Mutation site and control gene IBC of the rice ideal brittle stem mutant ibc and its application

Abstract

Provided are the mutation site of the ideal brittle stem mutant ibc in rice, the control gene IBC and its application, in the field of biotechnology, the nucleotide sequence of the gene IBC being (1) as shown in SEQ ID No. 1 or SEQ ID No. 2, or (2) the nucleotide sequence of a mutant, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides. Also provided are the encoded protein of gene IBC, recombinant constructs, recombinant host cells, and methods and applications of gene IBC for making the stem of rice at maturity brittle. In the allelic variants and gene-edited loss-of-function mutants of IBC, the stems show a brittle stem phenotype at late maturity, the leaf blades are not brittle, the productivity is good, other agronomic traits are not obviously changed, and at the harvesting season, the rice straw harvested in the field is easy to crush, easy to crush and return to the field, easy to crush and silage when the rice straw is used as feed, and easy to be chewed by animals.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, and more particularly to the mutation site of the ideal brittle stem mutant ibc in rice, the control gene IBC, and applications thereof. [Background technology]

[0002] Rice (Oryza sativa L.) is one of the most important food crops worldwide. The mechanical strength of its stem is one of the important agronomic traits, which is directly related to the lodging resistance of rice plants, and ultimately affects rice yield. Meanwhile, rice produces a large amount of rice straw every year, which can be used for livestock feed, papermaking raw materials, building materials, biomass energy, and organic fertilizer raw materials, etc. However, due to the decomposition resistance barrier of rice straw biomass, it is extremely difficult to reuse rice straw in fields and for other comprehensive utilization, so it is very necessary to break through the bottleneck problem of comprehensive utilization of rice straw in terms of the composition and structure of the rice straw itself.

[0003] The mechanical strength of rice stems and their biomass decomposition resistance barrier are both determined by the secondary cell wall. The secondary cell wall is a cell wall layer that accumulates continuously inside the primary cell wall after certain cells stop growing. The secondary cell wall is mainly composed of cellulose, hemicellulose and lignin. Any change in each component will affect the structural change of the secondary cell wall, and thus the mechanical strength of the plant. In rice, brittle stem mutants are important materials for studying the biosynthesis of rice secondary cell walls, and most of the reported brittle stem mutants have a reduced cellulose content, which ultimately affects the thickening of the secondary cell wall. In recent years, researchers at home and abroad have cloned several important genes that control the synthesis of rice secondary cell walls. For example, there are three genes encoding catalytic subunits of secondary cell wall cellulose synthase in rice, OsCESA4, OsCESA7, and OsCESA9, whose mutations all lead to changes in the mechanical strength of the stem and show a brittle stem phenotype, among which OsCESA9 has a mutation at amino acid position 387 from aspartic acid to asparagine, and shows a semi-dominant brittle stem phenotype. Specifically, patent publication no. CN110964733A also discloses a semi-dominant brittle stem control gene SDBC1 in rice, but the stems and leaf blades of rice plants expressing this gene are both prone to breaking. In addition, there are genes BC1 and BC12 that affect the assembly and arrangement of cellulose, gene BC3 that affects the vesicular transport of cellulose synthase, gene BC10 that affects the synthesis of hemicellulose, and gene CEF1 that controls the synthesis of secondary cell walls in rice. Research on these genes not only provides a new theory for the synthesis of secondary cell walls in rice, but these mutant materials also provide new genetic resources for the efficient use of rice straw.

[0004] In order to realize efficient comprehensive utilization of rice straw, it is necessary not only to change the compositional components of rice stalks, but also to consider the properties related to yield, and efficient utilization of rice straw cannot be improved at the expense of rice yield. Although the changes in cell wall components of most brittle stem mutants of rice have great potential to promote efficient utilization of rice straw, the cultivation of brittle stem mutants of rice faces many problems in the actual production process. For example, leaf blades are easily broken, which affects field operation, and stems are easily fallen, which affects final yield.

[0005] An ideal brittle stem rice suitable for large-scale cultivation should have the following characteristics: (i) high productivity, (ii) high lodging resistance, (iii) non-brittle leaf blades, and (iv) stems that become brittle late in maturity. Summary of the Invention [Problem to be solved by the invention]

[0006] One of the technical problems that the present invention aims to solve is that in the conventional technology, the leaf blades of brittle-stalk mutants of rice are easily broken, which affects field operations. However, the present invention provides a mutation site of ibc, an ideal brittle-stalk mutant of rice whose leaf blades are less likely to break and which does not fall over, a control gene IBC, a protein encoded by the IBC gene, a recombinant construct of the IBC gene, a host cell recombinant of the IBC gene, a method for making the stems of rice plants at maturity brittle, and applications of brittle-stalk rice or brittle rice straw as feed or fertilizer. [Means for solving the problem]

[0007] The present invention can solve the above technical problems by the following technical means. The mutation site of the ideal brittle stem mutant ibc in rice, the mutation site being a chromosome fragment inversion, the position of the chromosome fragment inversion being located in the LOC_Os03g18140 gene. The LOC_Os03g18140 gene is an IBC gene.

[0008] An ideal control gene IBC of the brittle stem mutant ibc in rice, the nucleotide sequence of said gene IBC being (1) as shown in SEQ ID No. 1 or SEQ ID No. 2, or (2) the nucleotide sequence of a mutant, allele or derivative thereof produced by adding, substituting, inserting or deleting one or more nucleotides.

[0009] Alternatively, it may hybridize under stringent conditions with the nucleotide sequence shown in SEQ ID No. 2 and simultaneously encode a nucleotide sequence that controls the mechanical strength of the stem of rice at maturity.

[0010] Stringent conditions refer to placing the hybridization membrane in prehybridization (0.25 mol / L sodium phosphate buffer, pH 7.2, 7% SDS) for 30 minutes at 65 ° C. Discard the prehybridization solution and add hybridization solution (0.25 mol / L sodium phosphate buffer, pH 7.2, 7% SDS, isotope-labeled nucleotide fragments) for 16 hours at 65 ° C. Discard the hybridization solution and add membrane washing solution I (20 mmol / L sodium phosphate buffer, pH 7.2, 0.1% SDS) to wash the membrane twice at 65 ° C, each time for 10 to 15 minutes, and add membrane washing solution II (10 mmol / L sodium phosphate buffer, pH 7.2, 0.1% SDS) to wash the membrane at 65 ° C for 10 to 15 minutes.

[0011] The nucleotide sequence SEQ ID No. 1 relates to the promoter, coding and control regions of the gene IBC.

[0012] The present invention utilizes heavy ion beam irradiation of Wuyun Japonica rice variety No. 7 (WYJ7) to obtain an ideal brittle culm ibc (idea brittle culm) mutant in which rice straw shows a brittle culm phenotype in the late ripening period without affecting agronomic traits related to yield, such as plant height, tiller number, panicle number, thousand-kernel weight, and lodging resistance.

[0013] In the present invention, we performed genetic analysis and population construction on the ibc mutant, and then performed genetic behavior analysis on it, and found that the brittle stem phenotype of the ibc mutant is a recessive control of the IBC single gene after mutation.

[0014] The beneficial effects are as follows: the present invention uses positional cloning to isolate and identify the IBC gene that controls the mechanical strength of rice stalks, and through material phenotypic analysis and genetic complementation experiments, it has proven the function of the IBC gene in controlling the mechanical strength and cell wall components of rice stalks, and has successfully used the gene to cultivate the brittle-stem rice variety "Kesong No. 7" suitable for large-scale production, truly resolving the problem of rice straw disposal from varieties.

[0015] IBC-encoded xylan acetyltransferase plays an important role in secondary cell wall hemicellulose polysaccharide modification. In IBC allelic and gene-edited loss-of-function mutants, the stems showed a brittle stem phenotype at late maturity, the leaf blades were not brittle, the productivity was high, other agronomic traits were not obviously changed, the rice straw harvested in the field at harvest time was easy to crush, easy to crush and return to the field, easy to crush and silage when rice straw is used as feed, and easy to be chewed by animals.

[0016] The IBC gene will provide theoretical basis, materials and genetic support for the future elucidation of the genetic basis of the control of secondary cell wall synthesis in rice at the molecular level and for the breeding of new environmentally friendly varieties based on molecular design of rice.

[0017] A protein encoded by the IBC gene, which is an ideal brittle stem mutant of rice, ibc, the amino acid sequence of which is (1) as set forth in SEQ ID No. 3, or (2) an amino acid sequence which differs from the amino acid sequence of SEQ ID No. 3 by substitutions, deletions and / or insertions of one or more (e.g. 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) amino acid residues, or (3) an amino acid sequence which has at least 70%, preferably at least 80%, more preferably at least 90%, in particular at least 95% or 98% or 99% identity to the amino acid sequence of SEQ ID No. 3, or (4) an active fragment of the amino acid sequence according to (1) or (2) or (3).

[0018] A recombinant construct comprising a nucleotide sequence of an IBC gene, which controls the mechanical strength of the stem at maturity in rice, and a vector used in the recombinant construct is a clone vector or an expression vector for expressing the nucleotide sequence.

[0019] A recombinant host cell, which is a host cell of the above-mentioned recombinant construct or contains a polynucleotide sequence in its genome, in which the gene IBC controlling the mechanical strength of rice stems according to the present invention is integrated. The host cell may be selected from a plant cell or a microbial cell, for example an E. coli cell or an Agrobacterium cell, preferably a plant cell, most preferably a rice cell. The cell may be isolated, in vitro, cultured or part of a plant.

[0020] The present invention discloses the application of brittle stem rice varieties obtained by ibc mutants and ibc mutation sites, as well as brittle stem rice varieties obtained by mutating the IBC gene by functional deletion through various means including physical mutagenesis, chemical mutagenesis and biological mutagenesis as mentioned above, and gene editing techniques, in rice straw processing, including feed and fertilizer products made from brittle rice straw.

[0021] Preferably, the host cell is a microbial cell.

[0022] Preferably, the microbial cell is an E. coli cell or an Agrobacterium cell.

[0023] A method for cultivating rice straw to make it brittle, the method comprising the step of rendering the regulatory gene IBC of the ideal brittle stem mutant ibc of rice described above functionally non-functional by mutagenesis means including physical mutagenesis, chemical mutagenesis and biological mutagenesis, or by gene editing technology to obtain a rice plant, the rice plant exhibiting a brittle stem phenotype.

[0024] A method for cultivating rice straw to make it brittle, the method comprising the steps of hybridizing the ideal brittle stem mutant ibc of rice having the above-mentioned mutation site with other rice varieties, and obtaining a rice line with a brittle stem phenotype by progeny segregation.

[0025] The present invention discloses the application of brittle stem rice varieties obtained by ibc mutants and ibc mutation sites, as well as brittle stem rice varieties obtained by mutating the IBC gene by functional deletion through various means including physical mutagenesis, chemical mutagenesis and biological mutagenesis as mentioned above, and gene editing techniques, in rice straw processing, including feed and fertilizer products made from brittle rice straw.

[0026] The brittle stem rice or brittle rice straw obtained by the above method is used as a feed or fertilizer.

[0027] The beneficial effects are as follows: When the brittle stem rice or brittle rice straw of the present invention is used as a raw material for feed, it contributes to chewing and digestion, and when used as fertilizer, it is easily decomposed in the field.

[0028] Another technical problem to be solved by the present invention is to provide a method for identifying a mutation site in the IBC gene, which controls the mechanical strength of rice stems at maturity.

[0029] The present invention can solve the above technical problems by the following technical means. A method for identifying a mutation site of the ideal brittle stem mutant ibc of rice, comprising the steps of: (1) carrying out PCR amplification with primers ibc-jd-1 and ibc-jd-2, the nucleotide sequence of the ibc-jd-1 forward primer ibc-jd-1-F being shown in SEQ ID No. 4, the nucleotide sequence of the ibc-jd-1 reverse primer ibc-jd-1-R being shown in SEQ ID No. 5, the nucleotide sequence of the ibc-jd-2 forward primer ibc-jd-2-F being shown in SEQ ID No. 6, and the nucleotide sequence of the ibc-jd-2 reverse primer ibc-jd-2-R being shown in SEQ ID No. 7; and (2) performing agarose gel electrophoresis detection on the amplification products of the two pairs of primers, and if the detection display shows that only the ibc-jd-1 primer has a target band, the ibc mutation site is homozygous; if the detection display shows that only the ibc-jd-2 primer has a target band, the ibc mutation site does not contain the ibc mutation site and is wild-type; and if the detection display shows that both the ibc-jd-1 and ibc-jd-2 primers have target bands, the ibc mutation site is heterozygous.

[0030] The beneficial effects are as follows: specific primers for PCR amplification are designed according to the unique mutation type of ibc, and the primers are used to perform PCR amplification on the rice DNA to be identified, and the rice materials containing homozygous, heterozygous, and ibc mutation sites can be clearly distinguished by agarose electrophoresis detection. This method can be used to follow up and identify new varieties of brittle stem rice using ibc mutation sites.

[0031] The advantages of the present invention are as follows: the present invention uses positional cloning to isolate and identify the IBC gene that controls the mechanical strength of rice stalks, and through material phenotype analysis and genetic complementation experiments, it has been proven that the IBC gene with an ideal brittle stem phenotype has the function of controlling rice stalk mechanical strength and cell wall components, and the gene has been successfully used to cultivate the brittle stem rice variety "Kesong No. 7" suitable for large-scale production, truly resolving the problem of rice straw disposal from varieties.

[0032] IBC-encoded xylan acetyltransferase plays an important role in secondary cell wall hemicellulose polysaccharide modification. In IBC allelic and gene-edited loss-of-function mutants, the stems showed a brittle stem phenotype at late maturity, the leaf blades were not brittle, the productivity was high, other agronomic traits were not obviously changed, the rice straw harvested in the field at harvest time was easy to crush, easy to crush and return to the field, easy to crush and silage when rice straw is used as feed, and easy to be chewed and digested by animals.

[0033] The IBC gene will provide theoretical basis, materials and genetic support for the future elucidation of the genetic basis of the control of secondary cell wall synthesis in rice at the molecular level and for the breeding of new environmentally friendly varieties based on molecular design of rice.

[0034] Specific primers for PCR amplification were designed according to the specific mutation type of ibc, and the PCR amplification was performed on the rice DNA to be identified using the primers. The rice material containing homozygous, heterozygous, and ibc mutation sites was clearly distinguished by agarose electrophoresis detection. This method can be used for the follow-up identification of new varieties of brittle stem rice using ibc mutation sites. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 is a comparative diagram of the strain types of the wild-type WT and the ibc mutant in Example 1 of the present invention. [Diagram 2]FIG. 2 is a comparative diagram of stem breakage in the wild type WT and the ibc mutant in Example 1 of the present invention. [Diagram 3] FIG. 3 is a comparative diagram of leaf blade breaking between the wild type WT and the ibc mutant in Example 1 of the present invention. [Figure 4] FIG. 4 shows the measurement results of the transverse strength of the stems of the wild type WT and the ibc mutant in Example 1 of the present invention. [Diagram 5] FIG. 5 shows the measurement results of transverse strength of leaf blades of the wild type WT and the ibc mutant in Example 1 of the present invention. [Figure 6] FIG. 6 shows the results of measuring the plant height of the wild type WT and the ibc mutant in Example 1 of the present invention. [Figure 7] FIG. 7 shows the results of measuring the number of tillers in the wild type WT and the ibc mutant in Example 1 of the present invention. [Figure 8] FIG. 8 shows the results of measuring the number of grains in panicles of the wild type WT and the ibc mutant in Example 1 of the present invention. [Figure 9] FIG. 9 shows the measurement results of panicle length of the wild type WT and the ibc mutant in Example 1 of the present invention. [Figure 10] FIG. 10 shows the measurement results of the fruit-setting rate of the wild-type WT and the ibc mutant in Example 1 of the present invention. [Figure 11] FIG. 11 is a diagram showing the measurement results of the thousand-grain weight of the wild-type WT and the ibc mutant in Example 1 of the present invention. [Figure 12] FIG. 12 is an electron microscope image of a cross section of the stem from the lowest leaf to the second node of the wild type WT and the ibc mutant in Example 1 of the present invention. [Figure 13] FIG. 13 is a diagram showing IBC gene localization in Example 2 of the present invention. [Figure 14] FIG. 14 is an analysis diagram of the IBC gene MutMap in Example 2 of the present invention. [Figure 15] FIG. 15 is a schematic structural diagram of the ibc mutation site in Example 2 of the present invention. [Figure 16] FIG. 16 shows the results of identifying the ibc mutation site in Example 2 of the present invention. [Figure 17] FIG. 17 is a structural diagram of the pIBCF expression vector in Example 2 of the present invention. [Figure 18] FIG. 18 is a diagram showing the phenotype of the genetic complementation strain in Example 2 of the present invention. [Figure 19] FIG. 19 is a sequence map of the CRISPR / Cas9-edited IBC gene in Example 3 of the present invention. [Figure 20] FIG. 20 is a diagram showing the brittle stem phenotype of the CRISPR / Cas9 gene-edited strain in Example 3 of the present invention. [Figure 21] FIG. 21 is a diagram showing the strain type of the CRISPR / Cas9 gene-edited strain in Example 3 of the present invention. [Figure 22] FIG. 22 shows rice straw milled after harvesting wild-type WT and ibc mutant materials in Example 4 of the present invention. [Figure 23] FIG. 23 is a comparison diagram of the grinding length of rice straw of wild-type WT and ibc mutant materials in Example 4 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be described clearly and completely below with reference to the embodiments of the present invention, but obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, any other embodiments obtained by a person skilled in the art without inventive efforts fall within the protection scope of the present invention.

[0037] Unless otherwise stated, all test materials, reagents, etc. used in the following examples are obtainable from commercial sources.

[0038] Any specific techniques or conditions not specified in the examples can be in accordance with the techniques, conditions or product instructions explained in the literature in the field.

[0039] Example 1 Phenotypic analysis of the ideal brittle stem mutant ibc (1) Analysis of agricultural traits Heavy ion beam 12 C 6+ A japonica rice variety, Wuyun 7 (WYJ7), was mutagenized with 120Gy (energy 80MeV, dose 120Gy) to obtain an ibc mutant. The phenotypic characteristics of the mutant were that in the early heading stage, plant height, tiller number, and growth period were not significantly different from those of the wild type, but in the later ripening stage, the stem showed a brittle stem phenotype, and there were no significant differences in leaf blades and other agronomic traits, such as panicle number, panicle length, fruit set rate, and thousand kernel weight, as shown in Figures 1 to 11.

[0040] (2) Genetic analysis of ibc mutants To study the molecular mechanism by which the ibc mutant forms brittle stems late in the ripening stage, we first performed genetic analysis. The ibc mutant was used to construct a backcross population by hybridization with the wild-type Wuyun 7. In the F2 generation segregating population of 480 plants, there were 112 plants with a brittle stem phenotype and 368 plants with normal stems, with a segregation ratio of 1:3 (χ 2 [1:3]=0.18<χ 2 0.05=3.84, P>0.05). In the F2 segregation population of JPEG0007624531000001.jpg12170600 plants, there were 159 plants with brittle stems and 441 plants with normal stems, and the segregation ratio of the number of brittle stems and normal stems was 1:3 (χ 2 [1:3]=0.24<χ 2 The results were consistent with those of the control group (p < 0.05 = 3.84, P > 0.05). These results indicate that the brittle stem trait of the ibc mutant is under the recessive control of a pair of single genes and is not affected by the genetic background.

[0041] (3) Observation of stem cross section using an electron microscope To further investigate the mechanism of brittle stem formation in the ibc mutant, we observed cross sections of the second node from the lowest leaf in the wild type and ibc mutant by scanning electron microscopy. The cell walls of the sclerenchyma in the ibc mutant were clearly thinner than those in the wild type (Fig. 12), indicating that the thinning of the secondary cell walls of the sclerenchyma is the cause of the brittle phenotype of the ibc mutant.

[0042] Example 2 Gene localization of the ideal brittle stem gene IBC (1) Construction of localized populations The ibc mutant was hybridized with varieties such as Indica 93-11, Huajing 74, and Nanjing 11, and the F1 generation of different hybridization combinations was selfed to obtain seeds of segregating populations. These seeds were cultivated in the field, and single plants that showed brittle stems at the late ripening stage were selected as localized single plants. Approximately 100 mg of leaf blades were taken from each single plant to extract DNA.

[0043] (2) Screening for simple sequence repeat (SSR) polymorphisms Using previously reported SSR primers that are uniformly distributed on chromosome 12 of rice, polymorphism screening was performed on the ibc mutant and Huajingsen 74 to obtain SSR primers with polymorphisms for the next step of the experiment.

[0044] (3) Localization of IBC genes First, 21 single lines with brittle stem phenotypes in the segregating population constructed by ibc mutant and Hua Jingsen 74 were randomly selected to perform initial localization of the IBC gene. SSR primers with polymorphisms obtained by screening were selected to perform linkage analysis on these 21 single lines. The results, as shown in Figure 13, showed that the molecular markers CSR7 and CSR18 at the end of the long arm of chromosome 3 were clearly linked to the mutant gene. The Recombinants in the figure indicate the recon, which further confirmed that the IBC gene was located between CSR7 and CSR18. The range was about 8.66 Mb.

[0045] In order to further narrow down the localization interval of the IBC gene, we searched for more primers with polymorphisms between the CSR7 and CSR18 molecular markers (the primer sequences used for IBC gene localization are shown in Table 1), and expanded the number of localization populations, and performed linkage analysis on the larger localization populations. As shown in Figure 13, Recombinants in the figure indicate recombinations. Finally, the IBC gene was finely localized between the Indel markers FM2 and FM5, i.e., within an interval of approximately 3.40 Mb, and the molecular markers FM3 and FM4 were simultaneously isolated.

[0046] Table 1 Primers used for IBC gene localization JPEG0007624531000002.jpg63162

[0047] (4) Identification of candidate genes and cloning of IBC genes With the development of technologies such as high-throughput second-generation sequencing and longer fragment reading third-generation sequencing, there are more options for localizing mutant genes of different mutation types. The fact that traditional positional cloning techniques cannot continuously reduce the localization interval for IBC genes may be due to their inherent mutation types. When we examined the localization of IBC genes by second-generation sequencing and third-generation sequencing, the MutMap localization results based on second-generation sequencing were consistent with the positional cloning results above (Figure 14). When the genome of the ibc mutant was combined at a specified position with the second-generation sequencing results by third-generation sequencing, it was found that the ibc mutant had a large chromosomal fragment inversion within the above localization interval (schematic diagram shown in Figure 15). Analysis of the genes at both ends of the inversion revealed that the breakpoint at one end was in the LOC_Os03g18140 gene, resulting in a loss-of-function mutation of the gene. LOC_Os03g18140 has attracted attention as a candidate gene for IBC because the breakpoint at the other end is not located in the coding region of the gene.

[0048] (5) Identification of the ibc mutation site According to the type of ibc mutation, corresponding identification primers are designed and PCR amplification is performed. The primer names are ibc-jd-1 (the ibc mutation site can amplify the band, but the wild type cannot) and ibc-jd-2 (the wild type can amplify the band, but the ibc mutation site cannot amplify the band). The identification results showed that the ibc mutant can amplify the band only using the ibc-jd-1 primer, while the wild type can amplify the band only using the ibc-jd-2 primer (Figure 16).

[0049] (6) Verification of functional complementation of IBC genes To verify that LOC_Os03g18140 is an IBC gene, RNA was extracted from the second node from the lowest leaf of the wild type, reverse transcribed, and amplified to obtain cDNA. The LOC_Os03g18140 expression vector pIBCF was constructed, driven by the promoter gene of LOC_Os03g18140 itself (3000 bp fragment upstream of ATG). The structure of the pIBCF expression vector is shown in Figure 17. As shown in Figure 18, all stems of the positive recombinant T0 generation plants obtained by transferring the pIBCF expression vector into the ibc mutant were restored to normal levels. This result proves that the LOC_Os03g18140 gene is an IBC gene.

[0050] The primers used to construct the expression vector pIBCF are: pIBC-F:5′-cggaattcTTCACTTTTGGGCATTGTTC-3′ pIBC-R:5′-cgggtaccCTCCGGAGCGCCCAGGAAGG-3′ IBC-CDS-F:5′-cgggtaccATGCAGCAGCGGCGGAAGTC-3′ IBC-CDS-R: 5′-cgggatccCTACTGGTCGGATGACCATG-3′.

[0051] The construction method of the expression vector pIBCF includes the following steps. (1) Using pIBC-F and pIBC-R primers, the wild-type Wuyun No. 7 DNA was used as a template to amplify the resulting PCR product, which was then double-digested with EcoRI and KpnI restriction endonucleases, and the pCAMBIA2300 backbone was double-digested using the enzymes. The amplified pIBC fragment was then inserted into the pCAMBIA2300 vector using T4 ligase to obtain the intermediate vector pCAMBIA2300-pIBC. (2) Using IBC-CDS-F and IBC-CDS-R primers, the resulting PCR product was amplified using wild-type Wuyun 7 cDNA as a template, and double-digested with KpnI and BamHI restriction endonucleases, and the intermediate vector pCAMBIA2300-pIBC obtained in step (1) was double-digested using the enzymes, and the amplified IBC-CDS fragment was then inserted into the pCAMBIA2300-pIBC vector using T4 ligase to obtain the final vector pCAMBIA2300-pIBC::IBC. This vector is called pIBCF. As shown in Figure 17.

[0052] Example 3 How to cultivate ideal brittle stem varieties of rice (1) Cultivate new brittle stem varieties using the ibc mutation site The ibc mutant was hybridized, backcrossed and selfed with normal stem rice varieties, such as 93-11, Hua Jing Sen 74, Dao Hua Xiang 2, etc., and during this process, the ibc mutation site and genetic background were selected using the identification primers ibc-jd-1 and ibc-jd-2, and finally a new brittle stem variety with homozygous ibc mutation gene was obtained in the background of 93-11, Hua Jing Sen 74, Dao Hua Xiang 2, etc. The specific implementation steps are as follows: (i) Recipient parents, such as 93-11, Huajingsen 74, Inakaka 2, etc., were hybridized with the ibc mutant as male parents to obtain F1. (i) The F1 was used as a female parent and backcrossed with a recipient parent, such as 93-11, Huajingsen 74, or Inakako 2, to obtain BC1F1. (iii) Cultivate BC1F1, detect the ibc genotype with the identification primers ibc-jd-1 and ibc-jd-2, and select the ibc heterozygous genotype. That is, the PCR amplification products of the above two pairs of primers both have the target band. (iv) Using molecular markers that are uniformly distributed across the 12 chromosome pairs of rice (including, but not limited to, SSR, SNP, InDel, EST, RFLP, AFLP, RAPD, and SCAR type markers) and have polymorphisms between the ibc mutant and the recurrent parent, the genetic background of the single line selected in step 3 was identified, and lines with high similarity to the recurrent parent genotype (e.g., higher than 75%) were selected. (v) The line selected in step 4 was backcrossed with the recipient parent, e.g., 93-11, Huajingsen 74, Inahana 2, etc., to obtain BC2F1. (vi) BC2F1 was cultivated, and steps 3 and 4 were repeated to select lines with heterozygous ibc genotypes and high recovery rates of the genetic background (e.g., higher than 95%), and self-fertilized seeds BC2F2 were harvested. (vii) BC2F2 was cultivated, and steps 3 and 4 were repeated to select the line with the highest homozygous genetic background rate and heterozygous ibc genotype, and BC2F3 was harvested. In the ibc homozygous line isolated from the BC2F3 progeny, the detection results of the identification primers ibc-jd-1 and ibc-jd-2 showed that only ibc-jd-1 could amplify the target band, and the stem phenotype was a brittle stem phenotype.

[0053] (2) Cultivate new brittle stem varieties using gene editing technology We used CRISPR / Cas9 technology to edit the IBC gene and generate mutants with a loss of IBC function. In several independent homozygous IBC gene knockout lines, except for stem brittleness at later stages (Figure 20), there were no obvious differences in other agronomic traits (Figure 21).

[0054] The construction and transformation methods of the CRISPR / Cas9 vector are as follows. The target primers designed based on the gDNA sequence of the IBC gene were: IBC-CRISPR-U3: 5′-CCTCTACAACGAGGACATCAAGT-3′ For the specific construction method, please refer to the paper published by Professor Liu Yaoguang of South China Agricultural University (A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. (2015) Molecular Plant, 8(8):1274-1284), and the vector was introduced into WYJ7 by Agrobacterium-mediated transformation (rice transformation was completed in the applicant's laboratory). Comparing the phenotypic analysis of wild-type and transgenic rice, it was found that several homozygous lines with IBC gene knocked out, crispr-ibc (sequencing peak map of mutation site is shown in Figure 19), all showed brittle stem phenotype at the later stage of stem (Figure 20), but there was no obvious difference in other agronomic traits (Figure 21).

[0055] Example 4 Evaluation of ibc brittle stem rice for feed use (1) Field crushing experiment of rice straw The wild type (WT) and ibc mutant materials were cultivated in a large area and harvested in the field by a combine harvester during the harvest season. As shown in Figure 22, the crushing degree of the rice straw of the ibc mutant after harvesting was obviously higher than that of the wild type (WT). According to the statistics of the crushing length of the rice straw of a pair of materials, the length of the rice straw after crushing of the ibc mutant was mostly concentrated in the range of less than 5 cm and 5 to 10 cm, as shown in Figure 23. This length contributes to the rice straw decomposing in the field in the later period and becoming a raw material for organic fertilizer, and also contributes to the chewing and digestion of cattle and sheep in feed production.

[0056] (2) Goat farming In order to evaluate the feeding value of brittle stalk rice, a study was conducted on the silage of brittle stalk rice straw and feeding it to meat sheep. A sheep farm with more than 10 years of breeding experience and a scale of raising more than 1,000 goats was selected to carry out the experimental work. First, 5 tons each of brittle stalk rice straw and general rice straw were collected, processed according to the silage method of whole stalk corn, and silaged in the form of a large bag, and the silage of whole stalk corn from the same period was used as a control. After silaging for two months, the bags were opened and samples were taken for detection.

[0057] The results showed that the appearance of the brittle stalk feed after silage was yellow-green, with a distinctly strong sour aroma, and it was soft to the touch and did not stick to the hands, which showed that it had excellent silage feed properties. The component detection results showed that the soluble sugar and starch contents of the silage brittle stalk were both higher than those of common rice straw and whole corn, showing excellent nutritional value. Thirty-six sheep of similar size were selected, numbered with notches on their ears, and divided into three feeding treatments, divided into three pens for each treatment, with four sheep per pen. They were fed a feeding method of mixing roughage and concentrated feed in a certain ratio in the preliminary test for two weeks, and in the main test for two months, and weighed once every 20 days.

[0058] The measurement results are shown in Table 2. The results showed that the feeding effect of silage brittle stem feed was the best, with an overall weight gain rate of 48.6%, and an average daily weight gain of 137 grams per sheep, which was 17.1% and 21.2% higher than silage corn and general rice straw, respectively, demonstrating good applicability.

[0059] Table 2. Results of feeding experiments after silage for brittle stem rice, ordinary rice, and whole corn JPEG0007624531000003.jpg21158

[0060] (3) Beef cattle breeding Forty Holstein beef cattle were used. The test cattle should be normal in growth and development, moderate in fatness, healthy and disease-free, in the relatively fast growth phase, about 4 months old, and in the weight range of 140-185 kg. They were randomly divided into four groups: ordinary rice straw (group A), brittle stalk rice straw (group B), silage brittle stalk rice straw (group C) and silage whole corn rice straw (group D). There were 10 cattle in each group, and they were fed separately, once in the morning and once in the evening. The regulated amount of concentrated feed was 40 kg per group every day, and there was no restriction on ordinary rice straw and other test materials. The feeding time was 8 weeks.

[0061] Regarding the effect of weight gain, feeding with ordinary rice straw (group A) resulted in an overall weight gain of 737.5 kg, feeding with brittle stalk rice straw (group B) resulted in an overall weight gain of 790.5 kg, feeding with silage brittle stalk rice straw (group C) resulted in an overall weight gain of 825.5 kg, and feeding with silage whole stalk rice straw (group D) resulted in an overall weight gain of 841 kg. This shows that silage brittle stalk rice straw has an effect close to that of silage whole stalk rice straw, and may be used as an alternative.

[0062] Regarding the palatability of the feed, the highest intake was silage brittle rice straw (group C) at 1,460.6 kg, followed by silage whole corn rice straw (group D) at 1,429.4 kg, and the lowest was general rice straw (group A). ​​The intake rate was silage brittle rice straw > silage whole corn rice straw > brittle rice straw > general rice straw.

[0063] The above embodiments are merely for illustrating the technical solutions of the present invention, and are not intended to limit the same. Although the above embodiments have been used to describe the present invention in detail, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified or some of the technical features therein can be replaced with equivalents, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

[Claim 1] A method for identifying a mutation site of an ideal brittle stem mutant ibc of rice, comprising the steps of: (1) performing PCR amplification with primers ibc-jd-1 and ibc-jd-2, the nucleotide sequence of the ibc-jd-1 forward primer ibc-jd-1-F being shown in SEQ ID No. 4, the nucleotide sequence of the ibc-jd-1 reverse primer ibc-jd-1-R being shown in SEQ ID No. 5, the nucleotide sequence of the ibc-jd-2 forward primer ibc-jd-2-F being shown in SEQ ID No. 6, and the nucleotide sequence of the ibc-jd-2 reverse primer ibc-jd-2-R being shown in SEQ ID No. 7; (2) performing agarose gel electrophoresis detection on the amplification products of the two pairs of primers, and if the detection display result shows that only the ibc-jd-1 primer has a target band, the ibc mutation site is homozygous, if the detection display result shows that only the ibc-jd-2 primer has a target band, the ibc mutation site does not contain the ibc mutation site and is wild-type, and if the detection display result shows that both the ibc-jd-1 and ibc-jd-2 primers have target bands, the ibc mutation site is heterozygous; The method for identifying a mutation site of an ideal brittle stem mutant ibc of rice, wherein the mutation site is a chromosome fragment inversion, and the position of the chromosome fragment inversion is located in the LOC_Os03g18140 gene.

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