastalternativer 

I Planta Genekspression og genredigering i Moso bambusblade

In this study, we used Agrobacterium as a vector to introduce foreign DNA fragments into bamboo leaves. Most DNA fragments are not integrated into chromosomes and undergo transient expression. We took advantage of this transient expression to carry out gene editing.The main challenge is the limitation of gene editing of bamboo leaves, which cannot regenerate. This propose difficulties in performing editing in regenerative organs, like young shoots, and obtaining edited branches or subsequent generations with desired genetic challenges. Its main advantage is the time-saving aspect of the principle.By being able to edit bamboo genes directly in bamboo leaves, it allows for quick verification of gene fractions. This eliminates the needs for time-consuming procedures, like tissue culture or transformation, significantly speeding up research on bamboo gene fractions. The primary research will be focused on investigating the molecular mechanism of bamboo biomass formation.This will involve understanding why bamboo grows rapidly, and using that knowledge to develop bamboo varieties which are suitable for plastic substituent.

Watch this Scientific Journal Video about In Planta Gene Expression and Measurement of Chlorophyll Fluorescence of Non-Photochemical Quenching Values in Bamboo Leaves at JoVE.com

In Planta Gene Expression and Measurement of Chlorophyll Fluorescence of Non-Photochemical Quenching Values in Bamboo Leaves  

I planta genekspression og måling af klorofylfluorescens af ikke-fotokemiske slukningsværdier i bambusblade

Begynd med at udvinde det genomiske DNA fra de vildtype- og Agrobacterium-inficerede bambusblade. Forstærk det genomiske DNA, der indeholder målstedet for målgenerne fra vildtype- og Agrobacterium-inficerede bambusblade ved hjælp af følgende PCR-betingelser. Efter PCR udføres endonukleaseenzymfordøjelse ved at fremstille en reaktionsblanding indeholdende en mikroliter alder-1, et mikrogram PCR-produkter og fem mikroliter 10X buffer.Derefter tilsættes vand til et endeligt volumen på 50 mikroliter. Inkuber fordøjelsesblandingen ved 37 grader Celsius i en time. Brug gelelektroforese til at analysere andelen af fordøjede DNA-fragmenter.Sammenlign de fordøjede fragmenter fra vildtype- og Agrobacterium-inficerede prøver for at vurdere genredigeringseffektiviteten. Udsæt bambusplanterne for forhold med høj lysintensitet for at øge mængden af absorberet lyskvantum og aktivering af bladets fotobeskyttelsessystem. Tænd derefter IMAGING-PAM-fluorometeret og indstil den aktiniske lysintensitet til måling af in vivo-fotosystem II klorofylfluorescens af bambusblade.Den røde beta-banefarve produceret af RUBY-genet indikerede vellykket Agrobacterium-medieret genekspression i bambusblade. Ud af de fire Agrobacterium-stammer forårsagede GV3101-stammen den mest betydningsfulde beta-baneakkumulering i inficerede bambusblade. Efter infektion med CRISPR-Cas9-konstruktionerne og højlysbehandlinger viste visse områder af bladbladene lavere ikke-fotokemiske slukningsværdier.Desuden bekræftede enzymfordøjelse og sekventeringsanalyse den vellykkede mutation af PeVDE-genet i de redigerede regioner. Sanger-sekventeringsresultater af PeVDE-fragmentet efter redigering af både sgRNA1 og sgRNA2 viste deletion af et langt fragment i PeVDE-genet. Efter 30 dages Agrobacterium-infektion udviste bladområderne transfekteret med sgRNA'er for både PeVDE og PeCCR5 lavere ikke-fotokemiske slukningsværdier.

Research

JoVE Journal

Biology

In Planta Gene Expression and Measurement of Chlorophyll Fluorescence of Non-Photochemical Quenching Values in Bamboo Leaves

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and ...