A gene sequence fragment related to red rice softness and a method for improving red rice softness

Abstract

The present invention relates to the field of genetic agriculture technology, specifically to a gene sequence fragment associated with the softness of red rice and a method for improving red rice softness. The present invention discloses a gene sequence fragment associated with the softness of red rice, the nucleotide sequence of which is shown in SEQ ID No. 1, located in the promoter region of the Wx gene. After knocking out or modifying the gene, the expression of the Wx gene moderately decreases, the amylose content of the Guizhou local high-quality red rice "Pi Gu Nian" decreases from 19% to approximately 14%, and the gel consistency increases from 66 mm to approximately 91 mm, thereby appropriately improving the softness of the Guizhou local high-quality red rice "Pi Gu Nian". The present invention can be applied to the precise regulation of rice amylose content and the molecular breeding of Guizhou local high-quality rice. The present invention provides a method for improving the softness of red rice by moderately reducing the expression of the Wx gene. Specific examples of the present invention demonstrate that by editing the promoter sequence of the Wx gene, the softness of the Guizhou local high-quality red rice "Pi Gu Nian" can be precisely improved, improving rice quality, facilitating the promotion and cultivation of this variety, and facilitating the preservation of high-quality germplasm resources.

Description

Technical Field

[0001] The present invention relates to the field of gene agricultural technology, and in particular to a gene sequence fragment related to the softness of red rice and a method for improving the softness of red rice. Background Art

[0002] Rice is the world's most important food crop, with over half of the world's population relying on it as their staple food. White rice consumed daily is typically polished, losing 80% of its nutrients during processing. Red rice, like colored rice, is rich in anthocyanins, unsaturated fatty acids, oryzanol, vitamins, and various minerals. It offers antioxidant, free radical scavenging, blood sugar control, and anti-aging benefits, making it a highly nutritious food. Red rice, primarily consumed as brown rice, lacks flavor and palatability compared to polished white rice, and is difficult to digest, making it rarely used as a staple food. Consequently, red rice is considered a coarse grain and only occasionally appears on the table. Targeted improvements to the softness of red rice, improving its palatability, and cultivating delicious and nutritious red rice varieties are crucial for promoting the development of the red rice industry.

[0003] "Pi Gu Nian" is a high-quality specialty germplasm resource from Guizhou Province, collected in Panzhou City, Guizhou Province. As a member of Panzhou red rice, it was designated a National Geographical Indication Product in 2016. Its rice is rosy in color, rich in nutrients, and has medicinal properties such as antioxidants and hypoglycemic properties, making it a dual-purpose food and medicine. However, "Pi Gu Nian" has a high amylose content (around 20%), and because red rice is primarily consumed as brown rice, it has a hard texture and is difficult to digest. This high nutritional quality but poor taste has limited its promotion and application. Improving the softness and palatability of "Pi Gu Nian" is a pressing issue.

[0004] Studies have shown that starch is the main component of rice endosperm (accounting for 90% of the dry weight). The starch content, composition, and structure determine the softness and palatability of rice. Generally speaking, cooked rice grains with low or medium amylose content have an elastic, sticky, and smooth texture and are more palatable (Li H, Prakash S, Nicholson TM, et al. The importance of amylose and amylopectin fine structure for textural properties of cooked rice grains. Food Chemistry, 2016, 196: 702-711.). The Wx gene is the main gene that controls the content of amylose. This gene encodes the GBSSI enzyme to catalyze the synthesis of amylose (Wang Z, Wu Z L, Xing YY, et al. Nucleotide sequence of rice waxy gene. Nucleic Acids Research, 1990, 18(19): 5898.). The diversity of amylose content is largely attributed to allelic variation at the Wx locus, and the glutinous and non-glutinous properties of rice are caused by variation in the Wx gene. To date, at least eight Wx alleles, Wxlv, Wxa, Wxin, Wxb, Wxop, Wxhp, Wxmq, and Wx, have been shown to be associated with five types of amylose content in rice varieties (Zhang C, Zhu J, Chen S, et al. Wxlv, the ancestral allele of rice Waxy gene. Molecular Plant, 2019, 12 (8): 1157-1166.). Different Wx loci exhibit different softness and cooking qualities (Lin L, Wang Y, Xu X, et al. Relationships between starch molecular components and eating and cooking qualities of rice using single-segment substitution lines with different Wx loci. Journal of Cereal Science, 2023, 114: 103765.). In addition to Wx, FLO19, BEIIb, and BEIIa also play a role in regulating rice amylose content. Too low an amylose content can cause the rice to become completely glutinous, hindering its softness.If the coding regions of these genes are knocked out, their gene functions will be completely lost, resulting in basically no synthesis of amylose, which is ultimately not conducive to improving the softness of rice.

[0005] How to coordinate the softness and excellent nutritional quality of rice, especially the "sticky skin" of Guizhou's local high-quality red rice, and improve the softness of the "sticky skin" of red rice without changing its nutritional quality is not only a practical need to improve the taste quality of rice, but also a need for sustainable agricultural development. Summary of the Invention

[0006] In order to solve the above problems existing in the prior art, the present invention provides a gene sequence fragment related to the softness of red rice and a method for improving the softness of red rice, which is specifically achieved through the following technical solutions.

[0007] A gene sequence fragment related to the tenderness of red rice is located in the region 1360-1337 bp upstream of the start codon of the promoter region of the starch granule-binding enzyme Wx gene, as well as in the region 1198-1175 bp upstream of the start codon of the Wx gene. Its amino acid sequence is shown in SEQ ID No. 3; its nucleotide sequence is shown in SEQ ID No. 1:

[0008] SEQ ID No. 1 (5'-3', underline indicates Target 3 and Target 4):

[0009]

[0010] The gene sequence fragment contains the CGCAACGGCGC region with an intermediate deletion of 1345-1335bp after gene editing.

[0011] The gene sequence fragment is used in rice improvement and breeding.

[0012] Furthermore, in terms of rice improvement and breeding, specifically knocking out or changing the gene sequence fragment can effectively improve the softness of rice; overexpressing the gene sequence fragment can effectively improve the hardness of rice.

[0013] Furthermore, in terms of rice improvement and breeding, specifically knocking out or changing the gene sequence fragment can effectively reduce the amylose content of rice; overexpressing the gene sequence fragment can effectively increase the amylose content of rice.

[0014] The method for editing a gene sequence fragment comprises the following steps:

[0015] The CRISPR / Cas9 system is used, the target sequence of the sgRNA is located in the promoter region, and the sequence connected to the 3' end of the target sequence is NGG; the length of the target sequence is 19-20 bp, and N is any nucleotide among A, T, G, and C.

[0016] Furthermore, the target sequence is located in the region 1360-1337 bp upstream of the start codon of the Wx gene and the region 1198-1175 bp upstream of the start codon of the Wx gene.

[0017] Furthermore, the sgRNAs are sgRNA1 and sgRNA2; the target sequence of sgRNA1 is shown as SEQ ID No. 4, and the target sequence of sgRNA2 is shown as SEQ ID No. 5:

[0018] SEQ ID No. 4: Target 1: 5'-ATCGCGCGGGCTTCACGCAACGG-3' (SEQ ID No. 4, as shown in the segment 1360-1337 bp away from the start codon ATG of the Wx gene in SEQ ID No. 1, with PAM being AGG);

[0019] SEQ ID No. 5: Target 2: 5′-GTGTTGTTCTGTTGTTCATCAGG-3′ (SEQ ID No. 5, as shown in the region 1198-1175 bp away from the start codon ATG of the Wx gene in SEQ ID No. 1, with PAM being CGG).

[0020] A method for accurately improving the softness of red rice, comprising knocking out or changing the gene sequence fragment.

[0021] Furthermore, the knockout or change of the gene sequence fragment is performed by gene editing using the above-mentioned method, and after gene editing, a mutant red rice plant is obtained, in which one or two target sequences on the red rice plant are mutated.

[0022] The final gene-edited red rice plants meet the following conditions: the Wx gene promoter region base sequence undergoes a mutation in one or both sgRNA target regions. Genetically modified red rice is understood to include not only first- and second-generation transgenic rice, but also their progeny. For transgenic rice, the gene can be inherited within the species or transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. Transgenic rice includes seeds, callus, whole plants, and cells.

[0023] A method for creating new rice germplasm with better softness is carried out by knocking out or changing the above-mentioned gene sequence fragment in the target rice.

[0024] The gene editing reagents / methods described in the present application can be used to inhibit the expression of the Wx gene, and / or to reduce the abundance of the Wx protein, and / or to knock out the Wx gene, and are used in rice molecular breeding; mutant plants are obtained after treatment with the reagents, and the relative expression level of the Wx gene in the Wx-2 material of the mutant plants is significantly reduced to about 0.6 compared with the wild-type Pi Gu Nien, while the relative expression levels of the Wx gene in the Wx-1 and Wx-3 materials do not show a significant decrease compared with the wild-type Pi Gu Nien.

[0025] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0026] The inventors of this application have discovered that the Wx gene is the primary gene that influences rice amylose content and contributes to its softness and palatability. Different Wx gene loci result in different amylose contents, but excessively high amylose content is not conducive to improving rice softness. Knocking out the coding region of the Wx gene completely disables its function, significantly reducing the rice's amylose content and ultimately hindering its softness. Using gene editing technology, the present inventors deleted multiple segments of the promoter sequence of the granule-bound starch synthase Wx gene in the rice variety "Pi Gu Nian." They found that deleting a specific segment upstream of the ATG moderately decreased Wx gene expression. The amylose content of "Pi Gu Nian" decreased from 19% to approximately 14%, and the gel consistency increased from 66 mm to approximately 91 mm, resulting in a moderate improvement in the softness of "Pi Gu Nian" red rice. This invention can be applied to the precise regulation of rice amylose content and the molecular breeding of high-quality local rice varieties in Guizhou. The present invention provides a method for improving the softness of red rice by moderately reducing Wx gene expression. The specific embodiments of the present invention demonstrate that by editing the promoter sequence of the Wx gene, the softness of the Guizhou local high-quality rice "Pi Gu Nian" can be accurately improved, the rice quality can be improved, the promotion and planting of this variety can be facilitated, and the preservation of high-quality germplasm resources can be facilitated. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure shows a comparison of the appearance phenotype, amylose content, total starch and gel consistency of different rice varieties in Example 1.

[0028] Figure 2 Schematic diagram of the promoter sequence knockout target design of the Wx gene in Example 1.

[0029] Figure 3 This is the vector skeleton diagram of Example 1.

[0030] Figure 4 Schematic diagram of sequencing after knockout of different sites in the promoter of the Wx gene.

[0031] Figure 5 The relative expression levels of the Wx gene in the mutants with different sequence knockout sites in the Wx gene promoter (mean±SD, n=4).

[0032] Figure 6 The amylose content of Wx gene promoter sequence knockout mutants at different sites (mean±SD, n=3).

[0033] Figure 7 The gel consistency of the Wx gene promoter sequence knockout mutants at different sites (mean±SD, n=3).

[0034] Figure 8This is the sequencing results of the Wx gene promoter sequence mutant strain Wx-2-5 in Example 2.

[0035] Figure 9 This is the sequencing results of the Wx gene promoter sequence mutant strain Wx-2-8 in Example 2.

[0036] Figure 10 This is the sequencing results of the Wx gene promoter sequence mutant strain Wx-2-9 in Example 2.

[0037] Figure 11 Comparison of appearance, brown rice rate, 1000-grain weight and proanthocyanidin content of three strains of the Wx gene promoter sequence mutants in Example 2 (mean±SD, n=3). DETAILED DESCRIPTION

[0038] The technical solutions of the present invention are further defined below with reference to specific embodiments, but the scope of protection claimed is not limited to the description. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are conventional methods, and the materials and reagents used are all commercially available.

[0039] The expression vectors pYLsgRNA-OsU3, pYLsgRNA-OsU6a, and the binary vector pYLCRISPR / Cas9Pubi-H are available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0040] The transcript used in the following examples is Wx-1, which serves as an example only and does not limit the editing site in the application. Unless otherwise specified, all examples were conducted according to conventional experimental conditions or product specifications. In the following examples, unless otherwise noted, the first position of each nucleotide sequence in the sequence listing refers to the 5'-terminal nucleotide of the corresponding DNA / RNA, and the last position refers to the 3'-terminal nucleotide of the corresponding DNA / RNA.

[0041] Example 1: Construction of promoter knockout vector

[0042] The applicant has previously collected materials of the high-quality red rice "Pi Gu Nian" from Guizhou Province (the materials were grown in Guiyang, Guizhou). Compared with other common red rices such as Xia Huang Nian, Niu Wei Nian, Chi Shi, and Hong Xu Nuo, it was found that the amylose content of "Pi Gu Nian" is higher ( Figure 1 AB, C), low total starch content ( Figure 1 AB, D), low viscosity ( Figure 1 D) Among them, Figure 1In the figure, A shows the appearance phenotypes of rice varieties Pi Gu Nien, Xia Huang Nien, Niu Wei Nien, Chi Shi, Hong Xu Nuo, and Weedy Rice 31; B shows the amylose content; C shows the total starch content; and D shows the gel consistency. Statistical data for B and D are mean ± SD, n = 3. Results were analyzed using the Duncan test for significant differences. Data are marked with lowercase letters above. Within the same figure, different lowercase letters indicate significant differences (p < 0.05). Same below.

[0043] 1. Target Sequence Selection

[0044] The coding sequence of "Pi Gu Nian" Wx is the sequence shown in SEQ ID No. 2 in the sequence listing, and its amino acid sequence is shown in SEQ ID No. 3; the promoter sequence of the Wx gene is the sequence shown in SEQ ID No. 1 in the sequence listing.

[0045] SEQ ID No. 1 (5'-3', underline indicates Target 3 and Target 4):

[0046]

[0047]

[0048]

[0049]

[0050] SEQ ID No.2(5'-3'):

[0051]

[0052] SEQ ID No.3:

[0053] MSA LTT SQL ATS ATG FGI ADR SAP SSL LRH GFQ GLKPRS PAG GDA TSL SVTTSA RAT PKQ QRS VQR GSR RFP SVVVYA TGA GMN VVF VGA EMA PWS KTG GLG DVL GGLPPAMAA NGH RVM VIS PRY DQY KDA WDT SVV AEI KVA DRYERV RFF HCY KRG VDR VFI DHPSFL EKV WGK TGE KIY GPDTGV DYK DNQ MRF SLL CQA ALE APR ILN LNN NPY FKG TYGEDVVFV CND WHT GPL ASY LKN NYQ PNG IYR NAK VAF CIHNIS YQG RFA FED YPE LNL SERFRS SFD FID GYD TPV EGRKIN WMK AGI LEA DRV LTV SPY YAE ELI SGI ARG CEL DNIMRLTGI TGI VNG MDV SEW DPS KDK YIT AKY DAT TAI EAKALN KEA LQA EAG LPV DRK IPLIAF IGR LEE QKG PDV MAAAIP ELM QED VQI VLL GTG KKK FEK LLK SME EKY PGK VRAVVKFNA PLA HLI MAG ADV LAV PSR FEP CGL IQL QGM RYGTPC ACA STG GLV DTV IEG KTGFHM GRL SVD CKV VEP SDVKKV AAT LKR AIK VVG TPA YEE MVR NCM NQD LSW KGPAKN WENVLL GLG VAG SAP GIE GDE IAP LAK ENV AAP*

[0054] The promoter sequence of Wx in "Pi Gu Ni" has been amplified and sequenced in the early stage. The double target of CRISPR vector was designed for the region where the cis-elements of the promoter sequence of the gene are more concentrated, and the promoter sequence knockout vector was constructed. For details, see Figure 2The carrier of the combination of Target 1 and Target 2 and the obtained material were named Wx-1, the carrier of the combination of Target 3 and Target 4 and the obtained material were named Wx-2, and the carrier of the combination of Target 5 and Target 6 and the obtained material were named Wx-3.

[0055] Among them, Wx-2 is used as an example for illustration, and its target sequence is as follows:

[0056] Target 3: 5'-ATCGCGCGGGCTTCACGCAACGG-3' (SEQ ID

[0057] No.4, as shown in SEQ ID No.1, is 1360-1337 bp away from the ATG start codon of the Wx gene.

[0058] , PAM is CGG);

[0059] Target 4: 5'-GTGTTGTTCTGTTGTTCATCAGG-3' (SEQ ID No. 5, as shown in the region 1198-1175 bp away from the start codon ATG of the Wx gene in SEQ ID No. 1, with PAM being AGG).

[0060] The sgRNA targeting Target 3 in the CRISPR / Cas9 method is recorded as sgRNA1, and the sgRNA targeting Target 4 in the CRISPR / Cas9 method is recorded as sgRNA2.

[0061] 2. Construction of sgRNA expression cassette

[0062] 1. Construction of sgRNA1 expression cassette pOsU3-WxgRNA

[0063] Using primers UF, gRNA-R, U3-OsWx-F, and U3-OsWx-R, and the pYLsgRNA-OsU3 plasmid as a template, fusion PCR was performed to amplify the pOsU3-WxgRNA expression cassette. The pOsU3-WxgRNA expression cassette, which encodes sgRNA1, was used for bacterial liquid PCR. Positive single clones with a band size of approximately 1.2 kb were selected for sequencing using the SP1 primer. The specific primer sequences used are as follows:

[0064] U3-OsWx-F:

[0065] 5′-ATCGCGCGGGCTTCACGCAAGTTTTAGAGCTAGAAAT-3′ (SEQ ID No. 6, the sequence indicated by the wavy line is Target 3, and the sequence indicated by the underline is the linker sequence);

[0066] U3-OsWx-R:

[0067] 5'-TTGCGTGAAGCCCGCGCGATTGCCACGGATCATCTGC-3' (SEQ ID No. 7, the sequence indicated by the wavy line is the reverse complement of Target 3, and the sequence indicated by the underline is the linker sequence);

[0068] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID No. 8);

[0069] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID No. 9).

[0070] The PCR product was diluted 10-fold and used as a template for the second round of PCR amplification. The second round of PCR amplification was performed using primers BL and B2 to introduce the BsaI enzyme recognition site. The primer sequences used are as follows:

[0071] BL: 5′-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAG CAAAGG-3′ (SEQ ID No. 10, the underlined sequence indicates the BsaI enzyme recognition site);

[0072] B2: 5′-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3′ (SEQ ID No. 11, the underlined sequence is the BsaI enzyme recognition site).

[0073] The amplified products were detected by agarose gel electrophoresis and the target fragments were purified and recovered.

[0074] 2. Construction of sgRNA2 expression cassette pOsU6a-WxgRNA

[0075] Using primers UF, gRNA-R, U6a-Wx-F, and U6a-Wx-R, and the pYLsgRNA-OsU6a plasmid as a template, fusion PCR was performed to amplify the pOsU6a-WxgRNA expression cassette. The pOsU6a-WxgRNA expression cassette encodes sgRNA2, which is the sgRNA2 expression cassette. The primer sequences used are as follows:

[0076] U6a-OsWx-F: 5′-GTGTTGTTCTGTTGTTCATCGTTTTAGAGCT AGAAAT-3′ (SEQ ID No. 12, the sequence indicated by the wavy line is Target 5, and the sequence indicated by the underline is the linker sequence);

[0077] U6a-OsWx-R: 5′-GATGAACAACAGAACAACACCGGCAGCCA AGCCAGCA-3′ (SEQ ID No. 13, the sequence indicated by the wavy line is the reverse complement of Target 5, and the sequence indicated by the underline is the linker sequence);

[0078] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID No. 8);

[0079] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID No. 9).

[0080] The PCR product was diluted 10-fold and used as a template for the second round of PCR amplification. The second round of PCR amplification was performed using primers B2' and BR to introduce the BsaI enzyme recognition site. The primer sequences used are as follows:

[0081] B2': 5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAA GG-3' (SEQ ID No. 14, the underlined sequence indicates the BsaI enzyme recognition site);

[0082] BR: 5′-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCC AAGCTC-3′ (SEQ ID No. 15, the underlined sequence is the BsaI enzyme recognition site).

[0083] The amplified products were detected by agarose gel electrophoresis and the target fragments were purified and recovered.

[0084] 3. Construction of recombinant expression vector CRISPR-OsWx-2Target

[0085] The second round PCR purification products obtained in step 1 and the second round PCR purification products obtained in step 2 were constructed into pYLCRISPR / Cas9Pubi-H (see the vector skeleton diagram) using the restriction endonuclease BsaI and ligase T4ligase according to the conventional molecular experimental operation "cut and ligate" method. Figure 3 ) and then transformed into DH5α, and single clones were picked.

[0086] The recombinant plasmid was sequenced to confirm that the correct expression vector was obtained. The sequencing primers were:

[0087] SP1: 5'-CCGACATAGATGCAATAACTTC-3' (SEQ ID No. 16).

[0088] The recombinant plasmid that was sequenced correctly was named CRISPR-OsWx-2Target. For ease of identification and distinction from other plasmids, we will abbreviate it as the Wx-2 plasmid. CRISPR-OsWx-2Target contains the sgRNA1 expression cassette pOsU3-WxgRNA, the sgRNA2 expression cassette pOsU6a-WxgRNA, and the cas9 encoding gene, capable of expressing sgRNA1 and sgRNA2, as well as Cas9. The Wx-1 and Wx-3 plasmids were obtained using the same method. These plasmids differ from the Wx-2 plasmid in their target sequences, as detailed above.

[0089] Example 2: Construction and phenotypic identification of the "Pi Gu Nien" plant with a specific segment of the Wx gene promoter knocked out

[0090] 1. Cultivation of "Pi Gu Ni" plants with a specific knockout region of the Wx gene promoter

[0091] The Wx-1, Wx-2, and Wx-3 plasmids obtained in Example 1 were transformed into Agrobacterium tumefaciens EHA105 using conventional electroporation methods. Calli from "Pi Gu Nien" were infected with Agrobacterium, and calli were selected with 50 mg / L hygromycin and differentiated. Transformed seedlings were obtained in approximately four months. After hardening the seedlings for one to two weeks, they were transplanted to soil. After approximately three months of growth, genomic DNA from the transformed seedlings was extracted and amplified using specific primers for the OsWx-1, OsWx-2, and OsWx-3 series.

[0092] The primer pair used to amplify OsWx-2 consists of OsWx-2-F and OsWx-2-R:

[0093] OsWx-2-F:5'-GAGTGCATGCAGATGCATG-3', (SEQ ID No. 17);

[0094] OsWx-2-R: 5'-AACATCGATCAGCCTAACC-3', (SEQ ID No. 18).

[0095] Genome editing materials were selected, their fragments were amplified to obtain products, and the products were sequenced to determine the mutation type. In this example, a "Pi Gu Ni" genetic transformation system was established and a series of mutant plants with reduced amylose content were obtained. Sequencing and identification were performed, and the results showed that all three plasmids had obtained mutant plants with fragment knockout. Figure 4 The results show the gene mutation status of different mutant plants. After collecting seeds from individual plants, they were planted to obtain T1 generation materials. RNA was extracted from rice leaves of each T1 generation material, reverse transcribed into cDNA, and subjected to real-time fluorescence quantitative PCR experiments on the Wx gene. The results showed that the relative expression level of the Wx gene in the Wx-2 material decreased to about 0.6 compared with the wild-type Pi Gu Nien, while the relative expression level of the Wx gene in the Wx-1 and Wx-3 materials decreased significantly compared with the wild-type Pi Gu Nien ( Figure 5 The amylose content of each material was counted and it was found that the amylose content in Wx-2 material was slightly reduced to about 14%, which was significantly lower than that of the wild-type skin drum sticky. The amylose content of Wx-1 and Wx-3 was reduced. Figure 6 ).

[0096] 2. The "sticky skin" phenotype of knockout of a specific region of the Wx gene promoter

[0097] Different Wx knockout materials were planted in the field, and the Wx gene resequencing results of three strains with double knockout of Wx gene promoter sequence targets 1, 2 and targets 3, 4 are as follows: Figure 8-10 The T1 generation lines with mutations at different sites are numbered Wx-2-5, Wx-2-8, and Wx-2-9. Figure 8 The sequencing results of the Wx-2-5 mutant were compared with the wild type, in which a sequence including targets 3 and 4 was knocked out; Figure 9 The sequencing results of the Wx-2-8 mutant were compared with the wild type, in which a sequence including targets 3 and 4 was knocked out; Figure 10 The sequencing results of the Wx-2-9 mutant are compared with the wild type, which lacks ATCGCGCGGG on target 3; Figure 11 The appearance, brown rice rate, 1000-grain weight and proanthocyanidin content among the three mutant lines are shown. Figure 8-10 The mismatch between the front and back sequences in the sequencing graph is due to sequencing errors at the beginning of the sequencing reaction.

[0098] The field phenotypes of these materials were observed. Taking the wild type of "Pi Gu Nien" in the first figure as the control, it can be clearly seen that the amylose content of the three strains of the Wx mutant changed ( Figure 11To more accurately identify the phenotype, the amylose content of the wild-type and three Wx mutant lines of "Pi Gu Nien" and the relative expression of the Wx gene in each line were measured and statistically analyzed. It was found that the relative expression of the Wx gene in the Wx-2-8 mutant was significantly lower than that in the wild-type line, and the expression levels in each line were between 0.6-0.8 times that of the wild-type line ( Figure 5 Among the three Wx mutant lines, the amylose content of the Wx-2-8 mutant was significantly lower than that of the wild-type material, and the gel consistency was significantly higher than that of the wild-type material ( Figure 6 、 7 The amylose and gel consistency of the three strains showed significant changes, especially the deletion of the CGCAACGGCGC base on target 3 of the Wx-2-8 mutant, which led to a significant change in amylose content. This indicates that the CGCAACGGCGC base on target 3 is a key site in the promoter sequence of the Wx gene that regulates the amylose content. It can be seen that the promoter region where target 3 is located is the core promoter region of the Wx gene, among which the CGCAACGGCGC base on target 3 (the segment 1345-1335bp away from the start codon ATG of the Wx gene) is the core promoter region that plays the most critical regulatory role.

[0099] Therefore, in the Guizhou local high-quality rice "Pi Gu Nian", by editing the special promoter segment of the Wx gene, the amylose content can be precisely reduced, and the softness of "Pi Gu Nian" can be significantly increased, solving the problem that the Guizhou local high-quality rice "Pi Gu Nian" has a hard taste and is difficult to promote its planting, resulting in difficulty in preserving its high-quality germplasm resources.

[0100] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and are subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. An application of a gene sequence fragment related to the softness of red rice in improving the softness and gel consistency of rice, characterized in that: The gene sequence fragments are SEQ ID No. 4: 5'-ATCGCGCGGGCTTCACGCAACGG-3' and SEQ ID No. 5: 5'-GTGTTGTTCTGTTGTTCATCAGG-3'.

2. A method for accurately improving the softness and consistency of red rice, characterized in that: Knock out the gene sequence fragment described in claim 1.

3. The method according to claim 2, wherein The knockout of the gene sequence fragment according to claim 1 is performed by gene editing, and after gene editing, a mutant red rice plant is obtained, and one or two target sequences on the red rice plant are mutated.

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