Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven
Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven
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百合高效遗传转化体系的建立及CRISPR/Cas9基因组编辑技术的应用。
DOI：
10.3390/ijms20122920
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发表时间：
2019-06
影响因子：
5.6
通讯作者：
Sun Hongmei
中科院分区：
文献类型：
--
作者：
Yan Rui;Wang Zhiping;Ren Yamin;Li Hongyu;Liu Na;Sun Hongmei
百合属是一种球根花卉，分布于世界各地，具有独特的地下器官。缺乏有效的百合遗传转化系统一直是国际上的障碍。由于现有的模式植物缺乏鳞茎，因此无法在模式植物中进行鳞茎相关基因功能验证研究。在这里，在两个百合属物种中建立了两个基于体细胞胚胎发生和不定芽再生的稳定高效的遗传转化系统。转基因植物和 T-DNA 插入系通过 β-葡萄糖醛酸酶 (GUS) 测定、聚合酶链式反应 (PCR) 和 Southern blot 进行确认。经过条件优化后，百合DC的转化效率分别提高到29.17%和4%。费施。和百合“白色天堂”，分别。为了进一步验证这些转化系统的有效性并将CRISPR/Cas9（成簇规则间隔短回文重复序列（CRISPR）相关蛋白9）技术应用于百合，我们敲除了两个百合物种中的LpPDS基因。观察到完全白化、浅黄色和白化-绿色嵌合突变体。转基因品系的序列分析揭示了各种突变模式，包括碱基插入、缺失和取代。这些结果验证了两种转化系统的可行性和高效性，并首次验证了CRISPR/Cas9系统在百合基因编辑中的成功应用。总体而言，本研究为百合基因功能研究和种质改良奠定了重要基础。
Lilium spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for Lilium has been an international obstacle. Because existing model plants lack bulbs, bulb-related gene function verification studies cannot be carried out in model plants. Here, two stable and efficient genetic transformation systems based on somatic embryogenesis and adventitious bud regeneration were established in two Lilium species. Transgenic plants and T-DNA insertion lines were confirmed by β-glucuronidase (GUS) assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in Lilium pumilum DC. Fisch. and the Lilium longiflorum ‘White Heaven’, respectively. To further verify the validity of these transformation systems and apply the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9) technology in Lilium, the LpPDS gene in the two Lilium species was knocked out. Completely albino, pale yellow and albino–green chimeric mutants were observed. Sequence analysis in the transgenic lines revealed various mutation patterns, including base insertion, deletion and substitution. These results verified the feasibility and high efficiency of both transformation systems and the successful application of the CRISPR/Cas9 system to gene editing in Lilium for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in Lilium spp.
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影响因子：
13.8
作者：
Lin CS;Hsu CT;Yang LH;Lee LY;Fu JY;Cheng QW;Wu FH;Hsiao HC;Zhang Y;Zhang R;Chang WJ;Yu CT;Wang W;Liao LJ;Gelvin SB;Shih MC
通讯作者：
Shih MC
影响因子：
2.4
作者：
P. Azadi;Ntui Valentine Otang;D. Chin;I. Nakamura;M. Fujisawa;H. Harada;N. Misawa;M. Mii
通讯作者：
P. Azadi;Ntui Valentine Otang;D. Chin;I. Nakamura;M. Fujisawa;H. Harada;N. Misawa;M. Mii
多个百合品种的再生和农杆菌介导的转化
DOI：
10.1007/s11240-012-0172-3
发表时间：
2012-05
期刊：
Plant Cell, Tissue and Organ Culture
影响因子：
--
作者：
Menzel, Tila;Maliepaard, Chris;Shen, Xiaohui;Krens, Frans
通讯作者：
Krens, Frans
第一代杨树中 CRISPR/Cas9 介导的高效定点诱变
影响因子：
4.6
作者：
Fan D;Liu T;Li C;Jiao B;Li S;Hou Y;Luo K
通讯作者：
Luo K
影响因子：
6.2
作者：
Bassuner, Burgund M.;Lam, Russell;Yeung, Edward C.
通讯作者：
Yeung, Edward C.