Articles
Establishment of a synchronized gene editing and complementation system of watermelon ClCENH3
Article number
1411_37
Pages
365 – 372
Language
English
Abstract
CENH3, a centromere-specific histone variant, plays a crucial role in centromere assembly and spindle attachment during cellular meiosis, ensuring accurate distribution of genetic information to daughter cells.
Studies in the model plant Arabidopsis have demonstrated that modified CENH3 can facilitate haploid production.
Consequently, the establishment of a one-step system for CENH3 gene editing and complementation can drastically reduce the production cycle of haploid induction lines.
However, such a system is currently lacking in watermelon, a globally important horticultural fruit crop.
In this study, we identified the ClCENH3 gene through homologous alignment analysis and successfully cloned it in watermelon.
To determine its tissue expression pattern, we performed qRT-PCR analyses of ClCENH3 transcripts in eight different tissues.
The results showed predominant expression in rapidly dividing tissues, such as shoot apices and young fruits, indicating its role in regulating cell division.
Subcellular localization experiments using GFP-tagged detection further validated the nuclear localization of the ClCENH3 protein.
To establish a synchronized gene editing and complementation system mediated by ClCENH3, we engineered the vector pBSE402-CENH3-αN-BH, containing a deletion of the αN helix of the ClCENH3 gene with edited ClCENH3 of two targets.
This construct was then transformed into watermelon germplasm ‘YL’ using the Agrobacterium rhizogenes (Ar.Qual)-mediated hairy root transformation technique, and successful transformations were screened based on GFP fluorescence.
DNA extraction, PCR analysis, and sequencing results confirmed the effective functioning of both targets.
In conclusion, we successfully constructed the ClCENH3-mediated synchronized gene editing and complementation system in watermelon.
This study introduces a novel experimental system, providing a valuable genetic resource and technical support for the future establishment of a ClCENH3-mediated haploid induction system in watermelon.
Studies in the model plant Arabidopsis have demonstrated that modified CENH3 can facilitate haploid production.
Consequently, the establishment of a one-step system for CENH3 gene editing and complementation can drastically reduce the production cycle of haploid induction lines.
However, such a system is currently lacking in watermelon, a globally important horticultural fruit crop.
In this study, we identified the ClCENH3 gene through homologous alignment analysis and successfully cloned it in watermelon.
To determine its tissue expression pattern, we performed qRT-PCR analyses of ClCENH3 transcripts in eight different tissues.
The results showed predominant expression in rapidly dividing tissues, such as shoot apices and young fruits, indicating its role in regulating cell division.
Subcellular localization experiments using GFP-tagged detection further validated the nuclear localization of the ClCENH3 protein.
To establish a synchronized gene editing and complementation system mediated by ClCENH3, we engineered the vector pBSE402-CENH3-αN-BH, containing a deletion of the αN helix of the ClCENH3 gene with edited ClCENH3 of two targets.
This construct was then transformed into watermelon germplasm ‘YL’ using the Agrobacterium rhizogenes (Ar.Qual)-mediated hairy root transformation technique, and successful transformations were screened based on GFP fluorescence.
DNA extraction, PCR analysis, and sequencing results confirmed the effective functioning of both targets.
In conclusion, we successfully constructed the ClCENH3-mediated synchronized gene editing and complementation system in watermelon.
This study introduces a novel experimental system, providing a valuable genetic resource and technical support for the future establishment of a ClCENH3-mediated haploid induction system in watermelon.
Publication
Authors
Shujuan Tian, Guoqi Xu, Gongli Ai, Jiao Jiang, Xiner Chen, Yangyuqi Zhang, Jinghao Jiang, Yuxuan Wei, Jiafa Wang, Man Liu, Xian Zhang, Li Yuan
Keywords
watermelon, qRT-PCR analyses, tissue expression pattern, subcellular localization, genetic transformation mediated by Agrobacterium rhizogenes, gene editing
Groups involved
Online Articles (37)