一种水稻叶片基因组DNA简易提取方法

doi:10.13560/j.cnki.biotech.bull.1985.2024-0796

摘要/Abstract

摘要：

【目的】为了应对日益增加的大规模遗传材料鉴定需求，急需建立一种快速、简易、高效、低成本的基因组DNA提取方法。【方法】根据水稻遗传鉴定的特点，对传统TPS法进行改进和简化。（1）将特制的钢珠加样条与全自动研磨仪结合使用，提高了研磨水稻叶片组织的速度；（2）使用TPS缓冲液室温研磨裂解，无需液氮冷冻研磨；（3）研磨后的组织匀浆仅需75℃水浴20 min即可完成裂解；（4）室温静置5 min后吸取粗提液上清5 μL至96孔PCR板内，使用排枪加入95 μL 灭菌的去离子水进行稀释，即完成基因组DNA的提取过程。【结果】简易TPS法提取基因组DNA的步骤减少至6步以内，省略了DNA沉淀、离心、洗涤、溶解等过程。提取过程简单、快速，可在30 min内完成96个样本的DNA提取。提取过程中使用的试剂种类较少且廉价易得、安全环保。通过优化DNA提取液的用量，用简易TPS法提取的基因组DNA开展的各类常规分子遗传鉴定的效果与CTAB法及传统TPS法相当。【结论】简易TPS法提高了水稻基因组DNA提取的效率，适合高通量的遗传鉴定。

关键词: 水稻叶片, 基因组DNA, CTAB法, 传统TPS法, 简易TPS法

Abstract:

【Objective】In order to cope with the increasing demand for large-scale genetic material identification, it is urgent to establish a fast, simple, efficient and low-cost genomic DNA extraction method.【Method】The traditional TPS method was improved and simplified according to the characteristics of rice genetic identification. 1）The tissue grinding speed of rice leaves was improved by combining special steel bar and automatic grinding instrument. 2）Directly grinding and lysing using the TPS buffer at room temperature instead of freezing grinding with liquid nitrogen. 3）The homogenized tissue after grinding was lysed in a 75℃ water bath for 20 min. 4）After standing at room temperature for 5 min, took 5 μL of the supernatant from the crude extract and transferred it to into a 96-well PCR plate, and added 95 μL sterilized deionized water for dilution using a pipette to complete the genomic DNA extraction process.【Result】The simplified TPS method reduced the steps of genomic DNA extraction to less than 6 steps, omitting DNA precipitation, centrifugation, washing, dissolution and so on. The extraction process is simple and fast, and the DNA extraction of 96 samples can be completed within 30 minutes. The reagents used in the extraction process are fewer, cheaper, easy to be obtained, safe and environmental protection. By optimizing the amount of DNA extraction solution, the effect of conventional molecular genetic identification using genomic DNA extracted by simplified TPS method is equivalent to that of CTAB method and traditional TPS method.【Conclusion】The simplified TPS method improves the efficiency of rice genomic DNA extraction and is suitable for rice high-throughput genetic identification.

Key words: rice leaf, genome DNA, CTAB method, traditional TPS method, simplified TPS method

金素奎, 国倩倩, 刘巧泉, 高继平. 一种水稻叶片基因组DNA简易提取方法[J]. 生物技术通报, 2025, 41(1): 74-84.

JIN Su-kui, GUO Qian-qian, LIU Qiao-quan, GAO Ji-ping. A Simplified Method for Extracting Genomic DNA from Rice Leaves[J]. Biotechnology Bulletin, 2025, 41(1): 74-84.

图/表 6

图1 简易TPS法提取水稻叶片基因组DNA的基本过程 A：使用2 mL离心管采集的水稻幼苗叶片；B：使用定制的钢珠加样条向离心管内加入研磨用的钢珠；C：使用全自动研磨仪振荡研磨叶片；D：裂解后的叶片组织匀浆

Fig. 1 Basic process of extracting genomic DNA from rice leaves using the simplified TPS method A: Rice seedling leaves were collected using a 2 mL centrifuge tube. B: Add grinding steel balls to the centrifugal tube using customized steel bar. C: Grinding the blade using an automatic grinding instrument. D: Homogenate of lysed leaf tissue

表1 本研究中所用引物的核苷酸序列

Table 1 Nucleotide sequences of primers used in this study

引物名称Primer name	正向引物序列Forward primer sequence（5'-3'）	反向引物序列Reverse primer sequence（5'-3'）	产物大小Product size/bp
OsACTIN1	CCTTCAACACCCCTGCTATG	TGAGTAACCACGCTCCGTCA	470
STS-1	CATGCTAGTAAGCAAAGGGCAACG	TTGCACGTCCAACTGTCCAAGC	229
HPT	GCTTTCAGCTTCGATGTAGGAGG	TTTCCACTATCGGCGAGTACTTC	884
OsNAP	AGTACCCACCCTCACAGCTC	AGTTGGTCTTGGTGCCCTTG	319
Wx pro	CTACTAGATCCGCTGCCGCC	GCAGGTCACAGCATTTATCTAAGC	661

表2 三种植物基因组DNA提取方法的比较

Table 2 Comparison of three methods for extracting genomic DNA from plants

方法 Method	使用试剂及仪器Reagents and instruments used												步骤 Step	耗时 Time/h	纯度 Purity
方法 Method	液氮研磨 Liquid nitrogen grounding	CTAB	Tris	HCl	EDTA	NaCl	氯仿 Chloroform	异戊醇 Isoamylol	KCl	异丙醇 Isopropanol	乙醇 Ethanol	离心机 Centrifuge	步骤 Step	耗时 Time/h	纯度 Purity
CTAB法CTAB method	√	√	√	√	√	√	√	√	×	√	√	√	≥10	3-4	高 High
传统TPS法Traditional TPS method	×	×	√	√	√	×	×	×	√	√	√	√	≥10	1-1.5	一般 Average
简易TPS法Simplified TPS method	×	×	√	√	√	×	×	×	√	×	×	×	≤6	≤0.5	低 Low

图2 简易TPS法提取水稻叶片基因组DNA的PCR条件探索与优化 A：以日本晴幼苗的叶片为材料提取基因组DNA，以水稻OsACTIN1基因片段作为PCR扩增目的基因，PCR反应体系及条件如材料方法中所述； M为DNA marker（GoldBand DL5000（翊圣生物））；顶端数字表示DNA粗提液稀释倍数；图右侧数字表示不同的单株重复；B：水稻OsACTIN1基因序列的结构示意图。其中橙色带方向框以及字母Ex-1/2/3/4表示外显子，黑色箭头以及字母P-F/R分别表示PCR扩增中使用的正向和反向引物，虚竖线及其下方的数字表示碱基位置；C：OsACTIN1的PCR产物进行测序所获得部分峰图。对基因组DNA稀释20倍条件下获得的PCR产物进行测序，测序引物为OsACTIN1基因PCR扩增的正向引物；D：对测序获得的序列与OsACTIN1的基因序列进行比对。黑色背景的字母表示相同的碱基

Fig. 2 Exploration and optimization of PCR conditions for extracting genomic DNA from rice leaves by the simplified TPS method A: The genomic DNA was extracted from the leaves of Nipponbare seedlings, and the rice OsACTIN1 gene fragment was used as the target gene for PCR amplification. The PCR reaction system and conditions were described in the Material and Method section. M stands for DNA marker（GoldBand DL5000（YEASEN BioTech））; the numbers at the top of the figure refers to the dilution ratio of DNA crude extract; the numbers on the left of the figure refer to band size（bp）of DNA marker; the numbers on the right of the figure refer to different individual replicates. B: Structural diagram of rice OsACTIN1 gene. The orange boxes with direction and the letters Ex-1/2/3/4 indicate exons, the black arrows with the letters P-F/R above indicate forward and reverse primers used in PCR amplification, respectively, and the dashed vertical lines with the number below indicate base positions. C: The partial peak maps of sequenced PCR products. The PCR products were obtained and sequenced under the 20-fold dilution of the genomic DNA, and the sequencing primer was the forward primer of OsACTIN1 gene used in PCR amplification. D: The sequences obtained by sequencing were aligned with the OsACTIN1gene. The letters on the black background indicate the same bases

图3 简易TPS法与CTAB法、传统TPS法提取的水稻基因组DNA用于遗传鉴定的比较 A：用Koshihikari（K）、Nona Bokra（N）及其F2个体提取基因组DNA，并用分子标记STS-1检测基因型。K和N分别表示2个亲本，数字1-12分别表示12个不同的单株；B：以日本晴背景的OsNAC24 gD-OE过表达转基因植株为材料，检测HPT基因。M表示DNA marker，本实验使用的DNA marker为DNA marker III（天根生化），数字1-12表示12个不同的单株，其中第2、7、8号3个单株为阴性单株；C：以野生型日本晴为材料，扩增OsNAP基因片段，并使用BamH I酶切PCR产物。M表示DNA marker，泳道顶端数字1-12表示12个不同的单株，每个单株左侧的泳道为未酶切的PCR产物，右侧的泳道为被BamHI酶切后的PCR产物；D：对日本晴中编辑Wx基因启动子的转基因植株进行的测序鉴定。图的上部是Wx基因的启动子结构示意图，图的下方左侧为测序后的序列，图的下右侧为对应的测序峰图。TSS表示转录起始位点，ATG表示起始密码子。NIP：日本晴

Fig. 3 Comparison of rice genomic DNA extracted by the simplified TPS method, CTAB method, and traditional TPS method in genetic identification A: The genomic DNA was extracted from Koshihikari（K）, Nona Bokra（N）and their F2 individuals, and the molecular marker STS-1 was used in genotyping. K and N refer to two parents respectively, and the number 1 to 12 refer to 12 individual plants. B: The HPT gene was detected in the OsNAC24 gD-OE transgenic plants under Nipponbare background. M refers to DNA marker, the DNA marker used in this experiment was DNA marker III（TIANGEN Biochemistry）, and the number 1 to 12 refer to 12 individual plants, among which, 3 plants No. 2, 7, and 8 were negative. C: The wild-type Nipponbare plants were used to amplify the OsNAP gene, then the PCR product was digested by BamH I. M refers to DNA marker, the DNA marker used in this experiment was GoldBand DL5000（YEASEN BioTech）; and the number from 1 to 12 at the top of the lane refer to 12 individual plants. The lane on the left of each single plant is the PCR product without digestion, and the lane on the right is the PCR product digested by the BamHI. D: Identification of transgenic plants with edited Wx promoter by sequencing in Nipponbare. The upper part of the figure is a schematic diagram of the Wx promoter, the lower left part of the figure are the sequences of target, and the lower right part of the figure is the corresponding sequencing peak. TSS stands for the transcription start site and ATG refers to the start codon. NIP: Nipponbare

图4 简易TPS法提取的基因组DNA的稳定性检测分别用新鲜提取的和在-20℃保存半年以上的日本晴基因组DNA检测OsACTIN1和OsNAP基因。M表示DNA marker，本实验使用的DNA marker为GoldBand DL5000（翊圣生物），数字1-12表示12个不同的单株

Fig. 4 Stability detection of genomic DNA extracted by simplified TPS method The detection of OsACTIN1 and OsNAP genes was performed using genomic DNA of Nipponbare freshly extracted and stored at -20℃ for more than six months, respectively. M refers to DNA marker, the DNA marker used in this experiment is GoldBand DL5000（YEASEN BioTech）, and the number 1 to 12 refer to 12 different individual plants

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