CN114717352B - Molecular marker of rice high temperature resistant regulatory gene Hsp70 and application thereof - Google Patents
Molecular marker of rice high temperature resistant regulatory gene Hsp70 and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker capable of identifying a rice high temperature resistant gene Hsp70, a forward and reverse primer thereof and application thereof in rice high temperature resistant screening and breeding. The marker has no genetic exchange through sequence difference design, and has high accuracy; the method is directly used for polyacrylamide gel electrophoresis detection, and is simpler and more convenient; can shorten the breeding period and improve the breeding efficiency.
Description
Technical Field
The invention belongs to the technical field of plant propagation, in particular relates to the technical field of agricultural biology, and in particular relates to a molecular marker of a rice high temperature resistant regulatory gene Hsp70 and application thereof.
Background
Rice is a staple food of nearly half of the population worldwide, and is also the most important food crop in China. However, as global climate warms, high temperature has serious influence on rice production, and when the rice is in high temperature weather in the heading and flowering period, the maturing rate of the rice is reduced, and the yield of the rice is reduced; the rice grain filling degree can be influenced by high-temperature weather in the rice grouting period, and the rice yield and the rice quality can be reduced. Therefore, the cultivation of the high-temperature-resistant rice variety has important significance for ensuring the rice yield and the rice quality under the high-temperature condition. A large number of researches show that the tolerance of rice to high temperature is regulated by an intrinsic genetic mechanism and external environmental factors. Therefore, the genetic law of high temperature resistance of rice is known, and the method has important significance for ensuring high and stable yield of rice and ensuring rice quality when the rice encounters high-temperature weather by improving the high-temperature resistance of the rice.
In recent years, with the development of genetic and molecular biological techniques, genes for regulating rice high temperature resistance TT1 (Li et al, nature Genetics,2015,47 (7): 827-33), SLG1 (Xu et al, nature Communications,2020,11 (1): 5441) and the like have been cloned. Among them, it has been shown that TT1 derived from African rice CG14 can be transferred into rice to improve the rice's tolerance to high temperatures (Li et al, nature Genetics,2015,47 (7): 827-33). The mutant slg is fine in stem, increased in grain length, reduced in-vivo tRNA thiolation level and sensitive to high temperature; the SLG1 overexpression can obviously improve the high-temperature tolerance of rice. SLG1 has differentiation between indica and japonica subspecies, and variation in promoter and coding region results in increased levels of thiolated tRNA and increased heat tolerance in indica varieties (Xu et al Nature Communications,2020,11 (1): 5441).
Molecular marker assisted selection is a modern breeding technology for selecting target traits by combining genotype and phenotype identification in offspring by utilizing markers closely linked with high temperature resistant genes or functional markers in genes. The method can not only greatly shorten the breeding period and improve the breeding efficiency, but also save a great deal of manpower and material resource costs. Therefore, the method has great application value in improving the high-temperature tolerance of rice, ensuring the high and stable yield of the rice in a high-temperature environment, and developing the functional marker of the high-temperature resistant gene has great significance in fully utilizing the gene, further improving the tolerance of the rice to high temperature and ensuring the grain safety.
Disclosure of Invention
Based on the molecular marker, the invention aims to provide a molecular marker of a rice high-temperature resistant gene Hsp70, and a forward primer and a reverse primer and application thereof. The specific technical scheme is as follows:
a molecular marker of a rice high temperature resistant gene Hsp70 is a high temperature resistant gene Hsp70-12, and a nucleotide sequence of a primer for amplifying the molecular marker is shown as SEQ ID NO.1 and SEQ ID NO. 2.
In some of these embodiments, the molecular marker is located on chromosome 1 of the rice genome.
In some of these embodiments, the size of the molecular markers is 131bp and/or 119bp.
In some of these embodiments, the nucleotide sequence of the molecular marker is SEQ ID No.3 and/or SEQ ID No.4.
The invention also provides a method for obtaining the molecular marker related to the heat-resistant property of rice, which comprises the following steps:
a method for obtaining a molecular marker related to heat resistance of rice comprises the following steps:
respectively taking genome DNA of rice variety Hanhui No.3 and Shanghai No. 1B as templates, and adopting primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 for PCR amplification;
and separating the PCR amplified product by electrophoresis to obtain an amplified fragment with the size of 131bp or an amplified fragment with the size of 119bp, namely the target molecular marker.
The invention also provides application of the molecular marker of the rice high temperature resistant gene Hsp70, which comprises the following specific steps:
the molecular marker of the rice on chromosome 1 is applied to the identification of the high temperature resistant characteristic of the rice and/or the auxiliary selective breeding of the high temperature resistant rice.
The invention also provides a method for identifying the rice high temperature resistant regulatory gene Hsp70 by using the molecular marker, which comprises the following specific technical scheme:
a method for identifying a rice high temperature resistant regulatory gene Hsp70 by using a molecular marker comprises the following steps:
1) Extracting genome DNA of a rice sample to be tested;
2) Performing PCR amplification on the genome extracted in the step 1) by using primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 to obtain an amplification product;
in some embodiments, detecting the PCR product obtained in the step 2) through 6% polyacrylamide gel electrophoresis, and if a 131bp band exists, indicating that the sample is the allele type of the high-temperature sensitive variety Hanhui No. 3; if 119bp bands are present, the genotype of the sample is indicated as the Shanghai drought 1B allele of the refractory variety.
In some of these embodiments, the PCR amplified amplification system comprises: taq enzyme, template DNA, dNTPs, and primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and PCR buffer.
In some embodiments, the PCR amplification is performed by a PCR reaction procedure comprising: pre-denaturation at 95+ -1deg.C for 4-6min, then carrying out 32 cycles at 98+ -1deg.C for 20s, 54.5+ -0.5deg.C for 15-25s, and 71+ -1deg.C for 20-30s, and finally extending at 71+ -1deg.C for 4-6min. The invention also provides a molecular marker assisted selection breeding method for high temperature resistant rice, which comprises the following steps:
a molecular marker assisted selection breeding method for high temperature resistant rice comprises the following steps:
1) Extracting genome DNA of a rice sample to be tested;
2) And (3) carrying out PCR amplification on the genome extracted in the step (1) by using primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 to obtain an amplification product.
In some of these embodiments, the PCR product obtained in step 2) is detected by 6% polyacrylamide gel electrophoresis, if there is a 131bp band, indicating that the sample is a high temperature sensitive variety allele; if 119bp bands exist, the genotype of the detected sample is indicated to be the allele of the high-temperature resistant variety.
The invention also provides a kit for identifying the rice high temperature resistant regulatory gene Hsp70, which has the following specific technical scheme:
a kit for identifying a rice high temperature resistant regulatory gene Hsp70, comprising: the nucleotide sequences are shown as a primer shown in SEQ ID NO.1 and a primer shown in SEQ ID NO.2, the primer is used for amplifying a molecular marker of a rice high temperature resistant regulatory gene Hsp70, and the molecular marker is Hsp70-12.
Based on the technical scheme, the invention has the following beneficial effects:
the molecular marker capable of identifying the rice high temperature resistant regulatory gene Hsp70, the forward and reverse primers thereof and the application thereof in screening and breeding of rice high temperature resistance. The marker has no genetic exchange through sequence difference design, and has high accuracy; the method is directly used for polyacrylamide gel electrophoresis detection, and is simpler and more convenient; can shorten the breeding period and improve the breeding efficiency.
Drawings
FIG. 1 is an electrophoresis detection diagram of 24 rice varieties by using the molecular marker Hsp70-12.
FIG. 2 shows the verification results of Hsp70-12 molecular markers in the high temperature resistant performance evaluation of different populations in the invention.
Detailed Description
In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It should be understood that the experimental methods in the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions suggested by the manufacturer. The various reagents commonly used in the examples are all commercially available products.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention is described in detail below by way of examples:
example 1 development of molecular markers for Hsp70 Gene, a high temperature resistant regulatory Gene of Rice
(1) Test material: high temperature sensitive variety: no.3 of arid hui; high temperature resistant variety: shanghai dry 1B.
(2) The extraction method of rice genome DNA comprises the following steps: in a mortar, 10mg of rice leaves are taken, a proper amount of liquid nitrogen is added, the rice leaves are ground into powder, 400 mu L of 1.5 xCTAB (1.5% CTAB,75mmol/L Tris-HCl,15mmol/L EDTA,1.05mol/L NaCl, ph=8.0) is added, and the mixture is ground into homogenate, and thenAdding 400 μL of 1.5×CTAB, sucking grinding fluid into 1.5ml centrifuge tube, adding 550 μL of chloroform, mixing, centrifuging at 12000r/min for 10min, collecting supernatant into another centrifuge tube, adding precooled equal volume isopropanol, centrifuging at 12000r/min for 5min, removing supernatant, drying precipitate, and adding 200 μL of ddH 2 O is dissolved.
(3) Development of gene molecular marker Hsp 70-12: hsp70 is located on chromosome 1 and is registered in the Japanese reference genome as Os01g0721000.
(4) According to the differential analysis of the nucleotide sequences of genes of a high temperature sensitive variety and a high temperature resistant variety, designing a primer Hsp70-12, wherein the forward and reverse primer sequences are as follows:
Hsp70-12 F：5’-CTCCTCGACAAGCTGATGTTC-3’(SEQ ID NO.1)；
Hsp70-12 R：5’-CTCGCGACGCCAACTATGT-3’(SEQ ID NO.2)。
(5) Carrying out PCR amplification on the rice variety to be tested, namely Hanhui No.3 and Shanghai No. 1B by using a functional marker Hsp70-12, wherein a PCR amplification system is as follows: 20 ng/. Mu.L of rice genome DNA template 1. Mu.L, 10. Mu.L of 2 XTaq Master mix (Nanjinouzan Biotechnology Co., ltd.), 10. Mu.M of each of the front and rear primers 1. Mu.L, and dd H was supplemented 2 O to 20. Mu.L. Pre-denaturation at 95+ -1deg.C for 4-6min, then 32 cycles at 98deg.C for 20s,54.5 deg.C for 20s, and 72 deg.C for 20s, and finally extension at 72 deg.C for 4min.
(7) 2. Mu.L of the PCR product was subjected to 6% polyacrylamide gel electrophoresis. As shown in FIG. 1, lane 23 is sensitive variety Hanhui No.3, and the band size is 131bp. Lane 24 is PCR product from Shanghai dry 1B with a band size of 119bp. From fig. 1, it can be seen that the electrophoresis bands are clear, and the difference is obvious.
(8) Electrophoresis cutting gel recycling/sequencing. The gel was recovered using a gel recovery kit (centrifugal column, catalog number: EG 101-02) from Beijing full gold biotechnology Co., ltd.) and sequencing was done by Shanghai Boshang biotechnology Co. The sequencing results are shown in Table 1.
Table 1: base comparison of corresponding molecular marker loci of Shanghai No.3 and Shanghai No. 1B
Dry recovery No. 3: (SEQ ID NO. 3)
CTCCTCGACAAGCTGATGTTCGGCCGGCAGAAGGAGGTGGAGCAGGTCGTTGGATTCCTGCTGCAGCCGGACGTCTGCGGCGCTGGCGCCGGCGCCGGCGCCGGCGTGCTGCACATAGTTGGCGTCGCGAG
Shanghai dry 1B (SEQ ID NO. 4)
CTCCTCGACAAGCTGATGTTCGGCCGGCAGAAGGAGGTGGAGCAGGTCGTTGGATTCCTGCTGCAGCCGGACGTCTGCGGCGCTGGCGCCGGCGTGCTGCACATAGTTGGCGTCGCGAG
Example 2: parental polymorphism detection of Hsp70-12 molecular marker
(1) The genome of the rice variety to be tested was extracted in the same manner as in example 1. The rice varieties tested are respectively as follows: dry land recovery 15, shanghai dry land 15, dry land rice No. 8, green land No.1, xiushui 115, peer C122, IRAT13, MILT1444, IAC1, keqing No.3, KU70-1, gongju No.3, CARTUNA, happy state, three grains, CICA4, BICO PRETO, mountain cereal, C418, MIFOR 6-2, oryza Glutinosa, ninghui 21.
(2) PCR amplification was performed as in example 1.
(3) Cleavage was performed as in example 1.
(4) Electrophoresis was performed as in example 1. The results are shown in FIG. 1.
Wherein M represents Marker:50bp DNA Ladder. Lanes 1 to 22, in order from left to right, correspond to rice variety drought-recovery 15 (heat resistance coefficient of 0.43), shanghai 15 (heat resistance coefficient of 0.50), gaddha 8 (heat resistance coefficient of 0.21), gaddha 1 (heat resistance coefficient of 0.80), xiushui 115 (heat resistance coefficient of 0.41), pec122 (heat resistance coefficient of 0.10), IRAT13 (heat resistance coefficient of 0.09), MILT1444 (heat resistance coefficient of 0.07), IAC1 (heat resistance coefficient of 0.26), keqing 3 (heat resistance coefficient of 0.62), KU70-1 (heat resistance coefficient of 0.15), mallotus 73 (heat resistance coefficient of 0.73), CARTUNA (heat resistance coefficient of 0.73), hairkuan early (heat resistance coefficient of 0.73), sanzhujiu (heat resistance coefficient of 0.72), CICA4 (heat resistance coefficient of 0.37), BICO TO (heat resistance coefficient of 0.41), mountain wine (heat resistance coefficient of 0.13), C (heat resistance coefficient of 0.418), and FOR 6 to FOR 0.08 (heat resistance coefficient of 0.8).
As can be seen, the sizes of electrophoresis bands of varieties including high temperature resistant alleles, namely, lane 1, lane 2, lane 1, lane 4, keqing 3 (Lane 10), mallotus 73 (Lane 12), CARTUNA (Lane 13), liangguo (Lane 14) and Sanyuan (Lane 15), are 119bp. The electrophoresis band sizes of the high temperature sensitive allele varieties of upland rice No. 8 (lane 3), xiushui 115 (lane 5), C122 (lane 6), IRAT13 (lane 7), MILT1444 (lane 8), IAC1 (lane 9), KU70-1 (lane 11), CICA4 (lane 16), BICO PRETO (lane 17), mountain valley (lane 18), C418 (lane 19), MIFOR 6-2 (lane 20), fragrant glutinous rice (lane 21) and Ninghui 21 (lane 22) are 131bp.
The summary comparison is as follows:
as can be seen from the comparison of the results, the heat resistance coefficient of the rice is related to the band size of the amplified product of the electrophoresis detection Hsp70-12 molecular marker, and the rice containing the allele with the electrophoresis band size of 119bp has high heat resistance coefficient and good heat resistance property. And the rice containing the allele with the electrophoresis band of 131bp has low heat resistance coefficient and is sensitive to high temperature.
Example 3: verification and application of Hsp70-12 molecular marker
(1) High temperature resistant performance verification of Shanghai No. 1B/Shanghai No.3 recombinant inbred line population: by means ofHsp70 gene molecular marker Hsp70-12 is used for the 174 recombinant inbred lines (F) of Shanghai 1B of rice high temperature resistant variety and Shanghai 3 of high temperature sensitive variety 10 ) The PCR amplification is carried out on 6% polyacrylamide gel, the PCR products are detected by electrophoresis, 56 strains respectively contain the allele type (131 bp band) of the Shanghai No.3 and 118 strains contain the allele type (119 bp band) consistent with Shanghai No. 1B, and the heat resistance coefficient identification result shows that the heat resistance coefficient (relative setting rate) of the strain containing the allele type of the Shanghai No.3 is extremely smaller than the heat resistance coefficient (relative setting rate) of the strain containing the allele type of Shanghai No. 1B (statistical test P=2.04E-06 in 2018 and statistical test P=2.33E-08 in 2019) (shown in A of fig. 2). RSSR is heat resistance coefficient (relative setting rate) =setting rate of rice in high temperature greenhouse/setting rate of rice in normal paddy field.
(2) High temperature resistance identification performance verification of rice germplasm resources: hsp70 gene molecular marker Hsp70-12 is utilized to carry out Hsp70 genotype identification on 150 parts of genomic DNA of rice germplasm resources, and the PCR product is subjected to electrophoresis detection in 6% polyacrylamide gel, wherein 79 parts of the genomic DNA are found to be 119bp band type, 71 parts of the genomic DNA have a band size of 131bp, and the heat resistance coefficient of the germplasm resources containing 119bp band type is extremely obviously larger than that of the germplasm resources containing 131bp band type (P=9.44E-04) (fig. 2 and B).
Wherein in the figure, HSP70-in represents the genotype corresponding to the 131bp band type, and HSP70-de represents the genotype corresponding to the 119bp band type.
Therefore, the molecular marker Hsp70-12 can be used for identifying the high temperature resistant gene Hsp70 of the rice and/or carrying out auxiliary selective breeding of the high temperature resistant rice.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
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<213> Artificial sequence (Artificial Sequence)
<400> 1
ctcctcgaca agctgatgtt c 21
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ctcgcgacgc caactatgt 19
<210> 3
<211> 131
<212> DNA
<213> Rice (Oryza sativa)
<400> 3
ctcctcgaca agctgatgtt cggccggcag aaggaggtgg agcaggtcgt tggattcctg 60
ctgcagccgg acgtctgcgg cgctggcgcc ggcgccggcg ccggcgtgct gcacatagtt 120
ggcgtcgcga g 131
<210> 4
<211> 119
<212> DNA
<213> Rice (Oryza sativa)
<400> 4
ctcctcgaca agctgatgtt cggccggcag aaggaggtgg agcaggtcgt tggattcctg 60
ctgcagccgg acgtctgcgg cgctggcgcc ggcgtgctgc acatagttgg cgtcgcgag 119
Claims (10)
1. Rice high temperature resistant geneHsp70The molecular marker is located on chromosome 1 of rice genome, is named Hsp70-12, and the nucleotide sequence of the primer for amplifying the molecular marker is shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. A method for obtaining a molecular marker related to heat resistance of rice, comprising the steps of:
respectively taking genome DNA of rice variety Hanhui No.3 and Shanghai No. 1B as templates, and adopting primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 for PCR amplification;
and separating the PCR amplified product by electrophoresis to obtain an amplified fragment with the size of 131bp or an amplified fragment with the size of 119 and bp, namely the target molecular marker.
3. The molecular marker gene of claim 1 or 2 for rice high temperature resistanceHsp70The genotype detection and/or the application in the identification of the high-temperature resistant characteristic of the rice and/or the application in the auxiliary selective breeding of the high-temperature resistant rice.
4. Molecular marker used for rice high temperature resistant geneHsp70The genotype detection method is characterized by comprising the following steps:
1) Extracting genome DNA of a rice sample to be tested;
2) Performing PCR amplification on the genome extracted in the step 1) by using primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 to obtain an amplification product;
3) Detecting the amplification product of the step 2) through electrophoresis, and if the amplification product with the size of 131bp exists, indicating that the tested rice contains a high-temperature sensitive allele; if there is an amplification product of 119bp in size, this indicates that the test rice contains a thermostable allele.
5. The method of claim 4, wherein the electrophoresis is a 6% polyacrylamide gel electrophoresis.
6. The method of claim 4 or 5, wherein the amplification system of PCR amplification comprises: taq enzyme, template DNA, dNTPs, and primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and PCR buffer.
7. The method according to claim 6, wherein the PCR amplification is performed by the following PCR reaction procedure: pre-denaturation at 95+ -1deg.C for 4-6min, then carrying out 32 cycles at 98+ -1deg.C for 20s, 54.5+ -0.5deg.C for 15-25s, and 71+ -1deg.C for 20-30s, and finally extending at 71+ -1deg.C for 4-6min.
8. The molecular marker assisted selective breeding method for the high temperature resistant rice is characterized by comprising the following steps of:
1) Extracting genome DNA of a rice sample to be tested;
2) Performing PCR amplification on the genome extracted in the step 1) by using primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 to obtain an amplification product;
3) Detecting the amplification product in the step 2) through electrophoresis, and if the amplification product with the size of 131bp exists, the rice to be tested is a high-temperature sensitive variety; if the amplified product with the size of 119bp exists, the rice to be tested is a high-temperature resistant variety.
9. The method for assisting selective breeding according to claim 8, wherein the 131bp amplification product is rice high temperature sensitive variety Hanhui No.3Hsp70Allele of the gene, the 119bp amplified product is Shanghai drought 1B of rice high temperature resistant varietyHsp70Alleles of genes.
10. Test for identifying high-temperature resistance characteristic of riceA kit, characterized in that it comprises: primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and the primers are used for amplifying rice high temperature resistant genesHsp70Is the Hsp70-12 located on chromosome 1 of rice according to claim 1.
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