Fluorescence labeling primer group of rice heat-resistant gene TT2 and application of fluorescence labeling primer group

Abstract

The invention relates to the field of molecular biology and genetic breeding, in particular to a fluorescence labeling primer group of a rice heat-resistant gene TT2 and application of the fluorescence labeling primer group. The primer group comprises a primer TT2-F, a primer TT2-A-R and a primer TT2-C-R; the nucleotide sequence of the primer TT2-F is as shown in SEQ ID NO: 1; the nucleotide sequence of the primer TT2-A-R is as shown in SEQ ID NO: 2; the nucleotide sequence of the primer TT2-C-R is as shown in SEQ ID NO: 3. The invention provides a detection primer for rapidly identifying TT2 excellent haplotypes, three specific fluorescent primers suitable for a PARMS amplification technology are designed based on TT2 gene 1637 site SNP difference, and an A / A heat-resistant type, a C / C heat-labile type and an A / C heterozygous type can be efficiently and accurately distinguished.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology and genetic breeding, and relates to a fluorescent labeling primer set of a rice heat-resistant gene TT2 and an application thereof. Background Art

[0002] As one of the most important food crops in the world, the production stability and yield of rice are of great significance to global food security. However, with the increasing trend of global warming, extreme high temperature weather events occur frequently, posing a serious threat to rice yield. The rice heat-resistant gene TT2 plays a key role in the mechanism of rice coping with high temperature environment. The Gγ subunit encoded by this gene locus regulates the heat tolerance of rice by means of changes in the SCT1-dependent wax biosynthesis pathway. In a high temperature stress environment, the natural allele of TT2 functional loss is closely associated with a higher retention of wax and enhanced heat tolerance. From the mechanism of action, the transcription factor SCT1 interprets Ca by enhancing its interaction with calmodulin. 2+ signal and exert negative regulatory functions on its target genes (such as wax synthesis regulator 2, OSWR2). Disruption of the TT2 gene leads to heat-triggered Ca 2+ The decrease in calmodulin levels in the SCT1 matrix weakened the interaction between calmodulin and SCT1, which reasonably explained the observed changes in wax content induced by heat.

[0003] The mutation of the 1637th base in the TT2 gene from C to A confers heat resistance to related rice materials. However, at present, the development of efficient and widely applicable molecular markers for the TT2 gene is still unsatisfactory, which greatly limits its precise application in heat-resistant rice breeding. The reported molecular markers are linked markers of the TT2 excellent haplotype, which cannot accurately reflect the genotype of the identified material, and there is a risk of genetic separation, or it is necessary to use a high-throughput SNP analyzer to detect its haplotype, which undoubtedly puts high demands on the experimental cost and instrument matching. Therefore, the development of a TT2 excellent haplotype detection marker that is simple, efficient, low-cost, environmentally friendly, and has low hardware requirements is of vital importance to the promotion of heat-resistant rice breeding. It helps to more accurately screen and cultivate rice varieties that adapt to high temperature environments, providing strong support for agricultural production. Summary of the invention

[0004] The object of the present invention is to provide a fluorescent labeling primer set for the heat-resistant gene TT2 of rice and its application, so as to solve the problems raised in the above-mentioned background technology.

[0005] The TT2 gene is a Gγ subunit protein. After the 1637th base of the TT2 gene mutates from C to A, the heat resistance of rice is enhanced, that is, the homozygous A / A type represents a favorable haplotype for heat resistance. The present invention specifically detects the polymorphism of the C / A base at the 1637th base of the TT2 gene based on the different fluorescence wavelengths of the amplified fragments by using the PARMS amplification technology.

[0006] The present invention provides the following technical solutions:

[0007] A fluorescent labeling primer set for a rice heat-resistant gene TT2, the primer set comprising a primer TT2-F, a primer TT2-AR and a primer TT2-CR;

[0008] The nucleotide sequence of the primer TT2-F is shown in SEQ ID NO: 1; the nucleotide sequence of the primer TT2-AR is shown in SEQ ID NO: 2; and the nucleotide sequence of the primer TT2-CR is shown in SEQ ID NO: 3.

[0009] Furthermore, the primer TT2-AR is added with a fluorescent signal label of carboxyfluorescein FAM at the 5' end, and the primer TT2-CR is added with a fluorescent signal label of hexachlorofluorescein phosphoramidate HEX at the 5' end.

[0010] The application of the fluorescent labeled primer set of the above-mentioned rice heat-resistant gene TT2 in detecting the SNP gene of the rice heat-resistant gene TT2.

[0011] Furthermore, the method for detecting the SNP gene of the rice heat-resistant gene TT2 comprises the following steps:

[0012] S1. Extracting genomic DNA of rice to be tested;

[0013] S2. Using the detection primer set as claimed in claim 1, and taking the genomic DNA of the rice to be tested as a template, performing PARMS amplification on it;

[0014] S3. Read the data of the amplified product using an ELISA reader and perform typing of different fluorescence.

[0015] Furthermore, the PARMS amplification system in step S2 is: 10 μL reaction system, 1 μL template DNA, 0.15 μL TT2-AR primer with a concentration of 10 mM, 0.15 μL TT2-CR primer with a concentration of 10 mM, 0.4 μL TT2-F primer with a concentration of 10 mM, 5 μL 2×PARMS master mix, 3.3 μL ddH2O;

[0016] The PARMS amplification program in step S2 is: 94°C for 3 min; 94°C for 20 sec, 65°C for 1 min, for a total of 10 cycles, each cycle at -0.8°C; 94°C for 20 sec, 57°C for 1 min, for 30 cycles.

[0017] A fluorescence detection kit for a rice heat-resistant gene TT2, the kit comprising the above primer set.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a detection primer for rapid identification of excellent haplotypes of TT2. Based on the SNP difference at position 1637 of the TT2 gene, three specific fluorescent primers suitable for PARMS amplification technology are designed, which can efficiently and accurately distinguish the A / A heat-resistant type, the C / C heat-intolerant type and the A / C heterozygous type. TT2 genotype identification was performed on 39 materials in rice, and it was found that the excellent heat-resistant haplotype bands could be accurately distinguished. The detection method of the present invention is low in cost, simple in operation, and environmentally friendly, and does not require enzyme cutting, electrophoresis, sequencing, etc., and the SNP type of TT2 is directly judged by the fluorescence typing result. The detection method of the present invention plays a role in molecular assisted selection in the current heat-resistant breeding process of rice, and can effectively shorten the breeding period. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 The fluorescence detection results of different SNP polymorphisms of TT2 gene of the present invention;

[0022] Figure 2 The fluorescence detection results of the present invention on the SNP polymorphism of TT2 gene of 39 rice materials. DETAILED DESCRIPTION

[0023] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0024] like Figure 1-2 As shown, the present invention provides a technical solution:

[0025] In the present invention, there is no special requirement for the source of each primer in the primer set, and the various raw materials required for the PARMS amplification system used are commercial products on the market, and can be selected according to sources known in the art. The rice materials used can be obtained from commercial channels or national germplasm banks, or are materials widely used and promoted by those skilled in the art.

[0026] Case study 1: Design of excellent haplotype-specific PARMS fluorescence detection primers for the heat-resistant gene TT2 in rice

[0027] 1. SNP differential site analysis

[0028] According to the report in Kan Y, Mu XR, Zhang H, Gao J, Shan JX, Ye WW, Lin HX. TT2 controls rice thermotolerance through SCT1-dependent alteration of wax biosynthesis. Nat Plants. 2022 Jan; 8(1): 53-67., the heat resistance of the TT2 gene is caused by the mutation of base 1637 of the genome from C to A, which is a gain-of-function mutation.

[0029] 2. PARMS fluorescent primer design

[0030] The primers were designed based on the C / A polymorphism located at the 1637th base of the TT2 genome. The homozygous A / A type represents a heat-resistant favorable genotype. The primer combination included TT2-F, TT2-AR and TT2-CR, wherein primer TT2-F is a universal primer located upstream of the SNP, the last base at the 3' end of primer TT2-AR is the complementary base T of the A type at the 1637th base of the TT2 gene, the last base at the 3' end of primer TT2-CR is the complementary base G of the C type at the 1637th base of the TT2 gene, the primer TT2-AR has a fluorescent signal label of carboxyfluorescein FAM at the 5' end, and the primer TT2-CR has a fluorescent signal label of hexachlorofluorescein phosphoamidate HEX at the 5' end.

[0031] The nucleotide sequences of the detection primers are as follows:

[0032] Primer TT2-F, SEQ ID NO: 1:

[0033] 5'-CATGCCCATCTCCCTCGTTTA-3';

[0034] Primer TT2-AR, SEQ ID NO: 2:

[0035] 5'-GAAGGTGACCAAGTTCATGCTCAGGCTGGCTTACTCTCTT-3';

[0036] Primer TT2-CR: SEQ ID NO: 3:

[0037] 5'-GAAGGTCGGAGTCAACGGATTCAGGCTGGCTTACTCTCTG-3'.

[0038] Case study 2: Establishment of an excellent haplotype-specific PARMS fluorescence detection method for the heat-resistant gene TT2 in rice

[0039] The genomic DNA of the rice to be tested was extracted by the CTAB method, and the genomic DNA of the rice to be tested was used as a template to perform PARMS amplification on the TT2 gene in the rice genome using primers TT2-F, TT2-AR and TT2-CR;

[0040] The PARMS amplification program was as follows: 94°C for 3 min; 94°C for 20 sec, 65°C for 1 min, for a total of 10 cycles, -0.8°C per cycle; 94°C for 20 sec, 57°C for 1 min, for 30 cycles.

[0041] The PARMS amplification system was as follows: 10 μL reaction system, 1 μL template DNA, 0.15 μL 10 mM TT2-AR primer, 0.15 μL 10 mM TT2-CR primer, 0.4 μL 10 mM TT2-F primer, 5 μL 2×PARMS master mix, 3.3 μL ddH2O;

[0042] The amplified product was read by an ELISA reader and different fluorescences were typed to obtain the genotype results.

[0043] 3. Fluorescence typing

[0044] The method for determining the TT2 genotype is as follows: if the SNP genotype of the amplified fragment is T / T, it is determined that the rice sample to be tested carries the A / A superior haplotype of the TT2 gene; if the SNP genotype of the amplified fragment is G / G, it is determined that the rice sample to be tested is the C / C haplotype of the TT2 gene; if the SNP genotype of the amplified fragment is T / G, it is determined that the rice sample to be tested contains a heterozygous A / C base, indicating that the TT2 gene is in a heterozygous state. For specific fluorescence typing, see Figure 1 .

[0045] Case study 3: Screening and verification of fluorescence detection primers in 39 rice materials

[0046] In order to verify the practicability of the fluorescence detection primers, 39 rice materials with known TT2 genotypes were used for verification, such as Figure 2 As shown, the fluorescent detection primer combination can clearly distinguish excellent haplotypes from non-excellent haplotypes. The results show that only three of the 39 rice samples do not carry the excellent haplotype of TT2, and 36 rice samples carry the excellent haplotype of TT2. The detection results are accurate and reliable, and are completely consistent with the expected results, indicating that the TT2 genotype fluorescence detection method of the present invention is fully applicable to the molecular assisted selection of heat-resistant rice breeding at this stage; the specific rice names are shown in Table 1 below, and the typing results are shown in Figure 2 shown.

[0047] Table 1: Specific rice names

[0048]

[0049]

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescent labeled primer set for a rice heat-resistant gene TT2, characterized in that: The primer set includes primer TT2-F, primer TT2-AR and primer TT2-CR; The nucleotide sequence of the primer TT2-F is shown in SEQ ID NO: 1; the nucleotide sequence of the primer TT2-AR is shown in SEQ ID NO: 2; and the nucleotide sequence of the primer TT2-CR is shown in SEQ ID NO:

3.

2. The fluorescent labeled primer set for the heat-resistant gene TT2 of rice according to claim 1, characterized in that: The primer TT2-AR has a fluorescent signal label of carboxyfluorescein FAM added to the 5' end, and the primer TT2-CR has a fluorescent signal label of hexachlorofluorescein phosphoramidate HEX added to the 5' end.

3. Use of the fluorescent labeled primer set of the heat-resistant rice gene TT2 according to any one of claims 1 to 2 in detecting the SNP gene of the heat-resistant rice gene TT2.

4. Use of the fluorescent labeled primer set of the heat-resistant rice gene TT2 according to claim 3 in detecting the SNP gene of the heat-resistant rice gene TT2, characterized in that: The method for detecting the SNP gene of the rice heat-resistant gene TT2 comprises the following steps: S1. Extracting genomic DNA of rice to be tested; S2. Using the detection primer set as claimed in claim 1, and taking the genomic DNA of the rice to be tested as a template, performing PARMS amplification on it; S3. Read the data of the amplified product using an ELISA reader and perform typing of different fluorescence.

5. Use of the fluorescent labeled primer set of the heat-resistant rice gene TT2 according to claim 4 in detecting the SNP gene of the heat-resistant rice gene TT2, characterized in that: The PARMS amplification system in step S2 is: 10 μL reaction system, 1 μL template DNA, 0.15 μL TT2-AR primer with a concentration of 10 mM, 0.15 μL TT2-CR primer with a concentration of 10 mM, 0.4 μL TT2-F primer with a concentration of 10 mM, 5 μL 2×PARMS mastermix, 3.3 μL ddH2O; The PARMS amplification program in step S2 is: 94°C for 3 min; 94°C for 20 sec, 65°C for 1 min, for a total of 10 cycles, each cycle at -0.8°C; 94°C for 20 sec, 57°C for 1 min, for 30 cycles.

6. A fluorescence detection kit for the heat-resistant gene TT2 of rice, characterized in that: The kit comprises the primer set according to any one of claims 1 to 2.

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