A Simplified Method for Extracting Genomic DNA from Rice Leaves

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

Abstract

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

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.

Figures/Tables 6

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

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

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

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

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

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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