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On-line Access: 2022-04-11

Received: 2022-02-25

Revision Accepted: 2022-03-11

Crosschecked: 2022-04-19

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Wenbo PAN

https://orcid.org/0000-0001-9734-3090

Wanggen ZHANG

https://orcid.org/0000-0003-4861-5488

Huawei ZHANG

https://orcid.org/0000-0002-8893-1429

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Journal of Zhejiang University SCIENCE B 2022 Vol.23 No.4 P.339-344

http://doi.org/10.1631/jzus.B2200119


Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators


Author(s):  Wenbo PAN, Zhentao CHENG, Zhiguo HAN, Hong YANG, Wanggen ZHANG, Huawei ZHANG

Affiliation(s):  School of Advanced Agricultural Sciences, Peking University, Beijing 100871, China; more

Corresponding email(s):   wanggen.zhang@pku-iaas.edu.cn, huawei.zhang@pku-iaas.edu.cn

Key Words:  Genetic transformation, Developmental regulator, GRF5, Watermelon, Cucurbitaceae, Genome editing


Wenbo PAN, Zhentao CHENG, Zhiguo HAN, Hong YANG, Wanggen ZHANG, Huawei ZHANG. Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators[J]. Journal of Zhejiang University Science B, 2022, 23(4): 339-344.

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author="Wenbo PAN, Zhentao CHENG, Zhiguo HAN, Hong YANG, Wanggen ZHANG, Huawei ZHANG",
journal="Journal of Zhejiang University Science B",
volume="23",
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pages="339-344",
year="2022",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B2200119"
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%A Wenbo PAN
%A Zhentao CHENG
%A Zhiguo HAN
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%A Wanggen ZHANG
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T1 - Efficient genetic transformation and CRISPR/Cas9-mediated genome editing of watermelon assisted by genes encoding developmental regulators
A1 - Wenbo PAN
A1 - Zhentao CHENG
A1 - Zhiguo HAN
A1 - Hong YANG
A1 - Wanggen ZHANG
A1 - Huawei ZHANG
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Abstract: 
cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.

生长调节基因介导的西瓜高效遗传转化与CRISPR/Cas9基因编辑体系

目的:克服西瓜遗传转化瓶颈,建立西瓜高效遗传转化体系,并以此为基础构建西瓜基因编辑体系,促进西瓜功能基因组学和基因编辑育种研究。
创新点:通过比较多个生长调节基因和农杆菌菌株,选取生长调节基因AtGRF5和农杆菌菌株GV3101,在西瓜中建立了高效且不依赖于基因型的转化体系,实现了西瓜突变体的快速创制。
方法:通过测试生长调节因子基因对西瓜转化率的影响,发现多个生长调节基因可以提高西瓜的遗传转化效率。其中AtGRF5可将西瓜的转化效率0.88%左右提高至24.73%。此外,筛选了不同的农杆菌菌株,发现使用GV3101表达AtGRF5可获得最高的转化效率。进一步使用AtGRF5辅助转化的方式对西瓜进行基因编辑,成功在T0代对PDS基因进行敲除并获得纯合突变体。
结论:使用农杆菌GV3101过表达AtGRF5可将西瓜的转化效率从0.88%左右提高至24.73%,且在两个基因型的西瓜中作用类似。该方法也可结合基因编辑技术用于西瓜突变体的快速获取。

关键词:遗传转化;生长调节基因;GRF5;西瓜;葫芦科;基因编辑

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

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