SILENCING THE Carica papaya β-1,3-GLUCANASE GENE BY CRISPRCas9

Name: Mirielson Loures da Silva
Type: MSc dissertation
Publication date: 18/03/2021
Advisor:

Namesort descending Role
Patricia Machado Bueno Fernandes (M/D) Advisor *

Examining board:

Namesort descending Role
Jose Aires Ventura (M/D) Internal Examiner *
Patricia Machado Bueno Fernandes (M/D) Advisor *
Silas Pessini Rodrigues External Examiner *

Summary: SILVA, M.L. Silencing of the Carica papaya β-1,3-Glucanase gene by CRISPR-Cas9.
2021. 75f. Dissertation (Master in Biotechnology) – Postgraduation Biotechnological
Programme, UFES, Espírito Santo. Brazil.
Callose deposition in plasmodesma is directly associated with viral movement. The
prediction of micro RNAs from papaya infected by the papaya meleira virus viral
complex (PMeV complex) revealed a high presence of miRNAs repressing the posttranscriptional expression of genes involved in the synthesis of β-1,3-glucanases. In
this sense, our study aimed to evaluate the silencing of the β-1,3-glucanase enzyme
gene as a form of resistance to the PMeV complex that causes the papaya sticky
disease. For this purpose, the new CRISPR/Cas9 gene-editing technology was used.
To attest to the functioning of the CRISPR/Cas9 technique in papaya tissues, we
sought to develop a visual marker interrupting chlorophyll biosynthesis by silencing the
phytoene desaturase gene in papaya leaves, cells and protoplasts. For the
transformation of papaya tissues by agroinfiltration, the binary vector pKSE401 was
used, which contains the Cas9 enzyme gene and a customizable sequence referring
to the structure of the guide RNA. The new plasmids generated were inserted by
electroporation into Agrobacterium tumefaciens GV3101, which was renamed
AtpKSPD and AtpKSBG for silencing of phytoene desaturase and β-1,3-glucanase,
respectively. As a result, all papaya leaves that were submitted to agroinfiltration by
AtpKSPD showed depigmentation due to the inactivation of the phytoene desaturase
gene and the same was observed in embryos regenerated from transformed cells. The
agroinfiltrated tissues expressed the customized gRNA together with Cas9. The same
method was used for AtpKSBG. The results obtained attest that the use of
CRISPR/Cas9 technology can be successfully applied in Carica papaya. We
emphasize that so far there is no established protocol for editing C. papaya by CRISPR
technology, thus, our work is a pioneer in the application of the technique in papaya,
also highlighting the pioneering in β-1,3-glucanase and phytoene desaturase gene
silencing in this crop, the second being a promising visual marker of the technique`s
functioning for C. papaya.
Keywords: Genetical enhancement. Genetic engineering. Gene inactivation.
Agribusiness. Cisgenic.

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