CN117487718A - 一种根瘤菌感受态制作技术及试剂配方 - Google Patents
一种根瘤菌感受态制作技术及试剂配方 Download PDFInfo
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- FGDZQCVHDSGLHJ-UHFFFAOYSA-M rubidium chloride Chemical compound [Cl-].[Rb+] FGDZQCVHDSGLHJ-UHFFFAOYSA-M 0.000 claims 2
- 241000589174 Bradyrhizobium japonicum Species 0.000 claims 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims 1
- 241000589194 Rhizobium leguminosarum Species 0.000 claims 1
- 241000589196 Sinorhizobium meliloti Species 0.000 claims 1
- 239000001110 calcium chloride Substances 0.000 claims 1
- 229910001628 calcium chloride Inorganic materials 0.000 claims 1
- 235000011148 calcium chloride Nutrition 0.000 claims 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 4
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- 108091029865 Exogenous DNA Proteins 0.000 abstract description 3
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- 241000589151 Azotobacter Species 0.000 abstract 1
- 241000589173 Bradyrhizobium Species 0.000 abstract 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract 1
- 241001135312 Sinorhizobium Species 0.000 abstract 1
- 238000004178 biological nitrogen fixation Methods 0.000 abstract 1
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Abstract
本发明属于生物技术领域，具体涉及一种根瘤菌感受态制作技术及试剂配方。根瘤菌是一类生物固氮的重要细菌，广泛用于共生固氮的研究。然而，根瘤菌的转化一直以来用的都是三亲结合法，我们可以通过制备感受态细胞，简化研究步骤。本发明提供了一种根瘤菌感受态制作技术，该技术包括以下步骤：(1)用培养液培养根瘤菌；(2)用含有钙、铷离子的处理试剂处理根瘤菌来改变其通透性；(3)使用外源DNA对处理后的根瘤菌进行转化；(4)用相关抗性的培养基进行筛选。本发明还提供了培养液和处理试剂的具体配方。本发明的技术适用于多种常见的根瘤菌菌株，如中华根瘤菌、缓生根瘤菌等，转化效率可达10^2^‑10^3^cfu/μg质粒。本发明的技术操作简单，成本低，可为固氮菌研究提供便利。
Description
技术领域
本发明涉及微生物学和生物技术领域，具体涉及一种根瘤菌感受态制作技术及试剂配 方。根瘤菌是一类能够与植物共生形成根瘤的微生物，在自然界中广泛存在。本发明提供的 根瘤菌感受态制作技术及试剂配方，能够高效地制备根瘤菌感受态，对于进一步研究根瘤菌 的分子生物学、遗传学和共生机制具有重要意义。
背景技术
在微生物学领域，感受态是指微生物在一定生理条件下吸收外源 DNA 并能够进行转 化和重组的一种状态。根瘤菌感受态是根瘤菌细胞处于一种能够高效摄取和利用外源 DNA 的状态，是进行遗传操作的基础。传统的根瘤菌感受态制作方法通常需要繁琐的操作步骤和 大量的实验条件摸索，且制备效率低下，难以满足研究需求。因此，开发一种简单、高效的 根瘤菌感受态制作技术及试剂配方，对于根瘤菌的遗传操作和共生机制研究具有重要意义。
发明内容
本发明提供了一种根瘤菌感受态制作技术及试剂配方。所述技术包括以下步骤：a. 准 备合适状态的根瘤菌细胞；b. 将所述根瘤菌细胞使用化学试剂进行破壁处理；c.在破壁后的 根瘤菌细胞中加入渗透压稳定剂；d. 将所述根瘤菌细胞在-80℃的低温下冷冻保存；e. 将所 述 冷冻的根瘤菌细胞进行热激处理；f. 在所述热激处理后的根瘤菌细胞中加入高浓度待转质 粒； g. 将所述根瘤菌细胞加入营养液并在适宜的温度下培养；h.在所述培养后的根瘤菌细 胞进行 涂板转化。本发明的根瘤菌感受态制作技术及试剂配方，能够高效地制备根瘤菌感受 态，操 作简单，实验条件易于控制，能够满足根瘤菌遗传操作和共生机制研究的需求。
具体内容：将根瘤菌划于 YMA 平板（注意其本身的本底抗性，如有需要培养基需加 入其本底抗性的抗生素），待其生长出来（3-5 天，依据菌种而定）；小摇：挑单克隆于30ml YMA，30℃ 200rpm 摇床培养；大摇：取 8ml 的小摇浑浊菌液至 200ml YMA ( 1000ml三 角瓶）30℃至 OD 值=0.5（记得保留 一批原本的培养基来当空白对照）（感受态效率与此 步骤的摇菌温度有关，一般来说温度越 接近 10℃效果越好，但是摇晃时间需要越久）；将 菌放置再冰上，冰浴 15min，同时预约大型离心机并预冷 4℃，同时预冷 50ml 离心管（需 要用灭菌的圆底离心管，菌液摇起来简单）；在将菌液分装至 50ml 离心管，4000~5000rpm，5min， 4℃ 弃上清；每管离心管加入 4*16ml buffer1 冰上轻轻摩擦（可以先加入 4ml buffer1，摇匀，后再补齐液 体，同时可以将液体集中起来），悬浮细菌沉淀，在冰上放置15min，4800rpm 离心 5 min， 4 ℃倒掉上清液（此步骤根据需求可以重复进行 2 次）；加入 4*（4ml buffer2+300ul DMSO），冰上悬浮沉淀；分装 100ul/管，液氮冷激，-70 ℃保存。
Claims (7)
1.一种根瘤菌感受态制作技术及试剂配方。
2.一种根瘤菌感受态制作技术及试剂配方，其特征在于，包括以下步骤：a. 准备合适状态的根瘤菌细胞；b. 将所述根瘤菌细胞使用化学试剂进行破壁处理；c. 在破壁后的根瘤菌细胞中加入渗透压稳定剂；d. 将所述根瘤菌细胞在-80℃的低温下冷冻保存；e. 将所述冷冻的根瘤菌细胞进行热激处理；f. 在所述热激处理后的根瘤菌细胞中加入高浓度待转质粒；g. 将所述根瘤菌细胞加入营养液并在适宜的温度下培养；h. 在所述培养后的根瘤菌细胞进行涂板转化。
3.根据权利要求1所述的根瘤菌感受态制作方法，其特征在于，所述根瘤菌细胞为Bradyrhizobium japonicum、Rhizobium leguminosarum或Sinorhizobium meliloti中的一种或多种混合。
4.一种根瘤菌感受态制作试剂配方，其特征在于，包括渗透压稳定剂、离子溶液和缓冲液。
5.根据权利要求1、2或3所述的根瘤菌感受态制作方法，其特征在于，所述渗透压稳定剂为甘油和DMSO。
6.根据权利要求1、2或3所述的根瘤菌感受态制作方法，其特征在于，所述离子溶液为CaCl2、MnCl2或RbCl中的一种或多种混合。
7.根据权利要求1、2或3所述的根瘤菌感受态制作试剂配方，其特征在于，所述缓冲液为KAC或Mops。
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