使用基于循环 RT-PCR 的策略对玉米 Mitochondrion 中初级和已处理转录的区分和映射
Summary
通过结合循环RT-PCR、定量RT-PCR、RNA 5’聚磷酸酶处理和北方斑点,提出了基于RT-PCR的循环RT-PCR策略。该协议包括一个标准化步骤,以尽量减少不稳定5’三磷酸酯的影响,它适用于对玉米线粒体中稳定积累的初级和加工转录转录进行鉴别和映射。
Abstract
在植物线粒体中,一些稳态转录本具有从转录启动(主转录)衍生的5’三磷酸盐,而其他含有5’单磷酸盐产生的转录后(处理转录)。为了区分这两种类型的成绩单,已经制定了几种策略,其中大多数取决于存在/缺少5’三磷酸盐。然而,小学5年级的三磷酸盐是不稳定的,它阻碍了对两种类型的抄本的明显区分。为了系统地区分和绘制玉米线粒体中稳定积累的初级和加工转录本,我们开发了基于CRT-PCR(cRT-PCR)的循环RT-PCR(cRT-PCR)策略,将cRT-PCR、RNA 5’多磷酸酶处理、定量RT-PCR (RT-qPCR) 和北方污点。作为改进,该策略包括RNA正常化步骤,以尽量减少不稳定的5’三磷酸盐的影响。
在此协议中,富集线粒体RNA由RNA 5’聚磷酸酶预处理,将5’三磷酸酯转化为单磷酸盐。经过循环和逆转录后,从5’聚磷酸酶处理和未处理RNA衍生的两种cDNA由玉米26S成熟rRNA归一化,其加工为5’结束,对5’聚磷酸酶不敏感。规范化后,通过比较从处理过的和未经处理的RNA获得的cRT-PCR和RT-qPCR产物,对初级和加工的转录本进行区分。转录端验通过cRT-PCR产物的克隆和测序确定,然后由北方印块验证。
通过采用这一策略,确定了玉米线粒体中大多数稳态记录。由于一些线粒体基因的转录模式复杂,一些稳态转录图没有被区分和/或映射,尽管它们是在北方的污点中检测到的。我们不确定此策略是否适合在其他植物线粒体或石膏中区分和映射稳态记录。
Introduction
在植物线粒体中,许多成熟和前体RNA作为多种等形体积累,稳定状态转录法可以根据其5’末端1、2、3的差异被分成两组。 4.主转录有5’三磷酸盐末端,从转录启动派生。相比之下,经过处理的转录本具有5’单磷酸盐,通过转录后处理产生。两种类型的转录本的鉴别和映射对于解开转录和转录结束成熟的分子机制非常重要。
为了区分植物线粒体的主要转录和加工记录,已经开发了四个主要策略。第一种策略是用烟草酸焦磷酸酶(TAP)预处理线粒体RNA,将5’三磷酸盐转化为单磷酸盐,并使初级转录本通过RNA连带循环。然后通过快速扩增cDNA端(RACE)或圆形RT-PCR(cRT-PCR)2、3、4,比较TAP处理和未处理RNA样本的转录量。在第二种策略中,处理的转录本首先从线粒体RNA中耗尽,使用终结器5′-磷酸盐依赖外糖酶(TEX),然后通过引世扩展分析5,6映射所剩的主要转录本.第三种策略是使用甘蓝转移酶预盖初级转录,然后三磷酸盐5’termini的位置由引基延伸与核糖酸酶或S1核酸酶保护分析7,8确定 ,9.与基于5’三磷酸的存在/不存在的不同,第四种策略结合了体外转录、位点定向诱变和引物延伸分析,以表征启动子并确定转录启动站点8,10,11。通过使用这些策略,许多主要和经过处理的转录本在植物线粒体中已经确定。
然而,一些研究报告说,原发转录的5’三磷酸盐是不稳定的,他们很容易转换为单磷酸盐不明的原因2,4,12,13。这个问题阻碍了对两种类型的成绩单的明确区分,因为使用技术取决于存在/缺少 5′ 三磷酸,以及以前在工厂中系统地区分初级记录和加工抄本的努力线粒体失败2,12。
在此协议中,我们将cRT-PCR、RNA 5’聚磷酸酶处理、RT-qPCR和北方斑点相结合,系统地区分玉米(ZeaMays)线粒体中稳态积累的初级和加工转录(图1)。cRT-PCR允许同时映射RNA分子的5’和3’的四肢,它通常适应在植物2,12,14,15的图谱抄本术语。RNA 5′ 聚磷酸酶可以从三磷酸盐5’特尼中去除两种磷酸盐,这使得主转录本可用于RNA连结酶的自我结扎。先前的研究表明,玉米中成熟的26S rRNA已经加工了5’终点,对RNA5’聚磷酸酶1,16)不敏感。为了将不稳定的三磷酸盐对原发5’特尼的影响降至最低,5’聚磷酸酶处理和未处理RNA通过成熟的26S rRNA进行归一化,然后通过比较来区分原发和加工的转录本。从两个RNA样本中获得的cRT-PCR产物。cRT-PCR映射和鉴别结果分别由北方印子和RT-qPCR验证。最后,使用替代引液来放大在北方污点中检测到的转录本,而不是通过cRT-PCR检测到的。通过这种基于cRT-PCR的策略,对玉米线粒体中大多数稳态转录被区分和映射为1。
Protocol
Representative Results
Discussion
在先前的研究中,利用阿拉伯多普西细胞悬浮培养的组蛋白和线粒体RNA,用cRT-PCR绘制线粒体转录终点,结果相似,获得12.然而,在许多其他研究中,只有富集线粒体RNA被用于绘制线粒体转录终点尼的图谱。研究发现,线粒体RNA的富集是玉米中线粒体转录终点的cRT-PCR映射的重要一步。在?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金(授权号31600250,Y.Z.)、广州市科技项目(批号201804020015,H.N.)和中国农业研究系统(批号No.S.)的支持。CARS-04-PS09,H.N.)。
Materials
| Acetic acid | Aladdin, China | A112880 | To prepare 1x TAE buffer |
| Applied Biosystems 2720 Thermal Cycler | Thermo Fisher Scientific, USA | 4359659 | Thermal cycler for PCR amplification |
| Ascorbic acid | Sigma-aldrich, USA | V900134 | For preparation of extraction buffer |
| Biowest Agarose | Biowest, Spain | 9012-36-6 | To resolve PCR products and RNAs |
| Bovine serum albumin | Sigma-aldrich, USA | A1933 | For preparation of extraction buffer |
| Bromophenol blue | Sigma-aldrich, USA | B8026 | For preparation of loading buffer for agarose gel electrophoresis and Northern blot |
| DEPC | Sigma-aldrich, USA | V900882 | Deactivation of RNase |
| DIG Northern starter kit | Roche, USA | 12039672910 | For DIG-RNA labeling and Northern blot. This kit contains the reagents for transcription-labeling of RNA with DIG and T7 RNA polymerase, hybridization and chemiluminescent detection. |
| EDTA | Sigma-aldrich, USA | V900106 | For preparation of extraction buffer and 1x TAE buffer |
| EGTA | Sigma-aldrich, USA | E3889 | For preparation of wash buffer |
| Gel documentation system | Bio-Rad, USA | Gel Doc XR+ | To image the agarose gel |
| Glycerol | Sigma-aldrich, USA | G5516 | For preparation of loading buffer for agarose gel electrophoresis |
| GoldView II (5000x) | Solarbio,. China | G8142 | DNA staining |
| Hybond-N+, Nylon membrane | Amersham Biosciences, USA | RPN119 | For Northern blot |
| Image Lab | Bio-Rad, USA | Image Lab 3.0 | Image gel, and compare the abundance of PCR products. |
| KH2PO4 | Sigma-aldrich, USA | V900041 | For preparation of extraction buffer |
| KOH | Aladdin, China | P112284 | For preparation of extraction buffer |
| L-cysteine | Sigma-aldrich, USA | V900399 | For preparation of extraction buffer |
| Millex | Millipore, USA | SLHP033RB | To sterile extraction and wash buffers by filtration |
| Miracloth | Calbiochem, USA | 475855-1R | To filter the ground kernel tissues |
| MOPS | Sigma-aldrich, USA | V900306 | For preparation of running buffer for Northern blot |
| NanoDrop | Thermo Fisher Scientific, USA | 2000C | For RNA concentration and purity assay |
| NaOH | Sigma-aldrich, USA | V900797 | For preparation of wash buffer |
| pEASY-Blunt simple cloning vector | TransGen Biotech, China | CB111 | Cloning of the gel-recovered band. It contains a T7 promoter several bps upstream of the insertion site. |
| Phanta max super-fidelity DNA polymerase | Vazyme, China | P505 | DNA polymerase for PCR amplification |
| Polyvinylpyrrolidone 40 | Sigma-aldrich, USA | V900008 | For preparation of extraction buffer |
| Primer Premier 6.24 | PREMIER Biosoft, USA | Primer Premier 6.24 | To design primers for reverse transcription and PCR amplification |
| PrimeScript II reverse transcriptase | Takara, Japan | 2690 | To synthesize the first strand cDNA |
| PureLink RNA Mini kit | Thermo Fisher Scientific, USA | 12183025 | For RNA purificaion |
| RNA 5' polyphosphatase | Epicentre, USA | RP8092H | To convert 5' triphosphate to monophosphate |
| RNase inhibitor | New England Biolabs, UK | M0314 | A component of RNA self-ligation and 5' polyphosphatase treatment reactions, and it is used to inhibite the activity of RNase. |
| Sodium acetate | Sigma-aldrich, USA | V900212 | For preparation of running buffer for Northern blot |
| Sodium chloride | Sigma-aldrich, USA | V900058 | To prepare 20x SSC |
| SsoFas evaGreen supermixes | Bio-Rad, USA | 1725202 | For RT-qPCR |
| T4 RNA Ligase 1 | New England Biolabs, UK | M0437 | For RNA circularization |
| Tetrasodium pyrophosphate | Sigma-aldrich, USA | 221368 | For preparation of extraction buffer |
| TIANgel midi purification kit | Tiangen Biotech, China | DP209 | To purify DNA fragments from agarose gel |
| Tris | Aladdin, China | T110601 | To prepare 1x TAE buffer |
| TRIzol reagent | Invitrogen, USA | 15596026 | To extract mitochondiral RNA. |
| Universal DNA purification kit | Tiangen Biotech, China | DP214 | To recover linearized plastmids from the restriction enzyme digestion reaction |
| Xylene cyanol FF | Sigma-aldrich, USA | X4126 | For preparation of loading buffer for agarose gel electrophoresis |
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