【现学现卖】实验-制备真菌原生质体

概念

原生质体（protoplast）为细胞壁内的原生质，即指能够和细胞壁分开的那部分细胞物质，包括细胞膜、细胞质和细胞核。换言之原生质体就是除去细胞壁的被细胞膜包围的“裸露细胞”。

现在微生物学中，用物理或化学方法除去微生物细胞壁后得到的主要由一层细胞膜包裹着的圆球状渗透敏感细胞既是微生物原生质体，有时也叫做原生质球。

原生质体的特征

（1）无细胞障碍，能摄取DNA 质粒病毒细菌细胞器等外源物质，是细胞工程进行遗传操作基因转移的良好材料。

（2）具有全能性可再生细胞壁并再生成完整植株。

（3）适合进行融合形成杂种细胞，能克服不同种细胞间的不亲和障碍，培育新品种。

（https://www.omicsonline.org/articles-images/2153-0645-1-102-g003.html）

A novel virus thatinfecting hypovirulent strain XG36-1 of plant fungal pathogen Sclerotiniasclerotiorum(Zhang et al.,2009)关于衰弱核盘菌XG36-1的弱毒病毒的研究。

简介

所用材料衰弱型（含病毒）菌株XG36-1（A），野生型菌株XG-13（B），脱毒衰弱并且抗生素标记菌株XG36-1A34^R（ACR）。A在PDA培养基上生长速率很低，菌落形态异常，产生很少的菌核，且几乎不产生子囊盘。与B相比，A不能在接种的植物叶片上产生病斑。A的有性生殖的后代表现型等与B一样，原生质体重新产生的菌株有25.5%也被治愈了，变现与野生型B一样。A可以将弱毒表现通过菌丝融合传染给ACR。TEM电镜观察发现A的细胞质被破坏并且颗粒化，核膜和线粒体膜破裂，线粒体嵴空化。观察到几个病毒粒子（40nm）在真菌细胞里被一层膜包裹着存在。

原生质体的制备和再生菌株方法

1.将新鲜的菌饼接种在铺有玻璃纸膜的PDA上，培养2d，收集菌丝并用灭菌的研钵研磨。

To obtainprotoplasts of strain XG36-1, mycelial-agar discs (5-mm-diam.) cut fromactively growing colony margins of strain XG36-1 were transferred ontocellophane membranes overlaying PDA. After 2 days, the mycelia were collectedfrom cellophane membranes, and then ground with sterilized mortar and pestle tomake hyphal fragments.

2. 在80ml/250ml PDB培养基里放一些研磨的菌丝碎片（原文说1ml，其实也没有定量），150rpm，20℃摇培20h。用两层灭菌细纱布过滤，收集滤液。纱布上的菌丝体用100 ml 的potassium chloride (KCl) buffer (0.6mol/L)清洗两次。

Approximately1 ml of hyphal fragment mush was transferred into a 250 ml flask containing 80ml PDB (Potato Dextrose Broth, PDB), and shaken at 150 rpm for up to 20 h at20°C. The filtrate was collected by passing through two layers of sterilizedcheesecloth, and the mycelium was washed twice with 100 ml of potassiumchloride (KCl) buffer (0.6 mol/L).

3. 挤干菌丝体里面的液体，然后用digestionbuffer（含有1.5mg/ml Lysing enzymes (提取自Trichodermaharzianum (Sigma-Aldrich, Inc)）重悬菌丝体， 32℃培养3h。

Themycelial mass was squeezed to remove liquids, and then re-suspended withdigestion buffer which contained 1.5 mg/ml Lysing enzymes from Trichodermaharzianum (Sigma-Aldrich, Inc), and then incubated at 32°C for 3 h.

4.重悬液经过四层纱布过滤，再用双层滤纸过滤得到最终滤液。将滤液4000rpm离心10min收集原生质体。收集的原生质体用0.6 mol/L KCl溶液通过重悬，再4000rpm离心5min的方法清洗两次。得到的沉淀原生质体沉淀重悬在0.6 mol/L KCl 溶液中获得1到2×10^3原生质体/ml浓度的溶液。

The liquidwas filtered through four layers for sterilized cheesecloth and then passedthrough two layers of sterilized filter paper to remove the debris andundigested hyphal fragments. Protoplasts were collected by centrifugation for10 min at 4000 rpm. The precipitate was washed twice with 0.6 mol/L KClsolution by re-suspension and centrifugation at 4000 rpm for 5 min. The finalprecipitate was re-suspended with in 0.6 mol/L KCl solution to give 1 to 2 × 10^3protoplasts/ml for regeneration.

5. 得到的沉淀原生质体重悬在0.6mol/L KCl 溶液中获得1到2×10^3原生质体/ml浓度的溶液。100μl 原生质体溶液与20ml再生培养基RM (RM: sucrose 0.7 M;yeast extract 0.5 g/L, agar 1.5 g/L, 使用前加 KCl到0.6 mol/L浓度)轻轻混匀，倒在RM固体培养基上。

One hundredmicroliter of protoplast suspension was gently mixed with 20 ml regenerationmedium (RM: sucrose 0.7 M; yeast extract 0.5 g/L, agar 1.5 g/L, addingKCl to a final concentration 0.6 mol/L before use), and poured into a Petridish (diam. 90 mm).

6. 培养基在20–22°C条件下培养3–4d。小菌落转移到新的PDA培养基上。在PDA上的菌落即为由A菌原生质体再生的菌株。

The plateswere incubated at 20–22°C for 3–4 days, and then small colonies were observedon the RM plates and transferred onto fresh PDA plates. These subcultures wereconsidered as protoplast regenerants of strain XG36-1.

A geminivirus-related DNAmycovirus that confers hypovirulence to a plant pathogenic fungus(Yu et al., 2010)。

原生质体用于后续病毒粒子的侵染等实验，这篇文献在第一个的基础上做了改进。

前面的方法同上，但是在第四步，原生质体重悬在STC buffer (1.2 Msorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl2)里，制成终浓度为1× 10^8 原生质体/ml的原生质体溶液。用于保存在-80℃的原生质体溶液中再添加5%的冻存剂DMSO。

Protoplasts were resuspended at aconcentration of 1 × 108 protoplasts per mL in STC buffer (1.2 M sorbitol, 10mM Tris-HCl, pH 7.5, 10 mM CaCl2). The protoplast stock (containing 5% dimethylsulfoxide) was routinely stored at −80 °C.

参考文献

Yu, X., Li, B., Fu,Y., Jiang, D., Ghabrial, S.A., Li, G. et al. (2010) A geminivirus-related DNAmycovirus that confers hypovirulence to a plant pathogenic fungus. Proceedings of the National Academy ofSciences 107: 8387-8392.

Zhang, L., Fu, Y., Xie, J.,Jiang, D., Li, G., and Yi, X. (2009) A novel virus that infecting hypovirulentstrain XG36-1 of plant fungal pathogen Sclerotinia sclerotiorum. Virology journal 6: 96.