Mutant gene of rice wax gene Wx and its application

Abstract

The present invention discloses a mutant gene of a rice wax gene Wx and its application, wherein the nucleotide sequence of the mutant gene is shown in SEQ ID NO. 1. The present invention finds that a T→C base mutation at the 202nd position of the 9th exon of the rice wax gene Wx will increase the content of resistant starch in rice, and the mutant gene of the rice wax gene Wx is a gene closely related to high resistant starch content; a primer pair is designed according to the mutation site of the mutant gene, and then the rice genomic DNA is amplified, and nucleic acid electrophoresis is used to effectively detect whether the rice variety is a rice variety with a high resistant starch content, and the genotype of the rice seedlings before transplanting can be quickly identified, so that selective transplanting can be performed, which greatly improves the selection efficiency of rice varieties with high resistant starch content and accelerates the breeding process.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular genetics, and more specifically, relates to a mutant gene of a rice wax gene Wx and an application thereof. Background Art

[0002] The rice wax gene Wx encodes granule-bound starch synthase (GBSS), which catalyzes the synthesis of amylose, affects the amylose content (AC) of rice, and is the main gene that determines the amylose content.

[0003] Resistant starch (RS) was first proposed by Englyst et al. (1982). It is not absorbed in the small intestine of the human body, which can reduce blood sugar levels after meals, increase satiety, satisfy the desire of diabetics to eat rice, reduce the risk of obesity, improve insulin sensitivity in patients with type 2 diabetes, and prevent chronic kidney disease (Kieffer et al., 2016; Bindels et al., 2017; Emilien et al., 2017). Nearly 80% of edible rice is starch, but the RS content of most rice varieties is less than 3% (Yang et al., 2006; Hu et al., 2004), and high RS varieties are scarce. Therefore, cultivating high RS rice varieties has important application value.

[0004] Studies have shown that the ratio of RS to amylose is positively correlated (Cai et al., 2015; Lin et al., 2016; Chen et al., 2017; Xia et al., 2018), and there is a significant correlation between the wax gene Wx and RS content in rice (Cai et al., 2015; Lin et al., 2016; Chen et al., 2017; Xia et al., 2018). Wx is the main gene regulating RS content and also the main gene determining RS content in different rice varieties (Raben et al., 1994; Fitzgerald et al., 2011).

[0005] Bao et al. (2017) obtained four candidate genes related to rice RS through genome-wide association analysis, all of which are genes related to the synthesis of amylose and amylopectin: Wx, SSⅡa, ISA1 and AGPS1. The rice SBE3 gene promotes the synthesis of amylopectin by introducing branches. Studies have found that a single base mutation in the SBE3 gene causes the leucine at position 599 to be converted to proline, and the RS content increases from 0.41% to 11.67% (Yang et al., 2012). Zhou et al. (2016) found that the mutation of the SSⅢa gene significantly increased the RS content in rice, and its regulation of RS depends on the high expression of the Wx gene.

[0006] Wx a The gene is the most common allele in indica rice, and this type of variety has higher amylose content (Cheng Taihui and Yang Wu, 2017), while Wx a The C / T site in exon 10 of the gene determines the consistency and hardness of rice glue (Tran et al., 2011).

[0007] The breeding of high RS rice varieties inevitably requires the determination and screening of RS content in a large number of intermediate materials, but the current RS content is mainly determined by chemical methods, which are cumbersome, inefficient, costly, and have poor repeatability. If molecular markers that are closely linked to the target trait can be developed and applied to the screening of high RS content plants in hybrid offspring, breeding efficiency will be greatly improved.

[0008] Conventional breeding methods based on phenotypic selection have disadvantages such as low selection efficiency and long breeding cycle. It is urgent to inject modern molecular technology means, assisted by efficient genotype directional selection, in order to quickly and efficiently breed excellent new rice varieties. With the rapid development of molecular biology and genomics, the application of molecular marker technology has become more extensive. PCR-based molecular markers such as microsatellites or SSR (simple sequence repeat) have the characteristics of high polymorphism rate, relative stability, simple and fast detection method and easy operation, and are widely used. Since molecular marker-assisted selection is not easily affected by environmental factors and trait dominant and recessive interference, it can select target trait genes from the molecular level, and it is also possible to break the linkage between unfavorable genes and efficiently aggregate multiple excellent genes into one. Molecular marker-assisted multi-gene aggregation breeding technology has become a development trend in crop breeding research such as rice and corn. The key lies in whether practical molecular markers that are closely linked to target trait genes or major effect QTLs can be obtained.

[0009] At present, there are few reports on the development and use of molecular markers closely linked to resistant starch content. Only in 2008, Mou Fanggui et al. reported that in the hybrid combination II-32B / RS 111, RM72 and RM547 located on chromosome 8 had a certain linkage relationship with resistant starch, and in the F2 of Yixiang B / RS111, RM217 and RM225 linked to the Wx gene had a certain linkage relationship with resistant starch. In 2009, Wang Lin et al. used the BSA method to find an SSR marker Xbarc590 in wheat that was closely related to high resistant starch content. In 2012, Park Jong-taek et al. used the T→C base mutation at the 105th position of the 16th exon of the rice starch branching enzyme SBE3 gene to design a RFLP marker closely related to high resistant starch content.

[0010] However, due to their own characteristics, SSR markers cannot reach the ideal density in the genome, and the linkage relationship with the target gene is not very close, so the screening accuracy is often not high; and the SNP markers of the gene itself are often developed with the aid of enzyme cutting, so the process is cumbersome and the cost is high. Therefore, it is of great significance to explore more genes closely related to high resistant starch content, develop molecular markers closely linked to resistant starch content, use the developed molecular markers to select breeding materials early, accelerate the breeding process, and improve the nutritional quality of rice. Summary of the invention

[0011] Based on this, one of the objects of the present invention is to provide a mutant gene of the rice wax gene Wx, which is closely related to the high resistant starch content of rice.

[0012] The specific technical solutions for achieving the above-mentioned invention objectives include the following:

[0013] A mutant gene of a rice wax gene Wx, the nucleotide sequence of the mutant gene is shown as SEQ ID NO.1.

[0014] The present invention also provides the use of the mutant gene of the rice wax gene Wx in screening rice with high resistant starch content.

[0015] The present invention also provides a protein encoded by a mutant gene of a rice wax gene Wx, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0016] The present invention also provides the use of the protein encoded by the mutant gene of the rice wax gene Wx in screening rice with high resistant starch content.

[0017] The present invention also provides a primer pair for screening rice with high resistant starch content, wherein the primer pair comprises primers with nucleotide sequences as shown in SEQ ID NO.5 to SEQ ID NO.8.

[0018] The present invention also provides a primer pair for screening rice with high resistant starch content, the primer pair comprising primers with nucleotide sequences as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10.

[0019] The present invention also provides a kit for screening rice with high resistant starch content, the kit comprising primer pairs shown in SEQ ID NO.5 to SEQ ID NO.8; and / or the kit comprising primer pairs shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10.

[0020] In some embodiments, the kit further comprises Taq DNA polymerase, PCR buffer system and dNTP.

[0021] The present invention also provides a method for screening rice with high resistant starch content, the method comprising the following steps:

[0022] (1) Using the genomic DNA of the rice to be screened as a template and SEQ ID NO.5 to SEQ ID NO.8 or SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10 as primers, PCR amplification was performed;

[0023] (2) Detect the bands of the amplified products by nucleic acid electrophoresis to identify whether there is a T→C base mutation at position 202 of the 9th exon of the rice wax gene Wx.

[0024] The present invention also provides a method for screening rice with high resistant starch content, the method comprising the following steps:

[0025] (1) Using the genomic DNA of the rice to be screened as a template and SEQ ID NO.3 and SEQ ID NO.4 as primers, PCR amplification was performed;

[0026] (2) Sequencing the PCR amplification product to detect whether there is a T→C base mutation at position 202 of the 9th exon of the rice wax gene Wx.

[0027] In some of the embodiments, step (2) also includes detecting whether there is a C→T base mutation at position 115 of the 10th exon of the rice wax gene Wx in the amplified product, or whether there is a proline→serine amino acid mutation at position 415 of the amino acid sequence of the amplified product, thereby screening out rice varieties with better palatability and high resistant starch content.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The inventors of the present application have proved through a large number of experiments that the base mutation of T→C at the 202nd position of the 9th exon of the rice wax gene Wx will increase the content of resistant starch in rice. Therefore, the mutant gene of the rice wax gene Wx is a gene closely related to high resistant starch content; according to the mutation site of the mutant gene, a primer pair is designed based on the principle of hindered amplification, and then the rice genomic DNA is amplified. Through nucleic acid electrophoresis, it is possible to effectively detect whether the rice variety is a rice variety with a high resistant starch content, and the genotype of rice in the seedling stage can be quickly identified, so that it can be selectively transplanted, which greatly improves the selection efficiency of rice varieties with high resistant starch content and accelerates the breeding process.

[0030] 2. The method of the present invention for screening rice with high resistant starch content has simple and quick steps, reliable stability, high accuracy and good repeatability, and is suitable for resistant starch molecular marker-assisted breeding, which can omit the complicated resistant starch content determination process and save breeding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the nucleotide sequence of the 9th exon of the rice wax gene Wx in different rice varieties in Example 1 of the present invention, wherein the black background represents the same nucleotide sequence, and the gray background represents a base mutation of T→C at the 202nd position.

[0032] Figure 2 This is the nucleotide sequence at the 10th exon of the rice wax gene Wx in Example 1 of the present invention, wherein the black background indicates the same nucleotide sequence among different rice varieties, and the gray background indicates a base mutation of C→T at the 115th position.

[0033] Figure 3 This is the result of nucleic acid electrophoresis detection after amplifying genomic DNA of some individual rice plants using primer sets W9-IF1, W9-IR1, W9-OF1, and W9-OR1 in Example 2 of the present invention.

[0034] Figure 4This is the result of nucleic acid electrophoresis detection after amplifying genomic DNA of some individual rice plants using primer sets W9-IF1, W9-IR1, W9-OF2, and W9-OR2 in Example 2 of the present invention. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0036] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0037] In the first aspect of the present invention, a mutant gene of a rice wax gene Wx is provided, which can encode a rice bound starch synthase, the nucleotide sequence of the mutant gene is shown in SEQ ID NO. 1, and the amino acid sequence encoded by the mutant gene is shown in SEQ ID NO. 2. Compared with the rice wax gene Wx, the mutant gene has a base mutation of T→C at position 202 of the 9th exon (a synonymous mutation that does not cause a change in the amino acid sequence), and a base mutation of C→T at position 115 of the 10th exon (causing a mutation of proline to serine).

[0038] SEQ ID NO.1 (3039 bp): covers the coding region and non-coding region of the rice wax gene Wx from the 4th exon to the 14th exon. Compared with the rice wax gene Wx, it lacks 4 bases (indicated by "-"), has a T→C base mutation at the 202nd position of the 9th exon, has a C→T base mutation at the 115th position of the 10th exon, and also includes several intronic base mutations.

[0039]

[0040] In a second aspect of the present invention, a protein encoded by a mutant gene of a rice wax gene Wx is provided, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. Compared with the protein encoded by the rice wax gene Wx, the protein encoded by the mutant gene has an amino acid mutation of proline→serine at position 415.

[0041] SEQ ID NO.2

[0042] MSALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQQRSVQRGSRRFPSVVVYATGAGMNVVFVGAEMAPWSKTGGLGDVLGGLPPAMAANGHRVMVISPRYDQYKDAWDTSVVAEIKVADRYERVRF FHCYKRGVDRVFIDHPSFLEKVWGKTGEKIYGPDTGVDYKDNQMRFSLLCQAALEAPRILNLNNNPYFKGTYGEDVVFVCNDWHTGPLASYLKNNYQPNGIYRNAKVAFCIHNISYQGRFAFEDYPELNLSERFRSSFDFIDGYDTPVEGRK INWMKAGILEADRVLTVSPYYAEELISGIARGCELDNIMRLTGITGIVNGMDVSEWDPSKDKYITAKYDATTAIEAKALNKEALQAEAGLPVDRKIPLIAFIGRLEEQKGSDVMAAAIPELMQEDVQIVLLGTGKKKFEKLLKSMEEKYPGK VRAVVKFNAPLAHLIMAGADVLAVPSRFEPCGLIQLQGMRYGTPCACASTGGLVDTVIEGKTGFHMGRLSVDCKVVEPSDVKKVAATLKRAIKVVGTPAYEEMVRNCMNQDLSWKGPAKNWENVLLGLGVAGSAPGIEGDEIAPLAKENVAAP

[0043] In the third aspect of the present invention, the use of the mutant gene of the rice wax gene Wx or the protein encoded by it in screening rice with high resistant starch content is provided. The rice variety containing the mutant gene of the rice wax gene Wx or the protein encoded by it has a significantly higher resistant starch content than the rice variety not containing the mutant gene of the rice wax gene Wx or the protein encoded by it.

[0044] In the fourth aspect of the present invention, a primer pair for screening rice with high resistant starch content is provided, and the primer pair includes two groups: one group is SEQ ID NO.5 to SEQ ID NO.8; the other group is SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10. The primer pair is designed with the nucleic acid sequence at position 202 of the 9th exon of the rice wax gene Wx as the target. The primer pair can be used as a molecular marker for screening rice varieties with high resistant starch content. The primer pair can accurately screen rice varieties with high resistant starch content, thereby accelerating the selection progress of rice varieties with high resistant starch content and improving breeding selection efficiency.

[0045] Resistant starch content is a quantitative trait, which is controlled by many genes. If the contribution value of the major gene of the quantitative trait can reach 30%, it is considered very high. The inventors of the present invention found that the mutant gene of the rice wax gene Wx is the major gene of high resistant starch content. After screening rice varieties with primer pairs (i.e., molecular markers) for screening rice with high resistant starch content, homozygous negative and heterozygous single plants are eliminated, which at least account for more than 3 / 4 of the total number of single plants. The remaining homozygous positive single plants are theoretically high resistant starch single plants. Therefore, the workload and cost of rice variety screening can be greatly saved.

[0046] In a fifth aspect of the present invention, a kit for screening rice varieties with high resistant starch content is provided, the kit comprising one or both of the two groups of primer pairs mentioned above, the primer pairs being SEQ ID NO.5 to SEQ ID NO.8; and / or SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10; the kit also comprising Taq DNA polymerase, PCR buffer system and dNTP.

[0047] In a sixth aspect of the present invention, a method for screening rice with high resistant starch content is provided, the method comprising the following steps:

[0048] (1) Using the rice genomic DNA to be screened as a template and SEQ ID NO.5 to SEQ ID NO.8 as primers, PCR amplification was performed;

[0049] (2) The bands of the amplified products detected by nucleic acid electrophoresis are: if there is one band of 190 bp, then the rice variety has a low resistant starch content; if there are two bands of 350 bp and 215 bp, then the rice variety has a homozygous genotype with a high resistant starch content (the double strand of the 9th exon of the rice wax gene Wx has a base mutation of T→C at the 202nd position); if there are two bands of 190 bp and 350 bp, then the rice variety has a heterozygous genotype with a high resistant starch content (the single strand of the 9th exon of the rice wax gene Wx has a base mutation of T→C at the 202nd position).

[0050] Or the method comprises the following steps:

[0051] (1) Using the genomic DNA of the rice to be screened as a template and SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10 as primers, PCR amplification was performed;

[0052] (2) The bands of the amplified products detected by nucleic acid electrophoresis are: if there is a single band of 189 bp, the rice variety has a low resistant starch content; if there is a single band of 149 bp, the rice variety has a homozygous genotype with a high resistant starch content (the double strand of the 9th exon of the rice wax gene Wx has a T→C base mutation at position 202); if there are two bands of 189 bp and 149 bp, the rice variety has a heterozygous genotype with a high resistant starch content (the single strand of the 9th exon of the rice wax gene Wx has a T→C base mutation at position 202).

[0053] W9-IF1:GCATGGACGTCAGCGAGTGGGATACT(SEQ ID NO.5)

[0054] W9-IR1:ACTTGGCGGTGATGTACTTGTCCTTGATG(SEQ ID NO.6)

[0055] W9-OF1: GGAGGGCAGGAAGATCAACTGGATGAA (SEQ ID NO.7)

[0056] W9-OR1:TTGCCTGAAATTTGTCACTCATTCTTGCC(SEQ ID NO.8)

[0057] W9-OF2:CCCGTACTACGCCGAGGAGCTCATCT(SEQ ID NO.9)

[0058] W9-OR2:TGCCTGAAATTTGTCACTCATTCTTGCCTT(SEQ ID NO.10)

[0059] A seventh aspect of the present invention provides a method for screening rice with good palatability and high resistant starch content, the method comprising the following steps:

[0060] (1) Using the genomic DNA of the rice to be screened as a template and SEQ ID NO.3 and SEQ ID NO.4 as primers, PCR amplification was performed;

[0061] WL3: GCAGATCAAGGTTGCAGACA(SEQ ID NO.3)

[0062] WR: TGGCAATAAGCCACACACAT(SEQ ID NO.4)

[0063] (2) By sequencing, the amplified product is detected to determine whether there is a C→T base mutation at position 115 of the 10th exon of the rice wax gene Wx, or whether there is a proline→serine amino acid mutation at position 415 of the amino acid sequence of the amplified product. Varieties without such base mutations or amino acid mutations are rice varieties with good palatability and high resistant starch content.

[0064] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0065] In the following examples, the resistant starch content was determined using a resistant starch content determination kit provided by Megazyme (Megazyme, Co. Wicklow, Ireland) with slight improvements. The specific steps are as follows: accurately weigh 100 mg of rice flour sample, carefully put it into a plastic test tube with a screw cap, add α-amylase reaction solution and amyloglucosidase (AGM) in sequence, and incubate at 37°C with shaking for 16 hours, so that the non-resistant starch is dissolved and hydrolyzed into D-glucose; after the incubation, add an equal volume of anhydrous ethanol to terminate the reaction; centrifuge the above solution, discard the supernatant, and the flocculent mass remaining at the bottom is the resistant starch in the sample, and then wash the precipitate with 50% ethanol, centrifuge after washing, and repeat the washing and centrifugation once; invert the centrifuge tube, dissolve the precipitate with 2M KOH after the precipitate is dried, adjust the solution to neutral with acetate buffer, add AGM, and incubate in a 50°C water bath for 30 minutes to hydrolyze the resistant starch into glucose, and finally use glucose oxidase / peroxidase reagent (GOPOD) reagent to determine the glucose content, and calculate the resistant starch content (in weight percentage, referred to as RS (%)).

[0066] Example 1 Obtaining the Mutant Gene and Molecular Marker of Rice Wax Gene Wx

[0067] (I) Screening of rice resources with high resistant starch content

[0068] Several types of rice resources were collected from home and abroad, such as glutinous rice resources, medium-low amylose rice resources, high amylose rice resources, etc., with a focus on collecting rice resources with high amylose content, such as "rice powder rice" resources in Yunnan and other places, and "hand-pulled rice" resources in India, Bangladesh, Africa and other places. The resistant starch content was determined using a resistant starch content determination kit, and 10 rice resource materials with RS content greater than 6% were screened out. The amylose content (AC) and resistant starch (RS) content of 14 different rice varieties are shown in Table 1.

[0069] Table 1 Amylose content and resistant starch content in different rice varieties

[0070]

[0071] (II) Cloning of the rice wax gene Wx

[0072] (1) The genomic DNA of the 14 rice resources with different RS contents were extracted using the CTAB method.

[0073] The rice small amount DNA extraction method mainly refers to the report of McCouch et al. (1988), and the method is briefly described as follows:

[0074] 1) Cut a small piece of leaf 4-5 cm, add 700 μl 1.5X CTAB (containing 1.5% CTAB, 75 mM Tris-HCl, 15 mM EDTA, 1.05 M NaCl), and grind thoroughly;

[0075] 2) Transfer the homogenate into a 1.5 ml centrifuge tube, place in a 56°C water bath for 30 min, and then cool to room temperature;

[0076] 3) Add equal volumes of chloroform:isoamyl alcohol (24:1) and shake well;

[0077] 4) Centrifuge at the highest speed (12000 rpm) for 10 min;

[0078] 5) Transfer the supernatant into a new centrifuge tube, add 0.6 times the volume of pre-cooled isopropanol, let it stand for 20 minutes, and then centrifuge to collect DNA;

[0079] 6) Remove the supernatant, air-dry the DNA, dissolve it in 50-100 μl ultrapure water, and detect it in a UV spectrophotometer. Dilute the DNA to prepare a set of DNA working solution with a concentration of about 50-100 ng / μL, and store it in a refrigerator at 4°C for later use.

[0080] (2) Primers WL3 (SEQ ID NO. 3) and WR (SEQ ID NO. 4) were designed based on the rice Wx gene sequence in NCBI, and genomic DNA of 14 rice materials with different RS contents was used as template for amplification to obtain PCR products;

[0081] WL3: GCAGATCAAGGTTGCAGACA(SEQ ID NO.3)

[0082] WR: TGGCAATAAGCCACACACAT(SEQ ID NO.4)

[0083] (3) The PCR product was directly sent to Shanghai Bioengineering Co., Ltd. for sequencing to obtain a 3043 bp fragment. The sequencing results were compared with the sequences in the NCBI database to analyze the similarities and differences between the high-resistant starch rice materials and the low-resistant starch rice materials. Through comparison and analysis, the high-resistant starch rice materials lacked 4 bases in the wax gene Wx compared with the low-resistant starch rice materials, and all had a T→C base mutation at the 202nd position of the 9th exon; and most of the 10th exon had a C→T base mutation at the 115th position, while a few materials had no mutation; in addition, there were several intronic base mutations.

[0084] (III) Development of molecular markers for high-resistant starch

[0085] For the base mutation of T→C at position 202 of the 9th exon of rice wax gene Wx, an online design ( http: / / primer1.soton.ac.uk / primer1.html ) Two sets of primers for amplification blocked mutation system SEQ ID NO.5 to SEQ ID NO.8, and SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10 are used as molecular markers closely related to the detection of high resistant starch content. The sequences of the primer pairs are as follows:

[0086] W9-IF1:GCATGGACGTCAGCGAGTGGGATACT(SEQ ID NO.5)

[0087] W9-IR1:ACTTGGCGGTGATGTACTTGTCCTTGATG(SEQ ID NO.6)

[0088] W9-OF1: GGAGGGCAGGAAGATCAACTGGATGAA (SEQ ID NO.7)

[0089] W9-OR1:TTGCCTGAAATTTGTCACTCATTCTTGCC(SEQ ID NO.8)

[0090] W9-OF2:CCCGTACTACGCCGAGGAGCTCATCT(SEQ ID NO.9)

[0091] W9-OR2:TGCCTGAAATTTGTCACTCATTCTTGCCTT(SEQ ID NO.10)

[0092] Example 2 Verification of the molecular markers in Example 1

[0093] 1. Materials

[0094] Example 1: 40 individual plants of the F4 generation of the rice varieties in Table 1, including: Lan / CN-5, Lan / CN-9, Lan / Sx, QR / Sx, QR / Ky43, Li / BR-50, and QR / BR50 hybrids.

[0095] 2. Methods

[0096] (1) DNA of 40 F4 generation individual plant samples was extracted using the method of Example 1;

[0097] (2) Using the DNA from step (1) as a template and SEQ ID NO.5 to SEQ ID NO.8 or SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10 as primers, a PCR amplification reaction was performed; the reaction system was: 2 μl 10X PCR buffer (100 mM Tris-HCl pH 8.0, 15 mM MgCl 2 , 500mM KCl, 1% TritonX-100, 0.2mM dNTPs, 0.2μM each upstream primer, 0.2μM each downstream primer, 50-100ng sample DNA, 0.625U Taq enzyme; the reaction procedure was: pre-denaturation at 94℃ for 4min, 35 cycles (94℃ for 30s, 60℃ for 30s, 72℃ for 1min), and finally extension at 72℃ for 10min.

[0098] (3) The PCR amplification products were analyzed by 10% polyacrylamide gel electrophoresis to detect polymorphism. The offspring with the same genotype as the low-resistant starch parent were marked as "-", the offspring with the same genotype as the high-resistant starch parent were marked as "+", and the heterozygous type was marked as H.

[0099] a. After PCR amplification of rice genomic DNA using the primer pairs shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8; nucleic acid electrophoresis detection, if there is a band of 190 bp, it is a rice variety with low resistant starch content; if there are two bands of 350 bp and 215 bp, it is a homozygous genotype of a rice variety with high resistant starch content; if there are two bands of 190 bp and 350 bp, it is a heterozygous genotype of a rice variety with high resistant starch content.

[0100] b. After PCR amplification of rice genomic DNA using the primer pairs shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, and SEQ ID NO.10; nucleic acid electrophoresis detection, if there is a band of 189 bp, it is a rice variety with low resistant starch content; if there is a band of 149 bp, it is a homozygous genotype of a rice variety with high resistant starch content; if there are two bands of 189 bp and 149 bp, it is a heterozygous genotype of a rice variety with high resistant starch content.

[0101] 3. Results

[0102] The molecular markers of 40 individual plants in the F4 generation were verified, and the results of nucleotide electrophoresis detection of some individual plants are shown in Figure 3 and Figure 4 The results showed that there were three banding patterns in the individual plants of the segregating population, which were consistent with the low resistant starch parent, the high resistant starch parent and the heterozygous type. The banding patterns all corresponded to the results of the RS resistant starch content determination. The specific statistical results are shown in Table 1.

[0103] Table 1 Genotypes of 40 plants and corresponding resistant starch content determination results

[0104]

[0105]

[0106] From the results in Table 1, it can be seen that among the 40 F4 generation plants, 23 plants have the same genotype as the low-resistant starch parent (do not contain the mutant gene of the rice wax gene Wx), and their resistant starch content is 0.41% to 1.99% by weight, with an average of 0.88%; 8 plants have the same genotype as the high-resistant starch parent (contain the mutant gene of the rice wax gene Wx, and are homozygous), and their resistant starch content is 3.10% to 11.10%, with an average of 5.91%; 9 plants have a heterozygous genotype (one chain contains the mutant gene of the rice wax gene Wx, and the other chain does not contain the mutant gene of the rice wax gene Wx), and their resistant starch content is 1.42% to 4.00%, with an average of 3.00%.

[0107] The results of this example show that the molecular markers provided in Example 1 of the present invention can be used to accurately screen out homozygous plants with high resistant starch content.

[0108] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0109] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. Sequence Listing <110> Chongqing Academy of Agricultural Sciences <120> Mutant gene of rice wax gene Wx and its application <130> 1 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 3039 <212> DNA <213> Artificial Sequence <400> 1 gcagatcaag gttgcagaca ggtacgagag ggtgaggttt ttccattgct acaagcgtgg 60 agtcgaccgt gtgttcatcg accatccgtc attcctggag aaggtggagt catcattagt 120 ttaccttttt tgtttttact gaattattaa cagtgcattt agcagttgga ctgagcttag 180 cttccactgg tgatttcagg tttggggaaa gaccggtgag aagatctacg gacctgacac 240 tggagttgat tacaaagaca accagatgcg tttcagcctt ctttgccagg tcagtgatta 300 cttctatctg atgatggttg gaagcatcac gagtttacca tagtatgtat ggattcataa 360 ctaattcgtg tattgatgct actgcaggca gcactcgagg ctcctaggat cctaaacctc 420 aacaacaacc catacttcaa aggaacttat ggtgagttat aattgatctc aagatcttat 480 aactttcttc gaaggaatcc atgatgatca gactaattcc ttccggtttg ttactgacaa 540 caggtgagga tgttgtgttc gtctgcaacg actggcacac tggcccactg gcgagctacc 600 tgaagaacaa ctaccagccc aatggcatct acaggaatgc aaaggtctat gcttgttctt 660 gccataccaa ctcaaatctg catgcacact gcattctgtt cagaaactga ctgtctgaat 720 ctttttcact gcaggttgct ttctgcatcc acaacatctc ctaccagggc cgtttcgctt 780 tcgaggatta ccctgagctg aacctctccg agaggttcag gtcatccttc gatttcatcg 840 acgggtatga gtaagattct aagagtaact tactgtcaat tcgccatata tcgattcaat 900 ccaagatcct tttgagctga caaccctgca ctactgtcca tcgttcaaat ccggttaaat 960 ttcaggtatg acacgccggt ggagggcagg aagatcaact ggatgaaggc cggaatcctg 1020 gaagccgaca gggtgctcac cgtgagcccg tactacgccg aggagctcat ctccggcatc 1080 gccaggggat gcgagctcga caacatcatg cggctcaccg gcatcaccgg catcgtcaac 1140 ggcatggacg tcagcgagtg ggatcccagc areacaagt acatcaccgc caagtacgac 1200 gcaaccacgg tagaacgaa tgcattcttc acaagatatg caatctgaat tttctttgaa 1260 aaagaaatta tcatctgtca cttcttgatt gattctgaca aggcaagaat gagtgacaaa 1320 tttcaggcaa tcgaggcgaa ggcgctgaac aaagggcgt tgcaggcgga ggcgggtctt 1380 1440 tctgacgtca tggccgccgc catcccggag ctcatgcagg aggacgtcca gatcgttctt 1500 ctggtataat ataatacact acaagacaca cttgcacgat atgccaaaaa ttcagaacaa 1560 attcagtggc aaaaaaaaa ctcaaatatt agggaagaac ctaatatcaa ataattagaa 1620 ggggtgaggc tttgaaccca ggtcatctag cccaccacct tgtagagcta gccggaagag 1680 ctctgagcat ttctcgattc agtggcaaat gatgtgtata attttgatcc gtgtgtgttt 1740 cagggtactg gaaagaagaa gttcgagaag ctgctcaaga gcatggagga gaagtatccg 1800 ggcaaggtga gggccgtggt gaagttcaac gcgccgcttg ctcatctcat catggccgga 1860 gccgacgtgc tcgccgtccc cagccgcttc gagccctgtg gactcatcca gctgcagggg 1920 atgagatacg gaacggtata caatttccat ctatcaattc gattgttcga tttcatcttt 1980 gtgcaatgca atgcaattgc aaatgcaaat gcatgatgat tttccttgtt gatttctcca 2040 gccctgtgct tgcgcgtcca ccggtgggct cgtggacacg gtcatcgaag gcaagactgg 2100 tttccacatg ggccgtctca gcgtcgacgt aagcctatac atttacataa caatcagata 2160 tgacacattc tataccgat aagtcagtac actactacac atttacatgg ttgctggtta 2220 tatggtttt ttggcagtgc aaggtggtgg agccaagcga cgtgaagaag gtggcggcca 2280 ccctgaagcg cgccatcaag gtcgtcggca cgccggcgta cgaggagatg gtcaggaact 2340 gcatgaacca ggacctctcc tggaaggtat aaattacgaa acaaatttaa cccaaacata 2400 tactatatac tccctccgct tctaaatatt caacgccgtt gtctttttaa aatatgtttg 2460 accgttcgtc ttattaaaaa attaaataa ttataaatta ttttcctatc atttgattca 2520 ttgttaaata tacttatatg tatacatata gttttacata tttcataaaa gtttttgaac 2580 aagacgaacg gtcaaacatg tgctaaaaag ttaacggtgt cgaatattca gaaacggagg 2640 gagtataaac gtcttgttca gaagttcaga gattcacctg tctgatgctg atgatgatta 2700 attgtttgca acatggattt caggggcctg cgaagaactg ggagaatgtg ctcctgggcc 2760 tgggcgtcgc cggcagcgcg cggggatcg aaggcgacga gatcgcgcg ctcgccaagg 2820 agaacgtggc tgctccttga agagcctgag atctacatat ggagtgatta attaatatag 2880 cagtatatgg atgagagacg aatgaaccag tggtttgttt gttgtagtga atttgtagct 2940 atagccaatt atataggcta ataagtttga tgttgtactc ttctgggtgt gcttaagtat 3000 cttatcggac cctgaattta tgtgtgtggc ttattgcca 3039 <210> 2 <211> 609 <212> PRT <213> Artificial Sequence <400> 2 Met Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe 1 5 10 15 Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe 20 25 30 Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu 35 40 45 Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val 50 55 60 Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala Thr Gly 65 70 75 80 Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser 85 90 95 Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met 100 105 110 Ala Ala Asn Gly His Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln 115 120 125 Tyr Lys Asp Ala Trp Asp Thr Ser Val Val Ala Glu Ile Lys Val Ala 130 135 140 Asp Arg Tyr Glu Arg Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val 145 150 155 160 Asp Arg Val Phe Ile Asp His Pro Ser Phe Leu Glu Lys Val Trp Gly 165 170 175 Lys Thr Gly Glu Lys Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys 180 185 190 Asp Asn Gln Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala 195 200 205 Pro Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe Lys Gly Thr Tyr 210 215 220 Gly Glu Asp Val Val Phe Val Cys Asn Asp Trp His Thr Gly Pro Leu 225 230 235 240 Ala Ser Tyr Leu Lys Asn Asn Tyr Gln Pro Asn Gly Ile Tyr Arg Asn 245 250 255 Ala Lys Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe 260 265 270 Ala Phe Glu Asp Tyr Pro Glu Leu Asn Leu Ser Glu Arg Phe Arg Ser 275 280 285 Ser Phe Asp Phe Ile Asp Gly Tyr Asp Thr Pro Val Glu Gly Arg Lys 290 295 300 Ile Asn Trp Met Lys Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr 305 310 315 320 Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly 325 330 335 Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val 340 345 350 Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Lys Asp Lys Tyr Ile 355 360 365 Thr Ala Lys Tyr Asp Ala Thr Thr Ala Ile Glu Ala Lys Ala Leu Asn 370 375 380 Lys Glu Ala Leu Gln Ala Glu Ala Gly Leu Pro Val Asp Arg Lys Ile 385 390 395 400 Pro Leu Ile Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp 405 410 415 Val Met Ala Ala Ala Ile Pro Glu Leu Met Gln Glu Asp Val Gln Ile 420 425 430 Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys Ser 435 440 445 Met Glu Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn 450 455 460 Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala Asp Val Leu Ala Val 465 470 475 480 Pro Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg 485 490 495 Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr 500 505 510 Val Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp 515 520 525 Cys Lys Val Val Glu Pro Ser Asp Val Lys Lys Val Ala Ala Thr Leu 530 535 540 Lys Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val 545 550 555 560 Arg Asn Cys Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn 565 570 575 Trp Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly Ser Ala Pro Gly 580 585 590 Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala 595 600 605 Pro

Claims

1. Use of a primer pair in preparing a kit for screening rice with high resistant starch content, characterized in that: The primer pair includes primers whose nucleotide sequences are shown as SEQ ID NO.5 to SEQ ID NO.

8.

2. Use of a primer pair in preparing a kit for screening rice with high resistant starch content, characterized in that: The primer pair includes primers having nucleotide sequences as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.

10.

3. Use of a kit for screening rice with high resistant starch content, characterized in that: The kit comprises the primer pair shown in claim 1.

4. Use of a kit for screening rice with high resistant starch content, characterized in that: The kit includes the primer pair shown in claim 2.

5. The use according to claim 3 or 4, characterized in that: The kit also includes Taq DNA polymerase, PCR buffer system and dNTP.

6. A method for screening rice with high resistant starch content, characterized in that: The method comprises the following steps: (1) Using the genomic DNA of the rice to be screened as a template, SEQ ID NO.5 to SEQ ID NO.8 or SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.10 were used as primers for PCR amplification; (2) Detect the bands of amplified products by nucleic acid electrophoresis to identify rice wax genes Wx Exon 9 Whether there is a T→C base mutation at position 202.

7. A method for screening rice with high resistant starch content, characterized in that: The method comprises the following steps: (1) Using the genomic DNA of the rice to be screened as a template and SEQ ID NO.3 and SEQ ID NO.4 as primers, PCR amplification was performed; (2) Sequencing of PCR amplification products to detect rice wax gene Wx Whether there is a T→C base mutation at position 202 of exon 9.

8. The method for screening rice with high resistant starch content according to claim 7, characterized in that: Step (2) also includes detecting the rice wax gene in the amplified product Wx Whether there is a C→T base mutation at position 115 of exon 10, or detecting the rice wax gene in the amplified product Wx Whether there is an amino acid mutation from proline to serine at position 415 of the encoded amino acid sequence.

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