Method for rapidly detecting rice high-alkali elimination functional sites based on qRT-PCR (quantitative reverse transcription-polymerase chain reaction) technology

Abstract

The invention discloses a method for rapidly detecting a rice high-alkali elimination functional site based on a qRT-PCR (quantitative reverse transcription-polymerase chain reaction) technology. The method comprises the following steps: step 1, extracting genome DNA (deoxyribonucleic acid) of a rice plant; step 2, qRT-PCR (quantitative reverse transcription-polymerase chain reaction) amplification: adding the two primers ALK-TT-F and ALK-TT-R into a reaction system at the same time, and amplifying 215bp at the functional site of the ALK gene in the rice plant; and step 3, reading a Ct value. According to the present invention, based on the Quantitative Real Time PCR (qRT-PCR) technology and the 4327 and 4328 base variation sites of the ALK gene sequence, the primers (ALK-TT-F: AACCAGCTCTACGCCAT and ALK-TT-R: CCGCGCACCTGGTAA) capable of only amplifying the ALK high alkali elimination value functional sites are designed, and the Ct value is directly read through the qRT-PCR to obtain the genotype data containing the rice high alkali elimination value so as to screen the rice variety with the high cooking quality;

Description

Technical Field

[0001] The invention relates to the technical field of rice gene detection, and in particular to a method for rapidly detecting rice high alkaline elimination value functional sites based on qRT-PCR technology. Background Art

[0002] Rice is one of the most important food crops in my country. With the improvement of people's living standards, people's requirements for rice quality are getting higher and higher. The cooking and eating quality of rice mainly includes the content of amylose, gelatin consistency and gelatinization temperature. Among them, the alkali digestion value method is the most reliable for the determination of gelatinization temperature. The rice with high alkali digestion value also has higher taste quality. Therefore, developing a method for rapid detection of high alkali digestion value functional sites and using it in molecular marker-assisted selection breeding has important practical research significance for improving rice quality.

[0003] Gao et al. found that the ALK gene in rice is the main gene regulating the alkali absorption value of rice. Generally, under the haplotype where the 4327th and 4328th bases of the gene are both T, the alkali absorption value of the rice is higher, reaching 5.7-7.0. Liu Liping et al. conducted an association analysis on the ALK allele variation of the backbone parent materials of hybrid rice and the cooking and eating quality. The results showed that the 864 / 865 (GC / TT) mutation site of exon 8, that is, the 4327th and 4328th base mutation sites of the gene, had the largest association index with the alkali absorption value (Journal of Applied and Environmental Biology, 2023). Wang Jun et al. developed the functional markers ALKH5 and ALKH5 of the ALK gene based on HRM technology, and conducted ALK genotype detection on 81 indica rice varieties and 279 japonica rice varieties. The results showed that 212 japonica rice varieties and 62 indica rice varieties contained the G-TT genotype (Chinese Journal of Rice Science, 2023).

[0004] In the prior art, HRM technology is generally used to perform GC / TT genotyping on the 4327th and 4328th bases of the ALK gene, and the genotype is determined by the melting curve peak diagram. However, determining the TT and GC sites by melting curve is relatively complicated and time-consuming. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] The purpose of the present invention is to solve the problem that it is complicated and time-consuming to judge TT and GC sites through melting curves in the prior art, and to propose a method for quickly detecting rice high alkaline elimination value functional sites based on qRT-PCR technology.

[0007] 2. Technical solution

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for rapidly detecting rice high alkalinity elimination value functional sites based on qRT-PCR technology, comprising the following steps:

[0010] Step 1: Extract genomic DNA from rice plants;

[0011] Step 2: qRT-PCR amplification: Add two primers ALK-TT-F and ALK-TT-R to the reaction system at the same time, and amplify 215 bp at the functional site of the ALK gene in the rice plant;

[0012] Step 3: Read the Ct value.

[0013] Preferably, in step 1, the genomic DNA of rice is obtained by CTAB extraction method.

[0014] Preferably, the primers for qRT-PCR of the rice alkali-absorption regulating gene ALK in step 2 are as follows: ALK-TT-F: AACCAGCTCTACGCCAT, ALK-TT-R: CCGCGCACCTGGTAA.

[0015] Preferably, the PCR reaction procedure in step 2 is: 1) pre-denaturation: 95°C, 1 min; 2) 39 cycles of reaction: 95°C, 20 sec; 60°C, 30 sec.

[0016] Preferably, the qRT-PCR reaction system in step 2 is 25 μL: 12.5 μL 2×SYBR Green mastermix, 0.3 μL 10 mM ALK-TT-F primer, 0.3 μL 10 mM ALK-TT-R primer, 5 μL template DNA, and 6.9 μL ddH2O.

[0017] Preferably, in step 3, if the Ct value is less than 30, it is a high alkali elimination value functional site; if the Ct value is greater than 33, it is a low alkali elimination value functional site.

[0018] 3. Beneficial effects

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] In the present invention, based on the Quantitative Real Time PCR (qRT-PCR) technology and the base variation sites at positions 4327 and 4328 of the ALK gene sequence, primers that can only amplify the ALK high alkali consumption value functional site are designed (composed of two primers ALK-TT-F: AACCAGCTCTACGCCAT, ALK-TT-R: CCGCGCACCTGGTAA). Through qRT-PCR, the Ct value is directly read to obtain the genotype data containing the high alkali consumption value of rice, which is used to screen rice varieties with higher cooking quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of sequence difference analysis of high-base elimination value and low-base elimination value sites of the ALK gene proposed in the present invention, wherein TT is a high-base elimination value functional site; GC is a low-base elimination value functional site;

[0022] Figure 2 This is the qRT-PCR primer design diagram for the ALK gene high alkaline elimination value functional site proposed in the present invention. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0024] Embodiment 1:

[0025] Reference Figure 1-2 A method for rapidly detecting rice high alkalinity elimination value functional sites based on qRT-PCR technology comprises the following steps:

[0026] Step 1: Extract genomic DNA from rice plants;

[0027] Step 2: qRT-PCR amplification: Add two primers ALK-TT-F and ALK-TT-R to the reaction system at the same time, and amplify 215bp at the functional site of the ALK gene in the rice plant; the PCR reaction procedure is: 1) pre-denaturation: 95℃, 1min; 2) 39 cycles of reaction: 95℃, 20sec; 60℃, 30sec;

[0028] The qRT-PCR reaction system was 25 μL: 12.5 μL 2×SYBR Green master mix, 0.3 μL 10 mM ALK-TT-F primer, 0.3 μL 10 mM ALK-TT-R primer, 5 μL template DNA, 6.9 μL ddH2O;

[0029] The primers for qRT-PCR of rice alkali-absorption regulating gene ALK are as follows:

[0030] ALK-TT-F: AACCAGCTCTACGCCAT, ALK-TT-R: CCGCGCACCTGGTAA;

[0031] Step 3: Read the Ct value. If the Ct value is less than 30, it is a high-alkali elimination value functional site; if the Ct value is greater than 33, it is a low-alkali elimination value functional site.

[0032] In this embodiment, the high-alkali-dissipation-value Guangxi high-quality hybrid indica rice parent material Yexiang B and the low-alkali-dissipation-value hybrid indica rice Parent The ALK gene sequence alignment in material lilac B showed the presence of TT / GC at bases 4327 and 4328 ( Figure 1 ), primers that can only amplify the high alkali digestion value site sequence in qRT-PCR are designed. By reading the Ct value, it is possible to directly determine whether it is the functional site TT with high alkali digestion value, providing a fast and reliable technology for screening rice varieties with higher cooking quality.

[0033] In this example, based on the Quantitative Real Time PCR (qRT-PCR) technique and the 4327th and 4328th base variant sites of the ALK gene sequence ( Figure 1 ), designed primers that can only amplify the ALK high base elimination functional site (consisting of 2 primers ALK-TT-F: AACCAGCTCTACGCCAT, ALK-TT-R: CCGCGCACCTGGTAA) Figure 2 ), through qRT-PCR, the Ct value is directly read to obtain the genotype data including the high alkali consumption value of rice, which is used to screen rice varieties with higher cooking quality.

[0034] Embodiment 2:

[0035] In this embodiment, a method for rapidly detecting rice high alkalinity elimination functional sites based on qRT-PCR technology comprises the following steps:

[0036] Step 1: Extracting rice genomic DNA: 31 rice leaves grown in Nanning, Guangxi were taken respectively, and rice genomic DNA was obtained by CTAB extraction method (cetyltrimethylammonium bromide method);

[0037] Step 2: qRT-PCR: The qRT-PCR reaction system is 25ul: 12.5μL 2×SYBR Green master mix, 0.3μL 10mM ALK-TT-F primer, 0.3μL 10mM ALK-TT-R primer, 5μL template DNA, 6.9μL ddH2O. PCR reaction procedure: 1) Pre-denaturation: 95℃, 1min; 2) 39 cycles of reaction: 95℃, 20sec; 60℃, 30sec;

[0038] Step 3: Read the Ct value, as shown in Table 1. The alkali elimination value of 31 indica rice varieties was measured, and those with a Ct value less than 30 were functional sites of the ALK gene with high alkali elimination value, and their alkali elimination values ​​were significantly higher than those of other varieties (Table 1).

[0039] In this embodiment, it can be seen from Table 1 that 10 varieties with Ct values ​​less than 30 contain ALK high alkali elimination value functional sites. Combined with the results of rice alkali elimination value data analysis, the 10 varieties with Ct values ​​less than 30 have relatively high alkali elimination values, ranging from 6.4 to 7.0; while the 21 varieties with Ct values ​​greater than 33 have relatively low alkali elimination values, ranging from 2.9 to 5.0. Therefore, this method can be used to quickly screen high-quality rice varieties with high alkali elimination values;

[0040] Table 1 Alkali elimination value data of 31 indica rice parent materials

[0041]

[0042]

[0043] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for rapid detection of rice high alkalinity functional loci based on qRT-PCR technology, It is characterized in that The following steps are involved: Step 1: Extract genomic DNA from rice plants; Step 2: qRT-PCR amplification: Add two primers ALK-TT-F and ALK-TT-R to the reaction system at the same time, and amplify 215 bp at the functional site of the ALK gene in the rice plant; Step 3: Read the Ct value.

2. A method for rapidly detecting rice high alkalinity elimination functional sites based on qRT-PCR technology according to claim 1, It is characterized in that In the step 1, the genomic DNA of rice is obtained by the CTAB extraction method.

3. A method for rapidly detecting rice high alkalinity elimination functional sites based on qRT-PCR technology according to claim 1, It is characterized in that The primers for qRT-PCR of the rice alkali-absorption regulating gene ALK in step 2 are as follows: ALK-TT-F: AACCAGCTCTACGCCAT, ALK-TT-R: CCGCGCACCTGGTAA.

4. A method for rapidly detecting rice high alkalinity elimination functional sites based on qRT-PCR technology according to claim 1, It is characterized in that The PCR reaction procedure in step 2 is: 1) pre-denaturation: 95° C., 1 min; 2) 39 cycles of reaction: 95° C., 20 sec; 60° C., 30 sec.

5. A method for rapidly detecting rice high alkalinity elimination functional sites based on qRT-PCR technology according to claim 1, It is characterized in that The qRT-PCR reaction system in step 2 is 25 μL: 12.5 μL 2×SYBR Green mastermix, 0.3 μL 10 mM ALK-TT-F primer, 0.3 μL 10 mM ALK-TT-R primer, 5 μL template DNA, and 6.9 μL ddH2O.

6. A method for rapidly detecting rice high alkalinity elimination functional sites based on qRT-PCR technology according to claim 1, It is characterized in that In step 3, if the Ct value is less than 30, it is a high alkali elimination value functional site; if the Ct value is greater than 33, it is a low alkali elimination value functional site.

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