BioVector? pCAMBIA1300-35S-mCherry-PTS1 植物表達(dá)質(zhì)粒 / BioVector? pCAMBIA1300-35S-mCherry-PTS1 Plant Expression Plasmid

通用定義 / General Definition:BioVector? pCAMBIA1300-35S-mCherry-PTS1 是一種專(zhuān)為植物細(xì)胞設(shè)計(jì)的過(guò)氧化物酶體定位于報(bào)告質(zhì)粒。該載體基于經(jīng)典的 pCAMBIA1300 骨架，利用強(qiáng)力的 CaMV 35S 啟動(dòng)子 驅(qū)動(dòng)紅色熒光蛋白 mCherry 的表達(dá)。其核心特征是在 mCherry 的 C 端融合了 PTS1 (Peroxisomal Targeting Signal 1) 序列（通常為三肽 SKL）。PTS1 信號(hào)肽能被胞質(zhì)中的受體識(shí)別并引導(dǎo)融合蛋白精準(zhǔn)定位于過(guò)氧化物酶體。該質(zhì)粒是研究植物過(guò)氧化物酶體動(dòng)力學(xué)、細(xì)胞器相互作用以及作為植物轉(zhuǎn)染實(shí)驗(yàn)中亞細(xì)胞定位對(duì)照的核心工具。

BioVector? pCAMBIA1300-35S-mCherry-PTS1 is a peroxisomal-targeted reporter vector designed for plant systems. Built upon the pCAMBIA1300 backbone, it utilizes the robust CaMV 35S promoter to drive the expression of the red fluorescent protein mCherry. The defining feature of this construct is the C-terminal fusion of the PTS1 (Peroxisomal Targeting Signal 1) sequence (typically the tripeptide SKL). The PTS1 signal is recognized by cytosolic receptors, ensuring the precise translocation of mCherry into the peroxisomal matrix. This plasmid is a vital tool for studying plant peroxisome dynamics, organelle interactions, and serving as a sub-cellular marker in plant transformation assays.

BioVector? pCAMBIA1300-35S-mCherry-PTS1 技術(shù)說(shuō)明書(shū) (Technical Datasheet)

中文版說(shuō)明書(shū) (Chinese Datasheet)

1. 質(zhì)?；拘畔?/strong>

• 載體骨架： pCAMBIA1300（二元載體，適用于農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化）。

• 啟動(dòng)子： CaMV 35S（雙啟動(dòng)子或單啟動(dòng)子，確保在雙子葉植物中高效表達(dá)）。

• 報(bào)告基因： mCherry（紅色熒光蛋白，激發(fā)波長(zhǎng)約 587 nm，發(fā)射波長(zhǎng)約 610 nm）。

• 定位信號(hào)： PTS1 (C-terminal SKL sequence) – 過(guò)氧化物酶體基質(zhì)定位。

• 植物篩選抗性： 潮霉素 (Hygromycin B, 50 mg/L)。

• 細(xì)菌篩選抗性： 卡那霉素 (Kanamycin, 50 mg/L)。

2. 核心生物學(xué)功能

• 亞細(xì)胞標(biāo)記： 在植物細(xì)胞（如煙草葉片、擬南芥原生質(zhì)體）中產(chǎn)生鮮艷的紅色點(diǎn)狀熒光，代表過(guò)氧化物酶體。

• 細(xì)胞器動(dòng)力學(xué)： 用于實(shí)時(shí)觀察過(guò)氧化物酶體在細(xì)胞骨架上的移動(dòng)及分裂過(guò)程。

• 共定位分析： 與綠色熒光 (GFP/YFP) 標(biāo)記的目標(biāo)蛋白共轉(zhuǎn)，驗(yàn)證目標(biāo)蛋白是否定位于過(guò)氧化物酶體。

3. 實(shí)驗(yàn)應(yīng)用方案

• 煙草瞬時(shí)表達(dá)： 通過(guò)農(nóng)桿菌滲入法 (Agro-infiltration) 在本氏煙草葉片中快速表達(dá)。

• 擬南芥轉(zhuǎn)化： 配合花序浸染法 (Floral Dip) 建立穩(wěn)定轉(zhuǎn)基因植株。

• 原生質(zhì)體轉(zhuǎn)染： 利用 PEG 介導(dǎo)法在擬南芥或水稻原生質(zhì)體中進(jìn)行亞細(xì)胞定位研究。

4. 注意事項(xiàng)

• 熒光猝滅： 雖然 mCherry 較穩(wěn)定，但在強(qiáng)激光下長(zhǎng)時(shí)間連續(xù)掃描仍可能導(dǎo)致猝滅。

• 農(nóng)桿菌選擇： 建議使用 EHA105、GV3101 或 LBA4404 等常用農(nóng)桿菌菌株進(jìn)行轉(zhuǎn)化。

English Datasheet

1. Basic Plasmid Information

• Backbone: pCAMBIA1300 (Binary vector for Agrobacterium-mediated transformation).

• Promoter: CaMV 35S (Ensures constitutive high-level expression in most plant tissues).

• Reporter: mCherry (Red Fluorescent Protein; Ex ~587 nm, Em ~610 nm).

• Targeting Signal: PTS1 (C-terminal SKL sequence) – Directs protein to the peroxisomal matrix.

• Plant Selection: Hygromycin B (50 mg/L).

• Bacterial Selection: Kanamycin (50 mg/L).

2. Key Biological Functions

• Subcellular Marker: Produces distinct punctate red fluorescent signals within plant cells, identifying individual peroxisomes.

• Organelle Interaction: Used to monitor peroxisome distribution and their spatial relationship with other organelles like chloroplasts or mitochondria.

• Co-localization Assays: Acts as a reference channel for validating the peroxisomal localization of GFP-tagged candidate proteins.

3. Experimental Applications

• Transient Expression: Highly effective for tobacco leaf agro-infiltration (Nicotiana benthamiana).

• Stable Transformation: Suitable for creating transgenic lines (e.g., Arabidopsis) via the floral dip method.

• Protoplast Assays: Ideal for PEG-mediated transfection in Arabidopsis or rice protoplasts for rapid subcellular imaging.

4. Key Usage Notes

• Signal Specificity: The PTS1 pathway is highly conserved; however, protein folding speed can occasionally affect import efficiency. Ensure cells are imaged within the optimal window (typically 36-72 hours post-infiltration).

• Backbone Versatility: The pCAMBIA1300 series is low-copy in E. coli; use high-quality kits and larger culture volumes for plasmid extraction.

注意 / Note: 質(zhì)粒應(yīng)儲(chǔ)存在 -20 攝氏度。在進(jìn)行多色熒光成像時(shí)，請(qǐng)確保設(shè)置正確的濾光片以消除 mCherry 與葉綠體自發(fā)熒光（深紅色）之間的干擾。Store at -20°C. When performing multi-color imaging, ensure precise spectral unmixing or appropriate filter settings to distinguish mCherry signals from the far-red autofluorescence of chlorophyll.

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