作者感谢国家重点研发计划（批准号：2021YFD2200502）和国家自然科学基金（批准号：31971736）的资助。

1.竹苗的制备
<ol><li>使用在中国广西桂林收获的种子准备毛竹（Phyllostachys edulis）幼苗。首先将种子浸泡在水中 2-3 天，确保每天换水。接下来，通过将土壤和蛭石以 3：1 的比例混合来创建基质。</li>
	<li>将浸泡的种子播种到基质中发芽。在实验室条件下保持幼苗，将温度保持在18-25°C之间。 在光相期间，确保16小时光照/ 8小时暗光周期，光强度为250-350μmol/ m2 / s。</li>
	<li>保持约60%的相对湿度。对于 农杆菌感染，使用15日龄且高度为2-10厘米的幼苗，这是转化的最佳阶段。 注意：由于竹子开花是不可预测的，因此并非每年都有种子。种子通常在4°C的干燥环境中储存2-3年，仍能保持20%以上的活力。</li>
</ol>2. 质粒和农杆菌的制备
<ol><li>质粒：为了验证瞬时表达效果，使用pHDE-35S：：RUBY构建体，该构建体包含由CaMV 35S启动子10驱动的可见报告基因。对于基因编辑，使用 pCambia1300-Ubi：：Cas9 构建体，该构建体携带由玉米 Ubi 启动子11 驱动的 Cas9 基因。在 pCambia1300-Ubi：：Cas9 构建体9 中的两个 AarI 位点之间插入 PeVDE 和其他靶基因的 sgRNA 引导序列。</li>
	<li>在 pCambia1300-Ubi：：Cas9 构建体的两个 AarI 限制性核酸内切酶位点之间插入 CRISPR/Cas9 引导 RNA 序列，包括 PeVDE 和 PeCCR5 靶基因。</li>
	<li>将 GGCA 添加到 20 个核苷酸序列的 5' 端并合成单链 DNA 序列。反向补补 20 个核苷酸序列，并在 5' 端加入 AAAC，然后合成另一个单链 DNA 序列。</li>
	<li>在稀释的水中将两个单链DNA序列稀释至10nM / L的浓度，充分混合，并在95°C下加热5分钟。让混合物冷却至室温，从而形成双股适配器。</li>
	<li>将接头与使用 T4 DNA 连接酶消化的 AarI 核酸内切酶消化的线性 pCambia1300-Ubi：：Cas9 片段连接，并对构建的 CRISPR/Cas9 载体进行测序以获得所需的基因靶向构建体。</li>
	<li>要将质粒转化为 农杆菌，请使用如下冻融方法：将 1 μL 质粒（浓度：10 - 1,000 ng/μL）与 100 μL 农杆菌感受态细胞（翻译效率：> 1 x 104 个菌落形成单位/μg）混合并轻轻混合。将混合物放在冰上5分钟。将混合物转移到液氮中5分钟。</li>
	<li>将混合物在37°C水浴中解冻5分钟。向混合物中加入500μL的Luria-Bertani（LB）培养基，并在28°C，200rpm的振荡培养箱上孵育2-3小时。对于 pHDE-35S：：RUBY 质粒，将它们分别引入根癌农杆菌 （A. tumefaciens） 的 AGL1、GV3101、LBA4044 和 EHA105 菌株中12。对于 CRISPR/Cas9 质粒，将它们引入根癌不动杆菌的 GV3101 菌株中。</li>
	<li>在酵母提取物蛋白胨（YEP）培养基（10g牛肉提取物，10g酵母提取物和5g NaCl/L）中用相应的抗生素（用于35S：：RUBY的壮观霉素和用于CRISPR / Cas9的卡那霉素）在28°C下生长农杆菌。 培养后36-48小时观察单个菌落。</li>
	<li>挑选菌落并将其转移到液体YEP培养基（含有相应的抗生素）中以进一步生长。24-36小时后，使用RUBY-F和RUBY-R的引物（表1）进行PCR，以确认质粒成功转移到 农杆菌中。</li>
	<li>将1mL成功转化的 农杆菌 转移到100mL新鲜液体YEP培养基（含有相应的抗生素）中，然后在28°C下生长过夜至OD600 为0.8。</li>
	<li>将细菌悬浮液在4°C下以4,000× g 离心5分钟，用悬浮液浸润培养基（10mM MgCl2 和10mM MES-KOH [pH 5.6]）洗涤细菌沉淀一次，然后再次离心。将细菌颗粒重悬于悬浮渗透培养基中至OD600 为0.6，用于竹子转化。</li>
</ol>3. 农杆菌 介导 的植物 转化系统
<ol><li>为转型做好准备;小心地从基质上取下带有土壤附着根的幼苗，确保根部保持完整。用锡箔纸包裹幼苗以保持水分并防止土壤脱落（图1A）。</li>
	<li>将包裹的幼苗转移到湿度较高（相对空气湿度>90%）和较低光照（强度小于50μmol/ m 2 / s）的环境中，持续2小时。</li>
	<li>使用注射器中的锋利针头，将竹苗卷曲的未成熟叶子的上部（距顶部约1-2厘米）缠绕一次或两次（ 如图1A中的红色三角形所示）。</li>
	<li>之后，将受伤的幼苗上部浸入 农杆菌悬浮液中。从伤口到快速浸入 农杆菌 的整个过程，因为新鲜的伤口将提高接种效率。</li>
	<li>立即将幼苗转移到压力为25-27mmHg的真空室中2分钟（图1B）。</li>
	<li>吸尘后，小心地打开幼苗并将其重新种植到基质中。将幼苗置于昏暗的光线或黑暗（<50μmol/ m2 / s）中，在室温（18-25°C）下高湿度（RH>90%）2天。随后，在正常生长条件下培养幼苗，每 5-7 天浇水一次。观察它们的表型，为后续实验提供材料。</li>
</ol>4. 设计用于基因编辑的单向导RNA（sgRNA）
<ol><li>鉴定位于靶基因序列的特定和保守结构域附近的原间隔相邻基序 （PAM） 位点。确保该 CRISPR/Cas9 系统中的特定 PAM 序列为 NGG。通过对竹子基因组数据库执行 BlastN 搜索来验证所选序列的靶向特异性。确保 sgRNA 是唯一的，尤其是在上游区域和接近 PAM 9,11 的区域。 注意：这种比较将有效减少受基因编辑过程影响的基因组中潜在的脱靶位点。</li>
	<li>在 PeVDE 基因13 的第一个外显子上设计两个 sgRNA（sgRNA-1 和 sgRNA-2）。在 sgRNA-1 中包括 PAM 上游的 AgeI 限制位点，在 sgRNA-2 中包括 PAM 上游的 XbaI 限制性位点。在 PeCCR5 基因的第四个外显子上设计一个 sgRNA，该外显子编码 KNWYCYGK 的保守基序。该基序对 CCRs14 的催化至关重要。</li>
	<li>在 PAM 位点附近设计一个 20 个核苷酸间隔序列。该间隔序列将引导 Cas9 酶到靶位点进行 DNA 切割和随后的基因编辑。 注：建议在PAM位点内选择核酸内切酶切割位点上游的目标区域。这将有助于验证基因编辑效率。</li>
</ol>5. 引物设计和PCR
<ol><li>手动设计用于扩增 PeVDE 和 PeCCR5 片段的特异性引物。将上游和下游引物设计为位于靶位点外至少 100 bp 处，长度差异超过 100 bp，以便在电泳过程中实现明显的条带分离。设计用于在基因前 500 bp 内扩增 RUBY 的引物。 表1列出了所有使用的引物。</li>
	<li>使用高保真DNA聚合酶进行PCR。在这种情况下，利用高保真和高效扩增的DNA聚合酶进行基因克隆。</li>
	<li>制备PCR反应混合物如下：5x缓冲液（Mg2 + Plus）：4μL;dNTP 混合物（每个 2.5 mM）：1.6 μL;正向和反向引物（各 10 pmol）：各 1 μL;竹基因组DNA（约50 ng）;DNA 聚合酶 （2.5 U/μL）：0.2 μL;将水稀释至总体积为 20 μL。</li>
	<li>遵循PCR运行条件：在98°C下初始变性5分钟;在98°C下变性10秒;56°C退火5秒;在72°C下延伸30秒;重复变性以延长 32 个循环;在72°C下最终延伸5分钟;无限期保持在4°C。 注：PCR条件仅作为示例提供，可能需要针对特定应用或靶标进行优化。</li>
</ol>6. DNA提取、核酸内切酶消化和测序
<ol><li>使用剪刀将NPQ值较低的区域与新鲜的竹叶片分开，如成像-PAM荧光计所识别的那样（参见步骤7.4）。用液氮冷冻叶样品，并将冷冻叶样品转移到研钵中。使用研杵将样品研磨成细粉，在研磨过程中加入足够的液氮。此步骤有助于释放细胞内容，包括DNA。</li>
	<li>使用十六烷基三甲基溴化铵铵（CTAB）方法从粉末叶中提取基因组DNA。将 50 mg 叶粉样品加入 800 μL 2% CTAB 溶液中。充分混合样品并在65°C孵育30分钟，每5分钟轻轻摇动一次。</li>
	<li>加入等体积的氯仿/异戊醇（24：1，v / v）并剧烈摇动混合物。在8,000g离心8分钟后，将上清液转移到新管中。</li>
	<li>加入等体积的氯仿/异戊醇，重复步骤6.3。加入等体积的冰冷异丙醇，并在置于-20°C下30分钟之前将试管倒置数次。在以8,000 ×g 离心5分钟后，弃去上清液。</li>
	<li>在4°C下用75%乙醇洗涤DNA沉淀2次，然后溶解在50μL水中。</li>
	<li>使用步骤5.4中的方案扩增含有野生型和 农杆菌感染竹叶靶基因靶位点的基因组DNA。</li>
	<li>对PCR产物进行核酸内切酶消化。选择一种特定的酶，该酶可识别扩增的DNA片段中所需的限制性位点。使用 AgeI 和 XbaI 核酸内切酶进行消化。</li>
	<li>制备由 AgeI或 XbaI（20单位/μL）-1μL，1μgPCR产物，10x缓冲液-5μL组成的反应混合物，并加水至总体积为50μL。 在37°C孵育1小时。</li>
	<li>使用凝胶电泳分析消化的DNA片段的比例。比较野生型和 农杆菌感染样本的消化片段，以评估基因编辑效率。</li>
	<li>将转座酶接头 TCGTCGGCAGCGTCAGATGTGTGTATAAGAGACAG 和 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG 序列分别标记到正向和反向引物的 5' 端，用于扩增 PeVDE 和 PeCCR5 片段 9,15。使用步骤5.4中描述的方案进行扩增。准备PCR产物（消化前）进行深度测序。</li>
</ol>7. 叶片NPQ值叶绿素荧光的测定
<ol><li>在测量之前，将竹苗暴露在1200μmol/ m 2 / s的高光强度条件下2小时。这种曝光增加了吸收光量子的量，并激活了叶子中的光保护系统。</li>
	<li>使用成像-PAM荧光计测量竹叶的 体内 PS II叶绿素荧光。将光化光强度设置为800μmol/ m2 / s，进行6分钟荧光测量。在此期间，每30秒施加一次饱和脉冲以获得叶绿素荧光曲线。曲线的稳定值将用于计算13。</li>
	<li>使用以下公式计算非光化学猝灭 （NPQ）： NPQ = （F m - F m'） / F m' 其中 F m 表示暗适应状态下的最大荧光，Fm' 表示任何光适应状态下的最大荧光。</li>
	<li>从成像软件的可视化界面监控NPQ值。该软件允许实时分析和显示荧光数据，包括NPQ值。</li>
</ol>

在 Bamboo 中开发基因转化和编辑方法

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作者声明他们没有竞争利益。

与通常需要1-2年的传统基因转化方法相比，该方法显著缩短了所需的时间，并在5天内实现了外源基因的瞬时表达和内源基因的基因编辑。然而，这种方法有局限性，因为它只能转化一小部分细胞，而且基因编辑的叶子是嵌合的，缺乏再生成完整植物的能力。然而，这种 植物 基因表达和基因编辑技术为竹子内源基因的功能验证提供了有力的途径。
目前，在植物中基因?...

对竹子基因功能的研究为进一步了解竹子和释放其基因改造潜力带来了巨大的希望。一种有效的方法可以通过 农杆菌介导的竹叶感染过程来实现，其中含有外源基因的T-DNA片段被引入细胞中，随后导致基因在叶细胞内的表达。
竹子是一种宝贵的可再生资源，在制造、艺术和研究方面有着广泛的应用。竹子具有机械强度高、韧性好、刚度适中、柔韧性好等优良的木材性能1，现已广泛应用于各种家用和工业用品中，包括牙刷、吸管、纽扣、一次性餐具、地下管道、火力发电用冷却塔填料等。因此，竹子育种对于获得具有优良木材特性的竹子品种，在替代塑料、减少塑料使用、保护环境、应对气候变化以及产生重大经济价值方面发挥着至关重要的作用。
然而，传统的竹子育种由于营养生长阶段长、开花期不确定而面临挑战。虽然分子育种技术已经发展并应用于竹子育种，但由于愈伤组织诱导和再生过程，竹子基因转化过程耗时、劳动密集且复杂2,3,4,5。稳定的遗传转化通常需要农杆菌介导的方法，其中涉及组织培养过程，如愈伤组织诱导和再生。然而，竹子的愈伤组织再生能力较低，极大地限制了稳定遗传转化在竹子中的应用。农杆菌感染植物细胞后，T-DNA片段进入植物细胞，大部分T-DNA片段未整合在细胞中，导致瞬时表达。只有一小部分T-DNA片段随机整合到其染色体中，导致稳定表达。瞬时表达水平显示一个积累曲线，该曲线可能因从农杆菌递送的 T-DNA 表达的每个基因而异。在大多数情况下，最高表达水平发生在浸润后 3-4 天，并在 5-6 天后迅速下降 6,7。先前的研究表明，在没有抗性选择压力的情况下获得的基因编辑植物中，超过1/3的突变来自CRISPR/Cas9的瞬时表达，而其余不到2/3的突变来自DNA整合到基因组中的稳定表达8。这表明T-DNA整合到植物基因组中对于基因编辑不是必需的。此外，抗性的选择压力显著抑制了非转基因细胞的生长，直接影响了感染外植体的再生过程。因此，通过在竹子中利用无选择压力的瞬时表达抗性，可以实现外源基因的非整合表达，并直接在植物器官中研究基因功能。因此，可以开发一种简单且省时的竹子外源基因表达和编辑方法9。
开发的外源基因表达和基因编辑方法的特点是简单、经济高效，无需昂贵的设备或复杂的程序9.该方法以竹子内源性紫黄质脱环氧化酶基因（PeVDE）作为外源基因表达的报告基因，无选择压力。这是因为编辑后的竹叶 PeVDE 降低了高光下的光保护能力，并表现出非光化学猝灭（NPQ）值的降低，这可以通过叶绿素荧光成像来检测。为了证明该方法的有效性，利用该系统敲除了另一个竹子内源基因肉桂酰辅酶A还原酶基因（PeCCR5）9，并成功产生了该基因的突变体。该技术可用于竹叶中具有功能的基因的功能表征。通过在竹叶中瞬时过表达这些基因，可以提高它们的表达水平，或者通过基因编辑，可以敲低它们的表达，从而可以研究下游基因表达水平、叶表型和产品含量。这为竹子的基因功能研究提供了一种更加高效可行的方法。该技术可应用于竹叶中起作用的基因的功能表征。通过在竹叶中瞬时过表达这些基因，可以提高它们的表达水平，或者通过基因编辑，可以敲低它们的表达，从而可以研究下游基因表达水平、叶表型和产品含量。此外，需要注意的是，由于广泛的多倍体化，竹基因组中大多数具有商业重要性的基因都存在于多个拷贝中，导致遗传冗余。这给在竹子中进行多重基因组编辑带来了挑战。在应用稳定的遗传转化或基因编辑技术之前，快速验证基因功能至关重要。在解决多个基因拷贝的问题时，一种方法是分析转录组表达谱，以识别在特定阶段活跃表达的基因。此外，靶向这些基因拷贝的保守功能域允许设计共同的靶序列或将多个靶位点掺入同一 CRISPR/Cas9 载体中，从而能够同时敲除这些基因。这为竹子的基因功能研究提供了一种更加高效可行的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35S::RUBY</td>
			<td>Addgene, United States</td>
			<td>160908</td>
			<td>Plamid construct</td>
		</tr>
		<tr>
			<td>Agrobacterium competent cells of GV3101, EHA105,LBA4404, and AGL1</td>
			<td>Biomed, China</td>
			<td>BC304-01, BC303-01, BC301-01, and BC302-01</td>
			<td>For Agrobacterium infection</td>
		</tr>
		<tr>
			<td>CTAB</td>
			<td>Sigma-Aldrich, United States</td>
			<td>57-09-0</td>
			<td>DNA extraction</td>
		</tr>
		<tr>
			<td>Imaging-PAM fluorometer</td>
			<td>Walz, Effeltrich, Germany</td>
			<td></td>
			<td>Detect chlorophyll fluorescence of bamboo leaves</td>
		</tr>
		<tr>
			<td>ImagingWin</td>
			<td>Walz, Effeltrich, Germany</td>
			<td></td>
			<td>Software for Imaging-PAM fluorometer</td>
		</tr>
		<tr>
			<td>Paq CI or Aar I</td>
			<td>NEB, United States</td>
			<td>R0745S</td>
			<td>Incorporate the target sequence onto the CRISPR/Cas9 vector.</td>
		</tr>
		<tr>
			<td>PrimeSTAR Max DNA polymerase</td>
			<td>Takara, Japan</td>
			<td>R045Q</td>
			<td>For gene cloning</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB, United States</td>
			<td>M0202V</td>
			<td>Incorporate the target sequence onto the CRISPR/Cas9 vector.</td>
		</tr>
	</tbody>
</table>

in planta gene expression and gene editing in moso bamboo leaves

开发了一种针对竹子的植物基因转化方法，避免了费时费力的愈伤组织诱导和再生过程。该方法涉及农杆菌介导的基因表达，通过刺伤和真空对竹苗进行。它成功地证明了外源基因的表达，如RUBY报告基因和Cas9基因在竹叶中的表达。使用GV3101菌株对红宝石幼苗中甜菜碱的积累转化效率最高，感染后转化率为85.2%。虽然外源DNA没有整合到竹基因组中，但该方法在表达外源基因方面是有效的。此外，利用该方法，利用天然报告基因开发了一种基因编辑系统，从中编辑的竹叶黄质去环氧化酶基因（PeVDE）在竹叶中产生了原位突变体，突变率为17.33%。PeVDE的突变导致高光下非光化学猝灭（NPQ）值降低，荧光计可以准确检测。这使得编辑后的PeVDE成为竹子中外源和内源基因的潜在天然报告基因。利用PeVDE的报告基因，成功编辑了一个肉桂酰辅酶A还原酶基因，突变率为8.3%。该操作避免了组织培养或愈伤组织诱导的过程，对于竹子中表达外源基因和内源基因编辑是快速有效的。该方法可以提高基因功能验证的效率，有助于揭示竹子关键代谢途径的分子机制。

The investigation of gene function in bamboo holds great promise for the advanced understanding of bamboo and unlocking its potential for genetic modification. An effective way of this can be achieved through the process of Agrobacterium-mediated infection in bamboo leaves, whereby the T-DNA fragment containing exogenous genes is introduced into the cells, subsequently leading to the expression of the genes within the leaf cells.
Bamboo is a valuable and renewable resource with a wide range of applications in manufacturing, art, and research. Bamboo possesses excellent wood properties such as high mechanical strength, toughness, moderate stiffness, and flexibility1, which is now widely used in a variety of household and industrial supplies, including toothbrushes, straws, buttons, disposable tableware, underground pipelines, and cooling tower fillers for thermal power generation. Therefore, bamboo breeding plays a crucial role in obtaining bamboo varieties with excellent wood properties for replacing plastics and reducing plastic usage, protecting the environment, and tackling climate change, as well as generating significant economic value.
However, traditional bamboo breeding faces challenges due to the lengthy vegetative growth stage and uncertain flowering period. Although molecular breeding techniques have been developed and applied to bamboo breeding, the process of bamboo gene transformation is time-consuming, labor-intensive, and complicated due to the callus induction and regeneration processes2,3,4,5. Stable genetic transformation often requires Agrobacterium-mediated methods, which involve tissue culture processes such as callus induction and regeneration. However, bamboo has a low ability for callus regeneration, greatly limiting the application of stable genetic transformation in bamboo. After Agrobacterium infects plant cells, the T-DNA fragment enters the plant cells, with the majority of T-DNA fragments remaining non-integrated in the cells, resulting in transient expression. Only a small portion of T-DNA fragments randomly integrate into its chromosome, leading to stable expression. The transient expression levels show an accumulation curve that can vary for each gene expressed from an Agrobacterium-delivered T-DNA. In most cases, the highest expression levels occur 3-4 days after infiltration and quickly decrease after 5-6 days6,7. Previous studies have shown that more than 1/3rd of mutations in gene-edited plants obtained without selection pressure for resistance come from the transient expression of CRISPR/Cas9, while the remaining less than 2/3rd come from stable expression after DNA integration into the genome8. This indicates that T-DNA integration into the plant genome is not necessary for gene editing. Moreover, selection pressure for resistance significantly inhibits the growth of non-transgenic cells, directly affecting the regeneration process of infected explants. Therefore, by using transient expression without selection pressure for resistance in bamboo, it is possible to achieve non-integrated expression of exogenous genes and study gene function directly in plant organs. Hence, an easy and time-saving method can be developed for exogenous gene expression and editing in bamboo9.
The developed exogenous gene expression and gene editing method is characterized by its simplicity, cost-effectiveness, and the absence of expensive equipment or complex procedures9. In this method, the bamboo endogenous violaxanthin de-epoxidase gene (PeVDE) was used as the reporter for exogenous gene expression without selection pressure. This is because the edited PeVDE in bamboo leaves reduces the photoprotection ability under high light and demonstrates a decrease in the non-photochemical quenching (NPQ) value, which can be detected through chlorophyll fluorescence imaging. To demonstrate the effectiveness of this method, another bamboo endogenous gene, the cinnamoyl-CoA reductase gene (PeCCR5)9, was knocked out using this system and successfully generated mutants of this gene. This technique can be used for the functional characterization of genes that have functions in bamboo leaves. By overexpressing these genes transiently in bamboo leaves, their expression levels can be enhanced, or by gene editing, their expression can be knocked down, allowing for the study of downstream gene expression levels, leaf phenotypes, and product contents. This provides a more efficient and feasible approach for gene function research in bamboo. This technique can be applied to the functional characterization of genes that function in bamboo leaves. By overexpressing these genes transiently in bamboo leaves, their expression levels can be enhanced, or by gene editing, their expression can be knocked down, allowing for the study of downstream gene expression levels, leaf phenotypes, and product contents. Additionally, it is important to note that, due to extensive polyploidization, the majority of commercially important genes in bamboo genomes are present in multiple copies, resulting in genetic redundancy. This poses a challenge for performing multiplex genome editing in bamboo. Prior to the application of stable genetic transformation or gene editing techniques, it is crucial to quickly validate gene functions. In addressing the issue of multiple gene copies, one approach is to analyze transcriptome expression profiles to identify genes that are actively expressed during specific stages. Furthermore, targeting the conserved functional domains of these gene copies allows for the design of common target sequences or the incorporation of multiple target sites into the same CRISPR/Cas9 vector, enabling the simultaneous knockout of these genes. This provides a more efficient and feasible approach for gene function research in bamboo.

作者聚焦：推进竹叶基因编辑以获得可持续的塑料替代品 

在普兰塔 毛竹叶片基因表达与基因编辑

In this study, we used Agrobacterium as a vector to introduce foreign DNA fragments into bamboo leaves. Most DNA fragments are not integrated into chromosomes and undergo transient expression. We took advantage of this transient expression to carry out gene editing.The main challenge is the limitation of gene editing of bamboo leaves, which cannot regenerate. This propose difficulties in performing editing in regenerative organs, like young shoots, and obtaining edited branches or subsequent generations with desired genetic challenges. Its main advantage is the time-saving aspect of the principle.By being able to edit bamboo genes directly in bamboo leaves, it allows for quick verification of gene fractions. This eliminates the needs for time-consuming procedures, like tissue culture or transformation, significantly speeding up research on bamboo gene fractions. The primary research will be focused on investigating the molecular mechanism of bamboo biomass formation.This will involve understanding why bamboo grows rapidly, and using that knowledge to develop bamboo varieties which are suitable for plastic substituent.

农杆菌介导的 植物 基因在竹叶中的基因表达 RUBY 报告基因已被证明可有效可视化瞬时基因表达，因为它能够从酪氨酸10 中产生鲜艳的红色甜菜碱。在这项研究中，利用农杆菌介导的转化在竹叶中瞬时表达外源性RUBY基因（图1）。在感染后的第3天，在未成熟的折叠叶子中观察到红色，一旦叶子展开，第5天...

Watch this Scientific Journal Video about Advancing Gene Editing in Bamboo Leaves for Sustainable Plastic Alternatives at JoVE.com

本研究以农杆菌为研究对象，研究了一种新型的农杆菌介导的植物基因表达和基因编辑方法。该方法大大提高了竹子基因功能验证的效率，对加快竹子育种进程具有重要意义。

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and ...

在这项研究中，我们以农杆菌为载体，将外源DNA片段引入竹叶中。大多数DNA片段不会整合到染色体中，而是会经历瞬时表达。我们利用这种瞬时表达进行基因编辑。主要的挑战是竹叶基因编辑的局限性，竹叶不能再生。这提出了在再生器官（如幼芽）中进行编辑以及获得具有所需遗传挑战的编辑分支或后代的困难。它的主要优点是该原理的节省时间方面。通过能够直接在竹叶中编辑竹子基因，它可以快速验证基因组分。这消除了对耗时程序的需求，如组织培养或转化，大大加快了竹子基因组分的研究。主要研究方向是研究竹子生物量形成的分子机制。这将涉及了解竹子为什么生长迅速，并利用这些知识开发适合塑料替代物的竹子品种。

in-planta-gene-expression-and-gene-editing-in-moso-bamboo-leaves

Research

JoVE Journal

Biology

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

1.1K 次观看.  Key Laboratory of State Forestry and Grassland Administration/Beijing on Bamboo & Rattan Science and Technology. 在这项研究中，我们以农杆菌为载体，将外源DNA片段引入竹叶中。大多数DNA片段不会整合到染色体中，而是会经历瞬时表达。我们利用这种瞬时表达进行基因编辑。主要的挑战是竹叶基因编辑的局限性，竹叶不能再生。这提出了在再生器官（如幼芽）中进行编辑以及获得具有所需遗传挑战的编辑分支或后代的困难。它的主要优点是该原理的节省时间方面。通过能够...

视频: 作者聚焦：推进竹叶基因编辑以获得可持续的塑料替代品 

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and regeneration processes. This method involves Agrobacterium-mediated gene expression via wounding and vacuum for bamboo seedlings. It successfully demonstrated the expression of exogenous genes, such as the RUBY reporter and Cas9 gene, in bamboo leaves. The highest transformation efficiency for the accumulation of betalain in RUBY seedlings was achieved using the GV3101 strain, with a percentage of 85.2% after infection. Although the foreign DNA did not integrate into the bamboo genome, the method was efficient in expressing the exogenous genes. Furthermore, a gene editing system has also been developed with a native reporter using this method, from which an in situ mutant generated by the edited bamboo violaxanthin de-epoxidase gene (PeVDE) in bamboo leaves, with a mutation rate of 17.33%. The mutation of PeVDE resulted in decreased non-photochemical quenching (NPQ) values under high light, which can be accurately detected by a fluorometer. This makes the edited PeVDE a potential native reporter for both exogenous and endogenous genes in bamboo. With the reporter of PeVDE, a cinnamoyl-CoA reductase gene was successfully edited with a mutation rate of 8.3%. This operation avoids the process of tissue culture or callus induction, which is quick and efficient for expressing exogenous genes and endogenous gene editing in bamboo. This method can improve the efficiency of gene function verification and will help reveal the molecular mechanisms of key metabolic pathways in bamboo.

In this study, a novel in planta gene expression and gene editing method mediated by Agrobacterium was developed in bamboo. This method greatly improved the efficiency of gene function validation in bamboo, which has significant implications for accelerating the process of bamboo breeding.

科研

生物学

Preparation of Bamboo Seedlings and Agrobacterium-Mediated In Planta Transformation System for Gene Expression

Preparation of Bamboo Seedlings and Agrobacterium -Mediated In Planta Transformation System for Gene Expression  

To begin, sow the pre-soaked bamboo seeds into the substrate of soil and vermiculite for germination. For agrobacterium infection, carefully remove the 15 day old seedlings with soil attached roots from the substrate. Wrap the seedlings with tinfoil to maintain moisture and prevent soil detachment.Transfer the wrapped seedlings to the plant growth chamber with higher humidity and lower illumination for two hours. Next, using a sharp needle from a syringe, wound the upper part of curled immature leaves once or twice. Quickly dip the wounded part of the leaves into agrobacterium suspension.Then immediately transfer the seedlings to a vacuum chamber with a pressure of 25 to 27 millimeters of mercury for two minutes. After vacuuming, carefully unwrap the seedlings and replant them into the substrate. Allow the seedlings to grow in a plant growth chamber in dim light with high humidity for two days.Then culture the seedlings under normal growth conditions, watering them every five to seven days.

竹苗的制备及 农杆菌介导的植物转化系统及其基因表达

In Planta Gene Expression and Measurement of Chlorophyll Fluorescence of Non-Photochemical Quenching Values in Bamboo Leaves

Begin by extracting the genomic DNA from the wild-type and Agrobacterium-infected bamboo leaves. Amplify the genomic DNA containing the target site of the target genes from wild-type and Agrobacterium-infected bamboo leaves using the following PCR conditions. After PCR, perform endonuclease enzyme digestion by preparing a reaction mixture containing one microliter of age-1, one microgram of PCR products, and five-microliter 10X buffer.Then, add water to a final volume of 50 microliters. Incubate the digestion mixture at 37 degrees Celsius for one hour. Using gel electrophoresis, analyze the proportion of digested DNA fragments.Compare the digested fragments from the wild-type and Agrobacterium-infected samples to assess gene editing efficiency. Expose the bamboo seedlings to high light intensity conditions to increase the amount of absorbed light quantum and activation of the leaf photoprotection system. Next, turn on the IMAGING-PAM fluorometer and set the actinic light intensity to measure in vivo photosystem II chlorophyll fluorescence of bamboo leaves.The red beta lane color produced by the RUBY gene indicated successful Agrobacterium-mediated gene expression in bamboo leaves. Out of the four Agrobacterium strains, GV3101 strain caused the most significant beta lane accumulation in infected bamboo leaves. After infection with the CRISPR-Cas9 constructs and high light treatments, certain areas of the leaf blades showed lower non-photochemical quenching values.Furthermore, enzyme digestion and sequencing analysis confirmed the successful mutation of the PeVDE gene in the edited regions. Sanger sequencing results of the PeVDE fragment after editing by both sgRNA1 and sgRNA2 showed deletion of a long fragment in the PeVDE gene. After 30 days of Agrobacterium infection, the leaf areas transfected with sgRNAs for both PeVDE and PeCCR5 exhibited lower non-photochemical quenching values.