图1 柑橘WOX、GRF、GIF基因家族保守功能结构域与进化树以及顺式元件分析 A：最大似然法（ML）构建的WOX、GRF、GIF基因家族系统发育树，Cs：甜橙、At：拟南芥、Os：水稻。B：甜橙WOX、GRF、GIF基因家族顺式元件分析，数字表示每个基因对应的顺式作用元件的数量，颜色越深表示顺式作用元件的数量越多

Fig. 1 Phylogenetic，conserved domain and cis-acting elements analysis of the WOX，GRF，and GIF gene families in citrus A：Maximum-likelihood（ML）phylogenetic trees of the WOX，GRF，and GIF gene families. Cs：Citrus sinensis；At：Arabidopsis thaliana；Os：Oryza sativa. B：cis-Regulatory element analysis of the WOX，GRF，and GIF gene families in sweet orange. The numbers indicate the quantity of cis-acting elements associated with each gene，and darker colors represent a higher abundance of cis-acting elements

图2 甜橙悬浮细胞体细胞胚发生与分化发育过程中WOX、GRF、GIF基因家族的表达分析 A：甜橙悬浮细胞体细胞胚发生与分化过程。i：悬浮细胞诱导；ii：球形胚（绿色箭头）、心形胚（黄色箭头）、鱼雷形胚（蓝色箭头）、成熟胚（红色箭头）；iii：进一步分化的成熟胚；iv：形成绿色芽点的体细胞胚；v：4个发育期的胚，1对应球形胚或心形胚，2为子叶型胚，3为中后期胚，4为分化出胚根等组织的后期胚。B：体细胞胚分化过程中WOX、GRF、GIF基因家族表达动态。以CsACT2为内参基因，以未分化的悬浮细胞的基因表达量设定为1。Student’s t 检验，** P < 0.01

Fig. 2 Expression analysis of WOX，GRF，and GIF gene families during somatic embryogenesis and differentiation in sweet orange suspension cells A：Somatic embryogenesis and differentiation of sweet orange（Citrus sinensis）suspension cells. i：Suspension cells；ii：Globular embryos（green arrows），heart-shaped embryos（yellow arrows），torpedo-shaped embryos（blue arrows），and mature embryos（red arrows）；iii：Further differentiation of mature embryos；iv：Shoot regeneration of somatic embryos；v：Four developmental stages used for expression analysis，where stage 1 corresponds to globular or heart-shaped embryos，stage 2 to cotyledonary embryos，stage 3 to mid-to-late embryos，and stage 4 to late-stage embryos with differentiated tissues such as the embryonic radicle. B：Expression dynamics of WOX，GRF，and GIF gene families during somatic embryogenesis. Gene expression levels were normalized to CsACT2 as the internal reference gene，and the expression level in undifferentiated suspension cells was set to 1. Student’s t-test，with ** P < 0.01

图3 CRISPR/dCas9-TV系统在柑橘原生质体中实现靶基因表达的高效激活 A：CRISPR/dCas9-TV系统载体示意图（35S：花椰菜花叶病毒35S启动子；HygR：潮霉素抗性；ZmUbi-1pro：玉米泛素基因-1启动子；tRNA：拟南芥甘氨酸tRNA骨架；sgRNA：单链向导RNA；HSP：热激蛋白终止子；AtUBQ10pro：拟南芥泛素10启动子；2x flag-tag：2个拷贝的flag标签蛋白；NLS：细胞核定位序列；TAL：黄单胞菌TALE转录激活子；VP64：疱疹病毒VP64转录激活结构域；NOS：农杆菌胭脂碱合成酶终止子；LB/RB：农杆菌T-DNA左边界/右边界）。B：sgRNA设计示意图（每个柑橘基因设计2个靶向启动子区保守区域的sgRNA，采用PTG加工系统实现一个载体同时表达多个sgRNA）。C：柑橘叶片原生质体转染35S::GFP质粒验证转染效率。转染后20 h的原生质体在荧光显微镜下观察绿色荧光蛋白表达效率。D：蛋白质印迹（Western blot）分析柑橘原生质体转染后，CRISPR/dCas9-TV系统中Flag-dCas9-TV融合蛋白的表达水平。E：利用柑橘原生质体瞬时表达系统验证CRISPR/dCas9-TV对靶标基因的激活效果。以CsACT2为内参基因，以转染了空载质粒的基因表达量设定为1。Student’s t 检验，** P < 0.01

Fig. 3 Efficient activation of target gene expression in citrus protoplasts using the CRISPR/dCas9-TV system A：Schematic diagram of the CRISPR/dCas9-TV system vector. Key elements are labeled：35S（Cauliflower mosaic virus 35S promoter）；HygR （Hygromycin resistance）；ZmUbi-1pro（Zea mays ubiquitin gene-1 promoter）；tRNA（Arabidopsis thaliana glycine tRNA scaffold）；sgRNA （Single-guide RNA）；HSP（Heat shock protein terminator）；AtUBQ10pro（Arabidopsis thaliana ubiquitin 10 promoter）；2x flag-tag（Two copies of flag-tag protein）；NLS（Nuclear localization signal）；TAL（Xanthomonas TALE transcriptional activator）；VP64（Herpes simplex virus VP64 transcriptional activation domain）；NOS（Agrobacterium nopaline synthase terminator）；LB/RB（Left border/Right border of the Agrobacterium T-DNA）. B：Schematic of sgRNA design. Two sgRNAs were designed for each citrus gene to target conserved regions within the promoter. The PTG （Polycistronic tRNA-gRNA）processing system was employed to enable simultaneous expression of multiple sgRNAs from a single vector. C：Validation of transfection efficiency in citrus leaf protoplasts using a 35S：：GFP plasmid. GFP（Green Fluorescent Protein）expression efficiency was observed under a fluorescence microscope 20 hours post-transfection. D：Western blot analysis of Flag-dCas9-TV fusion protein expression. Protein levels were assessed in citrus protoplasts following transfection with the CRISPR/dCas9-TV system. E：Validation of target gene activation by the CRISPR/dCas9-TV system using a citrus protoplast transient expression assay. Relative gene expression levels are shown，with the expression level in protoplasts transfected with the empty vector set to 1. CsACT2 was used as the internal reference gene.** P < 0.01 by Student’s t-test

图4 激活CsWUS促进甜橙胚性愈伤悬浮细胞分化及再生 mpi：侵染后月份数。空载对照诱导培养5个月仍未分化成体细胞胚；转染dCas9-TV-CsWUS质粒在筛选培养基下1个月内快速增殖形成新愈伤（蓝色箭头），2个月分化出球形胚（红色箭头），4个月后分化绿色的芽，5个月后形成绿色的叶片

Fig. 4 Activation of CsWUS promoted differentiation and regeneration of embryogenic callus suspension cells in sweet orange mpi：Months post-infection. No somatic embryos were observed in the empty vector control after 5 months of induction culture. In contrast，protoplasts transfected with the dCas9-TV-CsWUS construct rapidly proliferated and formed new callus tissue（blue arrow）within one month on selection medium. These calli subsequently developed globular embryos（red arrow）by two months，differentiated green buds by four months，and ultimately produced green leaves after five months of culture

图5 激活CpWUS、CpWOX9、CpGRF4&CpGIF1提高了葡萄柚上胚轴段的再生

Fig. 5 Activation of CpWUS，CpWOX9，and CpGRF4&CpGIF1 improves regeneration of grapefruit epicotyl explants

图6 激活ClWUS、ClWOX9和ClWRKY42提升柠檬叶片毛状根再生率和转化率，并促进类胡萝卜素积累 A：转染DR5pro::RUBY的柠檬叶片诱导形成紫色毛状根再生植株。i：叶片诱导毛状根；ii：将毛状根切割成约1 cm长的小段后进一步组织培养，iii：毛状根段的伤口处再生出绿色芽，iv：继续培养可形成带根的植株。B：与对照组相比，转染dCas9-TV-ClWUS&ClWOX9&ClWRKY42的柠檬叶片诱导形成的毛状根数量更多，且因类胡萝卜素积累呈现黄色。C、D：对B中对照组与试验组毛状根数量和转化阳性率进行统计分析。E：转化阴性与转化阳性毛状根中ClWUS、ClWOX9 和 ClWRKY42的表达差异。Student’s t 检验，* P < 0.05，** P < 0.01

Fig. 6 Activation of ClWUS&ClWOX9&ClWRKY42 resulted in enhanced transformation and regeneration rates of hairy roots A：Lemon leaves transfected with DR5pro::RUBY induced the formation of purple hairy roots and subsequently regenerated into plantlets. i：Leaf explants inducing hairy roots；ii：Hairy roots excised into 1 cm segments for further tissue culture；iii：Green buds regenerating from the cut ends of hairy root segments；iv：Continued culture resulting in rooted plantlets. B：Compared with the control，lemon leaves transfected with dCas9-TV-ClWUS/ClWOX9/ClWRKY42 produced a significantly higher number of hairy roots，which exhibited a yellow coloration due to carotenoid accumulation. C，D：Statistical analysis of hairy root number and positive transformation rate for the control and experimental groups shown in picture B. E：Relative expression levels of ClWUS，ClWOX9 and ClWRKY42 in transformation-negative and transformation-positive hairy roots. Student’s t-test，* P < 0.05；** P < 0.01