WO2023029587A1 - 一种水稻理想脆秆突变体ibc的突变位点、控制基因IBC及其应用 - Google Patents

一种水稻理想脆秆突变体ibc的突变位点、控制基因IBC及其应用 Download PDF

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WO2023029587A1
WO2023029587A1 PCT/CN2022/092957 CN2022092957W WO2023029587A1 WO 2023029587 A1 WO2023029587 A1 WO 2023029587A1 CN 2022092957 W CN2022092957 W CN 2022092957W WO 2023029587 A1 WO2023029587 A1 WO 2023029587A1
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ibc
rice
gene
brittle
mutant
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French (fr)
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叶亚峰
刘斌美
张从合
吴跃进
陶亮之
严志
何立斌
申广勒
王慧
詹玥
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中国科学院合肥物质科学研究院
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Priority to US18/574,729 priority patent/US20240240265A1/en
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Definitions

  • the invention relates to the field of biotechnology, in particular to a mutation site of a rice ideal brittle stalk mutant IBC, a control gene IBC and an application thereof.
  • Rice (Oryza sativa L.) is one of the most important food crops in the world.
  • the mechanical strength of its stalk is one of the important agronomic traits, which is directly related to the lodging resistance of rice plants and ultimately affects the yield of rice.
  • rice also produces a large amount of straw every year, which can be used for livestock feed, papermaking raw materials, building materials, biomass energy and organic fertilizer raw materials, etc. Utilization is very difficult, and it is very necessary to break through the bottleneck problem of comprehensive utilization of rice straw from the composition and structure of rice straw itself.
  • the mechanical strength of rice stalks, as well as its biomass resistance to degradation barrier, are determined by the secondary cell wall, which is the cell wall layer that continues to accumulate inside the primary cell wall after specific cells stop growing. Secondary cell walls are mainly composed of cellulose, hemicellulose and lignin. Changes in each of these components will affect the structural changes in the secondary cell wall, thereby affecting the mechanical strength of the plant.
  • brittle culm mutants are important materials for studying the biosynthesis of rice secondary cell walls. In the reported brittle culm mutants, most of the reported brittle culm mutants ultimately affect the thickening of secondary cell walls due to the reduction of cellulose content.
  • OsCESA4, OsCESA7, and OsCESA9 encode the catalytic subunit of rice secondary cell wall cellulose synthase. Their mutations will lead to changes in the mechanical strength of the stalk, showing a brittle phenotype.
  • the 387th amino acid of OsCESA9 is composed of Aspartic acid is mutated into asparagine, showing a semi-dominant brittle stalk phenotype.
  • the patent with publication number CN110964733A also discloses a rice semi-dominant brittle stalk control gene SDBC1, but the rice stalk expressed by this gene And blades are very easy to break.
  • genes BC1 and BC12 that affect cellulose assembly and arrangement; gene BC3 that affects vesicle transport of cellulose synthase; gene BC10 that affects hemicellulose synthesis; and gene CEF1 that regulates secondary cell wall synthesis in rice.
  • the study of these genes not only provides a new theory for the synthesis of rice secondary cell wall, but also these mutant materials provide new gene resources for the efficient utilization of rice straw.
  • the ideal brittle stalk rice suitable for large-scale planting and production needs to have the following characteristics: 1. Excellent yield traits; 2. Strong lodging resistance; 3. The leaves are not brittle; 4. The stalk becomes brittle at the late maturity.
  • One of the technical problems to be solved by the present invention is that the leaves of rice brittle stalk mutants in the prior art are easy to break, which affects field operations.
  • a mutation site of rice ideal brittle stalk mutant ibc is an inversion of a chromosomal segment, and the position of the inversion of the chromosomal segment is located on the LOC_Os03g18140 gene.
  • the LOC_Os03g18140 gene is an IBC gene.
  • a control gene IBC of rice ideal brittle stalk mutant IBC the nucleotide sequence of said gene IBC, (1) as shown in SEQ ID No.1 or SEQ ID No.2; or (2) addition, replacement, Nucleotide sequences of mutants, alleles or derivatives produced by insertion or deletion of one or more nucleotides.
  • nucleotide sequence shown in SEQ ID No.2 can hybridize with the nucleotide sequence shown in SEQ ID No.2 under stringent conditions, and at the same time encode a nucleotide sequence that controls the mechanical strength of rice stems at the mature stage.
  • Stringent conditions refer to that the hybridization membrane is placed in pre-hybridization (0.25mol/L sodium phosphate buffer, pH7.2, 7% SDS), pre-hybridization at 65°C for 30 minutes; the pre-hybridization solution is discarded, and the hybridization solution (0.25mol /L sodium phosphate buffer, pH 7.2, 7% SDS, isotope-labeled nucleotide fragments), hybridized at 65°C for 16 hours; discard the hybridization solution, and add washing solution I (20mmol/L sodium phosphate buffer, pH 7. 2, 0.1% SDS), wash the membrane twice at 65°C, 10-15 minutes each time; add washing solution II (10mmol/L sodium phosphate buffer, pH7.2, 0.1%SDS), wash the membrane at 65°C for 10- 15 minutes.
  • the nucleotide sequence of SEQ ID No.1 relates to the promoter, coding region and regulatory region of gene IBC.
  • the present invention irradiates the Chinese japonica rice variety Wuyunjing No. 7 (WYJ7) with heavy ions to obtain an agronomic trait that does not affect yield, such as plant height, tiller number, grain number per panicle, thousand-grain weight, and lodging resistance.
  • WYJ7 Chinese japonica rice variety Wuyunjing No. 7
  • the present invention constructs a genetic analysis population of the ibc mutant and analyzes its genetic behavior, and finds that the brittle stalk phenotype of the ibc mutant is recessively controlled by a mutated IBC single gene.
  • the present invention isolates and identifies a gene IBC that controls the mechanical strength of rice stalks through the method of map-based cloning, and through phenotypic analysis of materials and genetic complementation experiments, it is confirmed that the IBC gene plays an important role in the mechanical strength of rice stalks and cell wall components. Regulatory functions, and using this gene to successfully breed the brittle-stalk rice variety "Kefujing 7" suitable for large-scale production, truly solve the problem of rice straw treatment from the source of the variety.
  • IBC encodes xylan acetyltransferase, which plays an important role in the modification of secondary cell wall hemicellulose polysaccharides.
  • allelic variation of IBC and the loss-of-function mutants of gene editing the stalks showed a brittle phenotype at the late maturity stage, the leaves were not brittle, the yield traits were good, and other agronomic traits did not change significantly.
  • the straws were easily crushed when harvested in the field , which is beneficial to crushing and returning to the field, and it is easy to crush silage in the feed of straw, and it is easy for animals to chew.
  • Gene IBC will provide theoretical basis, material and genetic support for elucidating the genetic basis of rice secondary cell wall synthesis regulation at the molecular level and for breeding environmentally friendly new varieties of rice based on molecular design.
  • a control gene IBC coded protein of rice ideal brittle stalk mutant ibc the amino acid sequence of said gene IBC coded protein, (1) as shown in SEQ ID No.3; or (2) due to one or more (such as 1 -25, 1-20, 1-15, 1-10, 1-5, 1-3) substitution, deletion and/or insertion of amino acid residues and shown in SEQ ID No.3 Amino acid sequences with different amino acid sequences; or (3) at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% or 98% or 99% identical to the amino acid sequence shown in SEQ ID No.3 or (4) an active fragment of the amino acid sequence described in (1) or (2) or (3).
  • a kind of recombination construct, described recombination construct contains the nucleotide sequence of rice stalk mechanical strength control gene IBC at mature stage, the vector used in the recombination construct is a cloning vector or is used to express the expression of the nucleotide carrier.
  • a recombinant host cell including the host cell of the above-mentioned recombinant construct, or the polynucleotide sequence of the rice stalk mechanical strength control gene IBC of the present invention integrated in its genome.
  • the host cells may be selected from plant cells or microbial cells, such as Escherichia coli cells or Agrobacterium cells, preferably plant cells, most preferably rice cells.
  • the cells may be isolated, ex vivo, cultured or part of a plant.
  • the present invention discloses the use of ibc mutants and rice brittle stalk varieties obtained through ibc mutation sites, and the use of various means, including the above-mentioned physical mutagenesis, chemical mutagenesis, biological mutagenesis and gene editing technology, to make the IBC gene function
  • various means including the above-mentioned physical mutagenesis, chemical mutagenesis, biological mutagenesis and gene editing technology, to make the IBC gene function
  • the application of rice brittle stalk varieties obtained by deletion mutations in straw treatment including feed and fertilizer products with brittle straw as raw materials.
  • the host cell is a microbial cell.
  • the microbial cells are Escherichia coli cells or Agrobacterium cells.
  • a method for cultivating rice stalks to become brittle comprising the following steps: utilizing mutagenesis means, said mutagenesis means including physical mutagenesis, chemical mutagenesis and biological mutagenesis, or making the above-mentioned rice stalks ideally brittle by gene editing technology
  • mutagenesis means including physical mutagenesis, chemical mutagenesis and biological mutagenesis
  • a rice plant with loss of function of the control gene IBC of the mutant ibc wherein the obtained rice plant exhibits a brittle culm phenotype.
  • a method for cultivating rice stalks to become brittle comprising the following steps: crossing the rice ideal brittle stalk mutant ibc with the above-mentioned mutation site with other rice varieties, and obtaining rice plants with brittle stalk phenotypes through offspring segregation.
  • the present invention discloses the use of ibc mutants and rice brittle stalk varieties obtained through ibc mutation sites, and the use of various means, including the above-mentioned physical mutagenesis, chemical mutagenesis, biological mutagenesis and gene editing technology, to make the IBC gene function
  • various means including the above-mentioned physical mutagenesis, chemical mutagenesis, biological mutagenesis and gene editing technology, to make the IBC gene function
  • the application of rice brittle stalk varieties obtained by deletion mutations in straw treatment including feed and fertilizer products with brittle straw as raw materials.
  • the brittle stalk rice or brittle straw of the present invention is used as a raw material, which is beneficial to chewing and digestion when used in feed, and easy to degrade in the field when used in fertilizer.
  • the second technical problem to be solved by the present invention is to provide a method for identifying the IBC mutation site of the rice stalk mechanical strength control gene at maturity.
  • a method for identifying the above-mentioned rice ideal brittle stalk mutant IBC mutation site comprising the following steps:
  • nucleotide sequence of described ibc-jd-1 forward primer ibc-jd-1-F is as SEQ ID No.4
  • nucleotide sequence of the ibc-jd-1 reverse primer ibc-jd-1-R is shown in SEQ ID No.5
  • the ibc-jd-2 forward primer ibc-jd The nucleotide sequence of -2-F is shown in SEQ ID No.6, and the nucleotide sequence of the ibc-jd-2 reverse primer ibc-jd-2-R is shown in SEQ ID No.7 ;
  • the amplified products of the two pairs of primers are detected by agarose gel electrophoresis. If only the ibc-jd-1 primer has the target band, the ibc mutation site is homozygous; if the result is only ibc- If the jd-2 primer has the target band, it does not contain the ibc mutation site and is wild type; if the test results show that both the ibc-jd-1 and ibc-jd-2 primers have the target band, the ibc mutation site is heterozygous. fit.
  • the advantage of the present invention is that: the present invention isolates and identifies a gene IBC that controls the mechanical strength of rice stalks through the method of map-based cloning, and confirms the IBC gene of the ideal brittle stalk phenotype through material phenotype analysis and genetic complementation experiments It has a function in the mechanical strength of rice stems and the regulation of cell wall components, and using this gene to successfully breed a brittle rice variety "Kefujing 7" suitable for large-scale production, truly solve the problem of rice straw treatment from the source of the variety.
  • IBC encodes xylan acetyltransferase, which plays an important role in the modification of secondary cell wall hemicellulose polysaccharides.
  • allelic variation of IBC and the loss-of-function mutants of gene editing the stalks showed a brittle phenotype at the late maturity stage, the leaves were not brittle, the yield traits were good, and other agronomic traits did not change significantly.
  • the straws were easily crushed when harvested in the field , It is beneficial to crushing and returning to the field. It is easy to crush silage in the feed of straw, and it is easy for animals to chew and digest.
  • Gene IBC provides theoretical basis, material and genetic support for elucidating the genetic basis of rice secondary cell wall synthesis regulation at the molecular level and for breeding environmentally friendly new varieties of rice based on molecular design.
  • Fig. 1 is the plant type comparative figure of wild-type WT and ibc mutant in the embodiment of the present invention 1;
  • Fig. 2 is a comparison diagram of stem breakage of wild-type WT and ibc mutants in Example 1 of the present invention
  • Fig. 3 is the blade breaking comparative figure of wild-type WT and ibc mutant in the embodiment 1 of the present invention
  • Figure 4 is a graph showing the results of determination of stem bending resistance of wild-type WT and ibc mutants in Example 1 of the present invention.
  • Fig. 5 is the leaf bending resistance measurement result figure of wild-type WT and ibc mutant in the embodiment 1 of the present invention
  • Fig. 6 is the plant height determination result figure of wild-type WT and ibc mutant in the embodiment 1 of the present invention.
  • Fig. 7 is the result figure of the tiller number measurement of wild-type WT and ibc mutant in the embodiment 1 of the present invention.
  • Fig. 8 is the result figure of the ear grain number measurement of wild-type WT and ibc mutant in the embodiment 1 of the present invention.
  • Figure 9 is a graph showing the results of ear length determination of wild-type WT and ibc mutants in Example 1 of the present invention.
  • Figure 10 is a graph showing the results of determination of seed setting rate of wild-type WT and ibc mutants in Example 1 of the present invention.
  • Fig. 11 is the thousand-grain weight determination result figure of wild-type WT and ibc mutant in the embodiment 1 of the present invention.
  • Fig. 12 is an electron micrograph of the cross-section of the second culm of the wild-type WT and the ibc mutant in Example 1 of the present invention.
  • Figure 13 is a map of IBC gene mapping in Example 2 of the present invention.
  • Fig. 14 is the IBC gene MutMap analysis figure in the embodiment of the present invention 2;
  • Figure 15 is a schematic diagram of the structure of the ibc mutation site in Example 2 of the present invention.
  • Fig. 16 is a figure showing the identification results of ibc mutation sites in Example 2 of the present invention.
  • Figure 17 is a structural diagram of the pIBCF expression vector in Example 2 of the present invention.
  • Figure 18 is the phenotype of genetically complemented plants in Example 2 of the present invention.
  • Fig. 19 is the sequencing map of IBC gene edited by CRISPR/Cas9 in Example 3 of the present invention.
  • Figure 20 is a diagram of the brittle stem phenotype of CRISPR/Cas9 gene-edited plants in Example 3 of the present invention.
  • Figure 21 is a plant type diagram of CRISPR/Cas9 gene edited plants in Example 3 of the present invention.
  • Figure 22 is the crushed stalks after harvesting wild-type WT and ibc mutant materials in Example 4 of the present invention.
  • Fig. 23 is a comparison chart of the crushed lengths of wild-type WT and ibc mutant materials in Example 4 of the present invention.
  • test materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
  • the ibc mutant was obtained from the japonica rice variety Wuyunjing 7 (WYJ7) by heavy ion 12 C 6+ mutagenesis (energy 80 MeV, dose 120 Gy). There was no significant difference between the growth period and the wild type; in the late filling stage, the stem phenotype was brittle, and there was no significant difference in leaves and other agronomic traits, such as the number of grains per ear, ear length, seed setting rate, and thousand-grain weight, etc., as shown in Figure 1- Figure 11 shows.
  • a backcross population was constructed by crossing the ibc mutant with the wild type Wuyunjing 7.
  • 112 plants had brittle stem phenotype, and 368 plants had normal stem.
  • the ibc mutant was crossed with the indica rice variety Huajingxian 74.
  • the ibc mutant was crossed with indica 93-11, Huajing indica 74, Nanjing 11 and other varieties, and the F1 generations of different hybrid combinations obtained by selfing were obtained to obtain seeds of segregated populations. These seeds were planted in the field and selected at the late stage of filling The individual plants with stalks showing brittle stems were used as positioning individual plants. About 100 mg of leaves were taken from each plant for DNA extraction.
  • the reported SSR primers evenly distributed on 12 rice chromosomes were used to screen the polymorphisms of the ibc mutant and Huajaponica 74, and the polymorphic SSR primers were obtained for the next experiment.
  • the primer sequences used for IBC gene mapping are shown in Table 1
  • the number of mapping populations was expanded, for larger Linkage analysis is performed on the mapping population, as shown in Figure 13, Recombinants in the figure represent recombinants, and finally the IBC gene is finely mapped between the Indel markers FM2 and FM5, within a segment of about 3.40Mb, and with the molecular markers FM3 and FM5 FM4 co-segregates.
  • Table 1 is the primers used for IBC gene localization
  • the ibc mutant had an inversion of a large segment of chromosome in the above-mentioned positioning interval (the schematic diagram is shown in Figure 15), and the genes at both ends of the inversion were analyzed. It was found that one end of the breakpoint was located on the LOC_Os03g18140 gene, resulting in a loss-of-function mutation of the gene, and the other end of the breakpoint did not occur in the coding region of the gene, so LOC_Os03g18140 was focused on as a candidate gene for IBC.
  • the primer names are ibc-jd-1 (the ibc mutation site can amplify the band, but the wild type cannot amplify the band) and ibc-jd-2 (The wild type can amplify the band, but the ibc mutation site cannot amplify the band).
  • the identification results show that the ibc mutant can only amplify the band with the ibc-jd-1 primer, while the wild type can only amplify the band with the ibc
  • the -jd-2 primer amplifies the band ( Figure 16).
  • LOC_Os03g18140 is the IBC gene.
  • the wild-type inverted internode was used as the material, RNA was extracted and reverse transcription amplified to obtain cDNA.
  • the structure diagram of the pIBCF expression vector is shown in Figure 17.
  • the pIBCF expression vector is transferred into the ibc mutant, and the stems of the positive transgenic T0 generation plants are obtained.
  • the culms all returned to normal levels, as shown in Figure 18. This result proves that the LOC_Os03g18140 gene is the IBC gene.
  • the primers used to construct the expression vector pIBCF are as follows:
  • pIBC-R 5'-cgggtaccCTCCGGAGCGCCCAGGAAGG-3'
  • IBC-CDS-F 5'-cgggtaccATGCAGCAGCGGCGGAAGTC-3'
  • IBC-CDS-R 5'-cgggatccCTACTGGTCGGATGACCATG-3'
  • the construction method of expression vector pIBCF comprises the following steps:
  • ibc mutants to hybridize, backcross and self-cross with rice varieties with normal stalks, such as 93-11, Huajingxian 74, Daohuaxiang 2, etc., and use identification primers ibc-jd-1 and ibc-jd-2 was selected for the ibc mutation site and genetic background, and finally 93-11, Huajingxian 74, Daohuaxiang 2 and other backgrounds with brittle stalks with homozygous ibc mutation genes were obtained.
  • the specific implementation steps are as follows:
  • F1 as the female parent and the recipient parent, such as 93-11, Huajingxian 74, Daohuaxiang 2, etc., and backcross to obtain BC1F1.
  • Plant BC1F1 use the identification primers ibc-jd-1 and ibc-jd-2 to detect the ibc genotype respectively, and select the ibc heterozygous genotype, that is, the PCR amplification products of the above two pairs of primers all have the target band.
  • step 4 Backcross the plants selected in step 4 with the recipient parents, such as 93-11, Huajingxian 74, Daohuaxiang 2, etc., to obtain BC2F1.
  • Plant BC2F1 repeat steps 3 and 4, select plants heterozygous for the ibc genotype, and have a high recovery rate of genetic background (eg greater than 95%), and harvest the inbred BC2F2.
  • Plant BC2F2 repeat steps 3 and 4, select the plants with the highest homozygous rate of ibc genotype and genetic background, and collect BC2F3.
  • the identification primers ibc-jd-1 and ibc-jd-2 showed that only ibc-jd-1 could amplify the target band, and the stalk phenotype was brittle stalk phenotype.
  • CRISPR/Cas9 vector The construction and transformation method of CRISPR/Cas9 vector is as follows:
  • the target primers were designed as follows:
  • IBC-CRISPR-U3 5'-CCTCTACAACGAGGACATCAAGT-3'
  • Wild-type (WT) and ibc mutant materials were planted on a large scale, and field harvesting was carried out by a combine harvester during the harvest period. As shown in Figure 22, the crushing degree of the ibc mutant straw after harvest was significantly better than that of the wild-type (WT). According to the statistics of the crushed straw lengths of a pair of materials, as shown in Figure 23, most of the crushed straw lengths of the ibc mutant are concentrated in the length ranges of less than 5 cm and 5-10 cm. This length is conducive to the degradation of the straw in the field in the later stage, as a raw material for organic fertilizer; it is also conducive to the chewing and digestion of cattle and sheep in the feed.
  • silage with brittle-stalked rice straws and feeding mutton sheep was carried out.
  • a sheep farm with more than 10 years of breeding experience and a scale of more than 1000 goats was selected to carry out the experimental work.
  • 5 tons of brittle-stalked rice straw and 5 tons of ordinary rice straw were collected, and they were treated according to the silage method of whole-plant corn, ensilaged in the form of large parcels, and silage of whole-plant corn was used as a control during the same period. After two months of silage, open the package and take samples for testing.
  • Table 2 shows the results of feeding experiments on brittle-stalk rice, common rice, and whole-plant corn after silage
  • the test cattle are required to have normal growth and development, medium fat condition, healthy and disease-free, and the growth rate is in a relatively fast period.
  • the age is about 4 months, and the weight range is 140-185 kg.
  • They were randomly divided into 4 groups, namely ordinary rice straw (group A), brittle rice straw (group B), silage brittle rice straw (group C) and silage whole plant corn straw (group D), with 10 heads in each group, divided into groups Feeding, feeding once in the morning and evening.
  • the limit of fine material is 40Kg per group per day, and ordinary rice straw and other test materials are not limited.
  • the feeding period was 8 weeks.
  • Weight gain effect The overall weight gain of ordinary rice straw (group A) was 737.5Kg, the overall weight gain of brittle rice straw (group B) was 790.5Kg, the overall weight gain of silage crisp rice straw (group C) was 825.5Kg and silage Whole-plant corn stalks (group D) were fed with a total weight gain of 841Kg. It shows that the silage of rice straw with brittle stalks is close to the effect of silage of whole-plant corn straw, and has the potential of alternative use.
  • Feed palatability The highest feed intake was silage crisp stalk rice straw (group C) 1460.6Kg, followed by silage whole plant corn straw (group D) 1429.4Kg, and the worst was common rice straw (group A). Feeding speed: silage brittle stalk rice straw>silage whole plant corn stalk>brittle stalk rice straw>common rice straw.

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Abstract

提供了一种水稻理想脆秆突变体ibc的突变位点、控制基因IBC及其应用，涉及生物技术领域，所述基因IBC的核苷酸序列，(1)如SEQ ID No.1或SEQ ID No.2所示；或(2)添加、取代，***或缺失一个或多个核苷酸而生成的突变体、等位基因或衍生物的核苷酸序列。还提供基因IBC的编码蛋白、重组构建体、重组宿主细胞以及利用基因IBC使水稻成熟期茎秆变脆的方法及应用。IBC的等位变异以及基因编辑的功能缺失突变体中，茎秆在成熟后期出现脆秆表型，叶片不脆，产量性状优良，其他农艺性状无明显变化，在收获期，田间收割秸秆易粉碎，有利于粉碎还田，秸秆的饲料化中易粉碎青贮、动物易咀嚼。

Description

一种水稻理想脆秆突变体ibc的突变位点、控制基因IBC及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种水稻理想脆秆突变体ibc的突变位点、控制基因IBC及其应用。
背景技术
水稻(Oryza sativa L.)是世界上最重要的粮食作物之一，其茎秆的机械强度是重要的农艺性状之一，直接关系到水稻植株的抗倒伏能力，从而最终影响水稻的产量。另一方面，水稻每年还产生大量的秸秆，可用于禽畜饲料、造纸原料、建筑材料、生物质能源以及有机肥原材料等，但由于秸秆生物质的抗降解屏障导致秸秆在还田以及其他综合利用上非常困难，从水稻秸秆自身成分和结构上突破水稻秸秆综合利用的瓶颈问题是十分必要的。
水稻茎秆的机械强度以及其生物质抗降解屏障都是由次生细胞壁所决定的，次生细胞壁是特定细胞在停止生长后，在初生细胞壁内侧继续积累的细胞壁层。次生细胞壁主要有纤维素，半纤维素和木质素组成。其中每一个成分的改变都会影响次生细胞壁的结构变化，从而影响植株的机械强度。在水稻中，脆秆突变体是研究水稻次生细胞壁生物合成的重要材料，在已报导的脆秆突变体中，大部分由于纤维素含量的降低最终影响到次生细胞壁的加厚。近些年来，国内外研究人员克隆了多个控制水稻次生细胞壁合成的关键基因。例如OsCESA4，OsCESA7，OsCESA9这3个基因编码水稻次生细胞壁纤维素合成酶催化亚基，他们的突变都会导致茎秆机械强度的改变，表现出脆秆表型，其中OsCESA9在氨基酸第387位由天冬氨酸突变成天冬酰胺，表现出半显性脆秆的表型，具体如公开号为CN110964733A的专利也公开一种水稻半显性脆秆控制基因SDBC1，但该基因表达的水稻茎秆和叶片都非常容易折断。除此之外，还有影响纤维素组装和排列的基因BC1和BC12；影响纤维素合成酶囊泡运输的基因BC3；影响半纤维素合成基因BC10；以及调控水稻次生细胞壁合成基因CEF1。这些基因的研究不仅为水稻次生细胞壁的合成提供新的理论，而且这些突变体材料为水稻秸秆高效利用提供了新的基因资源。
实现水稻秸秆的高效综合利用，除了改变水稻茎秆的组成成分以外，还需要兼顾其产量相关的性状，不能以牺牲水稻的产量来提高水稻秸秆的高效利用。虽然大部分水稻脆秆突变体的细胞壁成分的改变促使其秸秆高 效利用上具有非常高的潜力，但是在现实生产过程中，水稻脆秆突变体的种植面临着诸多问题。例如叶片易折断，影响田间操作；茎秆易倒伏，影响最终产量等。
寻找适合于大规模种植生产的理想脆秆水稻需要具备以下几个特点：1、产量性状优良；2、抗倒伏能力强；3、叶片不脆；4、茎秆在成熟后期变脆。
发明内容
本发明所要解决的技术问题之一在于现有技术中水稻脆秆突变体的叶片易折断，影响田间操作，提供一种叶片不易折断、无倒伏、水稻理想脆秆突变体ibc的突变位点、控制基因IBC、基因IBC编码蛋白、基因IBC重组构建体、基因IBC重组宿主细胞以及利用基因IBC使水稻成熟期茎秆变脆的方法、脆秆水稻或脆性秸秆作为原料在饲料或肥料中的应用。
本发明通过以下技术手段实现解决上述技术问题：
一种水稻理想脆秆突变体ibc的突变位点，所述突变位点为染色体片段倒位，所述染色体片段倒位的位置位于LOC_Os03g18140基因上。
LOC_Os03g18140基因为IBC基因。
一种水稻理想脆秆突变体ibc的控制基因IBC，所述基因IBC的核苷酸序列，(1)如SEQ ID No.1或SEQ ID No.2所示；或(2)添加、取代，***或缺失一个或多个核苷酸而生成的突变体、等位基因或衍生物的核苷酸序列。
或与SEQ ID No.2所示核苷酸序列能够在严谨条件下杂交，并同时编码具有控制水稻成熟期茎秆机械强度的核苷酸序列。
严谨条件是指，将杂交膜置于预杂交(0.25mol/L磷酸钠缓冲液，pH7.2，7％SDS)中，65℃预杂交30分钟；弃预杂交液，加入杂交液(0.25mol/L磷酸钠缓冲液，pH7.2，7％SDS，同位素标记的核苷酸片段)，65℃杂交16小时；弃杂交液，加入洗膜液Ⅰ(20mmol/L磷酸钠缓冲液，pH7.2，0.1％SDS)，65℃洗膜2次，每次10-15分钟；加入洗膜液Ⅱ(10mmol/L磷酸钠缓冲液，pH7.2，0.1％SDS)，65℃洗膜10-15分钟。
SEQ ID No.1核苷酸序列涉及基因IBC的启动子、编码区和调控区。
本发明利用重离子辐照中国粳稻品种武运粳7号(WYJ7)获得一个不影响产量相关的农艺性状，如株高、分蘖数、穗粒数、千粒重、抗倒伏等，在灌浆后期秸秆表现出脆秆表型的理想脆秆ibc( idea  brittle  culm)突变体。
本发明通过对ibc突变体进行遗传分析群体构建，对其进行遗传行为分析，发现ibc突变体的脆秆表型是有突变后的IBC单基因隐性控制。
有益效果：本发明通过图位克隆的方法分离鉴定出一个控制水稻茎秆机械强度的基因IBC，通过对材料的表型分析及遗传互补实验，证实了IBC 基因在水稻茎秆机械强度以及细胞壁成分调控方面的功能，并利用该基因成功培育适应于大规模生产的脆秆水稻品种“科辐粳7号”，真正从品种源头解决水稻秸秆处理的难题。
IBC编码木聚糖乙酰基转移酶，在次生细胞壁半纤维素多糖修饰上扮演重要角色。IBC的等位变异以及基因编辑的功能缺失突变体中，茎秆在成熟后期出现脆秆表型，叶片不脆，产量性状优良，其他农艺性状无明显变化，在收获期，田间收割秸秆易粉碎，有利于粉碎还田，秸秆的饲料化中易粉碎青贮、动物易咀嚼。
基因IBC为今后从分子水平阐明水稻次生细胞壁合成调控遗传基础，以及水稻基于分子设计的环境友好型新品种育种提供理论依据及材料和基因支持。
一种水稻理想脆秆突变体ibc的控制基因IBC编码蛋白，所述基因IBC编码蛋白的氨基酸序列，(1)如SEQ ID No.3所示；或(2)由于一或多个(例如1-25个、1-20个，1-15个，1-10个，1-5个，1-3个)氨基酸残基的替代、缺失和/或***而与SEQ ID No.3所示的氨基酸序列不同的氨基酸序列；或(3)与SEQ ID No.3所示的氨基酸序列具有至少70％、优选至少80％、更优选至少90％、尤其是至少95％或98％或99％同一性的氨基酸序列；或(4)(1)或(2)或(3)所述氨基酸序列的活性片段。
一种重组构建体，所述重组构建体含有水稻成熟期茎秆机械强度控制基因IBC的核苷酸序列，所述重组构建体所用的载体为克隆载体或用于表达所述核苷酸的表达载体。
一种重组宿主细胞，包括上述重组构建体的宿主细胞，或在其基因组中整合有本发明所述的控制水稻茎秆机械强度基因IBC的多核苷酸序列。所述宿主细胞可以选自植物细胞或者微生物细胞，例如大肠杆菌细胞或农杆菌细胞，优选植物细胞，最优选水稻细胞。所述细胞可以是分离的、离体的、培养的或者是植物的一部分。
本发明公开了利用ibc突变体和通过ibc突变位点获得的水稻脆秆品种，以及利用各种手段，包括上述的物理诱变、化学诱变和生物诱变以及基因编辑技术，使得IBC基因功能缺失突变而获得的水稻脆秆品种在秸秆处理上的应用，包括脆性秸秆作为原料的饲料及肥料产品。
优选地，所述宿主细胞为微生物细胞。
优选地，所述微生物细胞为大肠杆菌细胞或农杆菌细胞。
一种培育水稻秸秆变脆的方法，包括以下步骤：利用诱变的手段，所述的诱变手段包括物理诱变，化学诱变和生物诱变，或经基因编辑技术使得上述水稻理想脆秆突变体ibc的控制基因IBC功能缺失的水稻植株，其中所获得的水稻植株表现出脆秆表型。
一种培育水稻秸秆变脆的方法，包括以下步骤：将带有上述突变位点的水稻理想脆秆突变体ibc与其他水稻品种杂交，通过后代分离获得脆秆表 型的水稻植株。
本发明公开了利用ibc突变体和通过ibc突变位点获得的水稻脆秆品种，以及利用各种手段，包括上述的物理诱变、化学诱变和生物诱变以及基因编辑技术，使得IBC基因功能缺失突变而获得的水稻脆秆品种在秸秆处理上的应用，包括脆性秸秆作为原料的饲料及肥料产品。
一种采用上述方法获得的脆秆水稻或脆性秸秆作为原料在饲料或肥料中的应用。
有益效果：以本发明中的脆秆水稻或脆性秸秆作为原料，用于饲料有利于咀嚼和消化，用于肥料易于在田间降解。
本发明所要解决的技术问题之二在于提供一种鉴定水稻成熟期茎秆机械强度控制基因IBC突变位点的方法。
本发明通过以下技术手段实现解决上述技术问题：
一种鉴定上述水稻理想脆秆突变体ibc突变位点的方法，包括以下步骤：
(1)利用引物ibc-jd-1和ibc-jd-2进行PCR扩增，所述的ibc-jd-1正向引物ibc-jd-1-F的核苷酸序列如SEQ ID No.4所示，所述的ibc-jd-1反向向引物ibc-jd-1-R的核苷酸序列如SEQ ID No.5所示；所述的ibc-jd-2正向引物ibc-jd-2-F的核苷酸序列如SEQ ID No.6所示，所述的ibc-jd-2反向向引物ibc-jd-2-R的核苷酸序列如SEQ ID No.7所示；
(2)两对引物的扩增产物进行琼脂糖胶电泳检测，检测显示结果若只有ibc-jd-1引物有目的条带，则ibc突变位点为纯合型；检测显示结果若只有ibc-jd-2引物有目的条带，则不含有ibc突变位点，为野生型；检测显示结果若ibc-jd-1和ibc-jd-2引物均有目的条带，则ibc突变位点为杂合型。
有益效果：依据ibc独特的突变类型设计PCR扩增的特异性引物，利用该引物对待鉴定的水稻DNA进行PCR扩增，通过琼脂糖电泳的检测可以清楚地区分纯合、杂合以及不含有ibc突变位点的水稻材料，此方法可以用于利用ibc突变位点培育水稻脆秆新品种的跟踪鉴定。
本发明的优点在于：本发明通过图位克隆的方法分离鉴定出一个控制水稻茎秆机械强度的基因IBC，通过对材料的表型分析及遗传互补实验，证实了理想脆秆表型的IBC基因在水稻茎秆机械强度以及细胞壁成分调控方面的功能，并利用该基因成功培育适应于大规模生产的脆秆水稻品种“科辐粳7号”，真正从品种源头解决水稻秸秆处理的难题。
IBC编码木聚糖乙酰基转移酶，在次生细胞壁半纤维素多糖修饰上扮演重要角色。IBC的等位变异以及基因编辑的功能缺失突变体中，茎秆在成熟后期出现脆秆表型，叶片不脆，产量性状优良，其他农艺性状无明显变化，在收获期，田间收割秸秆易粉碎，有利于粉碎还田，秸秆的饲料化中易粉碎青贮、动物易咀嚼、消化。
基因IBC为今后从分子水平阐明水稻次生细胞壁合成调控遗传基础， 以及水稻基于分子设计的环境友好型新品种育种提供理论依据及材料和基因支持。
依据ibc独特的突变类型设计PCR扩增的特异性引物，利用该引物对待鉴定的水稻DNA进行PCR扩增，通过琼脂糖电泳的检测可以清楚地区分纯合、杂合以及不含有ibc突变位点的水稻材料，此方法可以用于利用ibc突变位点培育水稻脆秆新品种的跟踪鉴定。
附图说明
图1为本发明实施例1中野生型WT和ibc突变体的株型比较图；
图2为本发明实施例1中野生型WT和ibc突变体的茎秆折断比较图；
图3为本发明实施例1中野生型WT和ibc突变体的叶片折断比较图；
图4为本发明实施例1中野生型WT和ibc突变体的茎秆抗折力测定结果图；
图5为本发明实施例1中野生型WT和ibc突变体的叶片抗折力测定结果图；
图6为本发明实施例1中野生型WT和ibc突变体的株高测定结果图；
图7为本发明实施例1中野生型WT和ibc突变体的分蘖数测定结果图；
图8为本发明实施例1中野生型WT和ibc突变体的穗粒数测定结果图；
图9为本发明实施例1中野生型WT和ibc突变体的穗长测定结果图；
图10为本发明实施例1中野生型WT和ibc突变体的结实率测定结果图；
图11为本发明实施例1中野生型WT和ibc突变体的千粒重测定结果图；
图12为本发明实施例1中野生型WT和ibc突变体的倒二茎秆横切面的电镜图；
图13为本发明实施例2中IBC基因定位图；
图14为本发明实施例2中IBC基因MutMap分析图；
图15为本发明实施例2中ibc突变位点结构示意图；
图16为本发明实施例2中ibc突变位点鉴定结果图；
图17为本发明实施例2中pIBCF表达载体的结构图；
图18为本发明实施例2中遗传互补植株的表型；
图19为本发明实施例3中CRISPR/Cas9编辑IBC基因的测序图谱；
图20为本发明实施例3中CRISPR/Cas9基因编辑植株的脆秆表型图；
图21为本发明实施例3中CRISPR/Cas9基因编辑植株的株型图；
图22为本发明实施例4中野生型WT和ibc突变体材料收割后粉碎的秸秆；
图23为本发明实施例4中野生型WT和ibc突变体材料秸秆粉碎长度 比对图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下述实施例中所用的试验材料和试剂等，如无特殊说明，均可从商业途径获得。
实施例中未注明具体技术或条件者，均可以按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。
实施例1
理想脆秆突变体ibc的表型分析
(1)农艺性状分析
采用重离子 12C 6+诱变(能量80MeV，剂量120Gy)粳稻品种武运粳7号(WYJ7)获得ibc突变体，该突变体的表型特征为：在抽穗前期，株高、分蘖数以及生育期与野生型无明显差异；在灌浆后期，茎秆表型出脆秆表型，叶片以及其他农艺性状无明显差异，例如穗粒数、穗长、结实率、千粒重等，如图1-图11所示。
(2)ibc突变体的遗传分析
为研究ibc突变体在灌浆后期脆秆形成的分子机制，首先对其进行遗传分析。利用ibc突变体与野生型武运粳7号杂交构建回交群体，在480株F 2代的分离群体中，其中脆秆表型的植株有112株，茎秆正常的植株有368株，脆秆与正常茎秆的株数分离比符合1:3(χ 2[1:3]＝0.18<χ 20.05＝3.84；P>0.05)，利用ibc突变体与籼稻品种华粳籼74进行杂交，在600株的F 2代分离群体中，其中脆秆表型的植株有159株，茎秆正常的植株有441株，脆秆与正常茎秆的株数分离比符合1:3(χ 2[1:3]＝0.24<χ 20.05＝3.84；P>0.05)。上述结果表明ibc突变体的脆秆性状受一对单基因隐性控制，并且不受遗传背景的影响。
(3)茎秆横切面电镜观察
为进一步对ibc突变体脆秆形成的机制进行研究，通过扫描电镜对野生型和ibc突变体的倒二节间的横切面进行观察发现，ibc突变体的厚壁组织的细胞壁明显比野生型要薄(图12)，说明厚壁组织次生细胞壁变薄是导致ibc突变体脆性表型形成的原因。
实施例2
理想脆秆基因IBC的基因定位
(1)定位群体的构建
利用ibc突变体与籼稻93-11，华粳籼74，南京11等品种进行杂交，分别获得的不同杂交组合的F1代自交获得分离群体的种子，将这些种子进行田间种植，在灌浆后期选取茎秆表现出脆秆的单株作为定位单株。每个单株取100mg左右的叶片，用来提取DNA。
(2)简单重复序列(SSR，Simple Sequence Repeat)多态性筛选
利用已报导的均匀分布水稻12条染色体上的SSR引物对ibc突变体和华粳籼74进行多态性筛选，获得具有多态性的SSR引物进行下一步实验。
(3)IBC基因的定位
首先选用ibc突变体和华粳籼74构建的分离群体中脆秆表型单株中的随机21株进行IBC基因的初步定位。选用筛选获得的具有多态性的SSR引物对这21个单株进行连锁分析。结果发现在第3号染色体长臂端上的分子标记CSR7和CSR18与突变基因有明显的连锁，如图13所示，图中Recombinants表示重组子，进一步确定IBC基因位于CSR7与CSR18之间，该区间大约8.66Mb范围。
为进一步缩小IBC基因的定位区间，在CSR7与CSR18分子标记之间寻找更多的具有多态性的引物(IBC基因定位所用引物序列如表1所示)，并扩大定位群体数量，对更大的定位群体进行连锁分析，如图13所示，图中Recombinants表示重组子，最终将IBC基因精细定位在Indel标记FM2和FM5之间，大约3.40Mb的区段内，并且与其中分子标记FM3和FM4共分离。
表1 为IBC基因定位所用的引物
Figure PCTCN2022092957-appb-000001
(4)候选基因的获得与IBC基因的克隆
随着高通量的二代测序和读取片段更长的三代测序等技术的发展，使得对不同变异类型的突变体基因定位上有更多的选择性。利用传统的图位克隆技术不能将IBC基因继续缩小定位区间，可能由于其独特的变异类型所导致。我们考虑通过二代测序和三代测序对IBC基因进行定位，基于二代测序的MutMap定位结果与上述图位克隆结果一致(图14)。通过三代测序并结合二代测序结果对ibc突变体的基因组进行定点组装，结果发现ibc突变体在上述定位区间内存在染色体大片段的倒置(示意图如图15)，对倒置两端的基因进行分析，发现一端断点位置在LOC_Os03g18140基因上，造成该基因的功能缺失突变，另一端断点位置则没有发生在基因的编码区， 因此重点将LOC_Os03g18140作为IBC的候选基因。
(5)ibc突变位点的鉴定
根据ibc突变类型设计相应的鉴定引物进行PCR扩增，引物名称分别为ibc-jd-1(ibc突变位点能扩增出条带，野生型不能扩增出条带)和ibc-jd-2(野生型能扩增出条带，ibc突变位点不能扩增出条带)，鉴定结果显示ibc突变体只能用ibc-jd-1引物扩增出条带，而野生型只能用ibc-jd-2引物扩增出条带(图16)。
(6)IBC基因的功能互补验证
为了验证LOC_Os03g18140就是IBC基因，以野生型倒二节间为材料，提取RNA并反转录扩增获得cDNA。构建LOC_Os03g18140自身启动子(ATG上游3000bp片段)驱动的LOC_Os03g18140表达载体pIBCF，pIBCF表达载体的结构图如图17所示，将pIBCF表达载体转入ibc突变体中，获得阳性的转基因T0代植株的茎秆全部恢复到正常水平，如图18所示。这一结果证明LOC_Os03g18140基因就是IBC基因。
构建表达载体pIBCF所用的引物如下：
pIBC-F：5’-cggaattcTTCACTTTTGGGCATTGTTC-3’
pIBC-R：5’-cgggtaccCTCCGGAGCGCCCAGGAAGG-3’
IBC-CDS-F：5’-cgggtaccATGCAGCAGCGGCGGAAGTC-3’
IBC-CDS-R：5’-cgggatccCTACTGGTCGGATGACCATG-3’
表达载体pIBCF的构建方法包括以下步骤：
(1)利用pIBC-F和pIBC-R引物，以野生型武运粳7号的DNA为范本，扩增获得的PCR产物，通过EcoRI和KpnI限制性内切酶双酶切，同时利用该酶对pCAMBIA2300骨架进行双酶切，进而利用T4连接酶将扩增出的pIBC的片段***pCAMBIA2300载体中，获得中间载体pCAMBIA2300-pIBC。
(2)利用IBC-CDS-F和IBC-CDS-R引物，以野生型武运粳7号的cDNA为范本，扩增获得的PCR产物，通过KpnI和BamHI限制性内切酶双酶切，同时利用该酶对步骤(1)中获得的中间载体pCAMBIA2300-pIBC进行双酶切，进而利用T4连接酶将扩增出的IBC-CDS片段***pCAMBIA2300-pIBC载体中，获得最终载体pCAMBIA2300-pIBC::IBC，将该载体成为pIBCF，如图17所示。
实施例3
培育水稻理想脆秆品种的方法
(1)利用ibc突变位点培育新的脆秆品种
用ibc突变体与茎秆正常的水稻品种，如93-11，华粳籼74，稻花香2号等，进行杂交，回交和自交，并在此过程中用鉴定引物ibc-jd-1和ibc-jd-2进行ibc突变位点和遗传背景选择，最终获得93-11，华粳籼74，稻花香2号等背景下带有纯合ibc突变基因的脆秆新品种。具体实施步骤如下：
1、以受体亲本，如93-11，华粳籼74，稻花香2号等为父本与ibc突变体杂交获得F1。
2、以F1为母本与受体亲本，如93-11，华粳籼74，稻花香2号等，回交获得BC1F1。
3、种植BC1F1，分别使用鉴定引物ibc-jd-1和ibc-jd-2检测ibc基因型，选择ibc杂合基因型，即以上两对引物PCR扩增产物均有目的条带。
4、使用水稻12对染色体上分布均匀的(包括但不限于SSR、SNP、InDel、EST、RFLP、AFLP、RAPD、SCAR类型标记)，且在ibc突变体和轮回亲本之间存在多态性的分子标记，对步骤3中选出的单株进行遗传背景鉴定，选取与轮回亲本基因型相似度高(如大于75％)的植株。
5、用步骤4中选出的植株与受体亲本，如93-11，华粳籼74，稻花香2等，回交获得BC2F1。
6、种植BC2F1，重复步骤3和步骤4，选出ibc基因型杂合，遗传背景回复率高(如大于95％)的植株，收自交种BC2F2。
7、种植BC2F2，重复步骤3和步骤4，选出ibc基因型杂合，遗传背景纯合率最高的植株，收取BC2F3。BC2F3后代中分离的ibc纯合株，鉴定引物ibc-jd-1和ibc-jd-2检测结果只有ibc-jd-1能扩增出目的条带，且茎秆表型为脆秆表型。
(2)利用基因编辑技术培育新的脆秆品种
利用CRISPR/Cas9技术编辑IBC基因，创建IBC基因功能缺失的突变体，在IBC基因敲除的几个独立的纯合株系中，除茎秆后期变脆外(图20)，其他农艺性状无明显差异(图21)。
CRISPR/Cas9载体的构建与转化方法如下：
根据IBC基因的gDNA序列，设计靶点引物如下：
IBC-CRISPR-U3：5’-CCTCTACAACGAGGACATCAAGT-3’
具体构建方法参见华南农业大学刘耀光教授发表的文章(A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants.(2015)Molecular Plant,8(8):1274-1284)，采用农杆菌介导的转化法将该载体导入到WYJ7中(由申请人实验室完成水稻转化)。比较野生型和转基因水稻的表型分析发现，敲除IBC基因的几个纯合株系crispr-ibc(突变位点的测序峰图如图19)中，茎秆在后期都出现脆秆表型(图20)，其他农艺性状无明显差异(图21)。
实施例4
ibc脆秆水稻饲料化评价
(1)秸秆田间粉碎实验
大面积种植野生型(WT)和ibc突变体材料，收获期通过联合收割机进行田间收割，如图22所示，ibc突变体的秸秆在收割后的粉碎程度比野生型(WT)明显要好。根据对一对材料的秸秆粉碎长度进行统计，如图23 所示，ibc突变体粉碎后的秸秆长度大部分集中于小于5cm和5-10cm长度区间。这一长度利于秸秆后期在田间降解，作为有机肥的原材料；也有利于饲料化中牛羊的咀嚼和消化。
(2)山羊养殖
为了评价脆秆水稻的饲用价值，开展了脆秆水稻秸秆青贮以及饲喂肉羊的研究。选择了一个有10年以上养殖经验、规模在1000只山羊以上的羊场开展实验工作。首先收集了脆秆稻草和普通稻草各5吨，按照全株玉米的青贮方法进行处理，以大包裹形式青贮，同期全株玉米青贮作为对照。青贮两个月后开包取样检测。
结果表明青贮后的脆秆饲料外观呈黄绿色，酸香味十分明显，质地松软不粘手，具备优质青贮饲料特性。成分检测表明青贮脆秆可溶性糖和淀粉含量均高于普通稻草及全株玉米，显示出很好的营养价值。通过选取大小较为一致的36只羊进行打耳标编号，分成3个喂养处理，每个处理分3个栏，每栏4只羊，按照一定比例粗饲料搭配精粮的喂养方式，预试验饲喂2周，正式试验饲喂2个月，每隔20天进行一次称重。
测定结果如表2所示，结果表明：青贮脆秆饲料饲喂效果最好，整体增重率为48.6％，平均每只羊日增重为137克，比青贮玉米和普通秸秆分别提高17.1％、21.2％，显示出良好的应用潜力。
表2 为脆秆水稻、普通水稻、全株玉米青贮后饲养实验结果
Figure PCTCN2022092957-appb-000002
(3)肉牛养殖
40头荷斯坦肉牛：试验牛要求生长发育正常，膘情中等，健康无病，生长速度处于较快时期，年龄4月左右，体重范围140--185千克。随机分成4组，分别为普通水稻秸秆(A组)、脆秆水稻秸秆(B组)、青贮脆秆水稻秸秆(C组)和青贮全株玉米秸秆(D组)，每组10头，分组饲喂，早晚各投料一次。其中精料限量每日每组40Kg，普通水稻秸秆和其他试验料不限。饲喂时间为8周。
增重效果：普通水稻秸秆(A组)喂养总体增重737.5Kg、脆秆水稻秸秆(B组)喂养总体增重790.5Kg、青贮脆秆水稻秸秆(C组)喂养总体增重825.5Kg和青贮全株玉米秸秆(D组)喂养总体增重841Kg。表明青贮脆秆水稻秸秆与青贮全株玉米秸秆效果接近，具备替代使用的潜力。
饲料适口性：采食量最多为青贮脆秆水稻秸秆(C组)1460.6Kg，其次是青贮全株玉米秸秆(D组)1429.4Kg，最差是普通水稻秸秆(A组)。采 食速度：青贮脆秆水稻秸秆>青贮全株玉米秸秆>脆秆水稻秸秆>普通水稻秸秆。
以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (10)

  1. 一种水稻理想脆秆突变体ibc的突变位点，其特征在于：所述突变位点为染色体片段倒位，所述染色体片段倒位的位置位于LOC_Os03g18140基因上。
  2. 一种水稻理想脆秆突变体ibc的控制基因IBC，其特征在于：所述基因IBC的核苷酸序列，(1)如SEQ ID No.1或SEQ ID No.2所示；或(2)添加、取代，***或缺失一个或多个核苷酸而生成的突变体、等位基因或衍生物的核苷酸序列。
  3. 一种水稻理想脆秆突变体ibc的控制基因IBC编码蛋白，其特征在于：所述基因IBC编码蛋白的氨基酸序列如SEQ ID No.3所示。
  4. 一种重组构建体，其特征在于：所述重组构建体含有权利要求2所述水稻理想脆秆突变体ibc的控制基因IBC的核苷酸序列，所述重组构建体所用的载体为克隆载体或用于表达所述核苷酸的表达载体。
  5. 一种重组宿主细胞，其特征在于：包括权利要求4中重组构建体的宿主细胞，或在包含权利要求1所述的控制水稻茎秆机械强度基因IBC的核苷酸序列。
  6. 根据权利要求5所述的重组宿主细胞，其特征在于：所述宿主细胞为微生物细胞。
  7. 一种培育水稻秸秆变脆的方法，所述方法包括：利用诱变的手段，所述的诱变手段包括物理诱变，化学诱变和生物诱变，或经基因编辑技术使得如权利要求2所述的水稻理想脆秆突变体ibc的控制基因IBC功能缺失的水稻植株，其中所获得的水稻植株表现出脆秆表型。
  8. 一种培育水稻秸秆变脆的方法，其特征在于：将带有权利要求1所述的突变位点的水稻理想脆秆突变体ibc与其他水稻品种杂交，通过后代分离获得脆秆表型的水稻植株。
  9. 一种鉴定如权利要求1所述的水稻理想脆秆突变体ibc突变位点的方法，其特征在于：包括以下步骤：
    (1)利用引物ibc-jd-1和ibc-jd-2进行PCR扩增，所述的ibc-jd-1正向引物ibc-jd-1-F的核苷酸序列如SEQ ID No.4所示，所述的ibc-jd-1反向向引物ibc-jd-1-R的核苷酸序列如SEQ ID No.5所示；所述的ibc-jd-2正向引物ibc-jd-2-F的核苷酸序列如SEQ ID No.6所示，所述的ibc-jd-2反向向引物ibc-jd-2-R的核苷酸序列如SEQ ID No.7所示；
    (2)两对引物的扩增产物进行琼脂糖胶电泳检测，检测显示结果若只有ibc-jd-1引物有目的条带，则ibc突变位点为纯合型；检测显示结果若只有ibc-jd-2引物有目的条带，则不含有ibc突变位点，为野生型；检测显示结果若ibc-jd-1和ibc-jd-2引物均有目的条带，则ibc突变位点为杂合型。
  10. 一种如权利要求7或8所述方法获得的脆秆水稻或脆性秸秆作为原料在饲料或肥料中的应用。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862280B (zh) * 2021-08-31 2023-03-28 中国科学院合肥物质科学研究院 一种水稻理想脆秆突变体ibc的突变位点、控制基因IBC及其应用
CN115762641B (zh) * 2023-01-10 2023-04-07 天津极智基因科技有限公司 一种指纹图谱构建方法及***

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080313777A1 (en) * 2007-06-15 2008-12-18 Pioneer Hi-Bred International, Inc. Secondary Wall Forming Genes From Maize and Uses Thereof
CN110964733A (zh) * 2020-01-03 2020-04-07 中国科学院合肥物质科学研究院 一种水稻半显性脆秆控制基因、分子标记及应用
CN113862280A (zh) * 2021-08-31 2021-12-31 中国科学院合肥物质科学研究院 一种水稻理想脆秆突变体ibc的突变位点、控制基因IBC及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080313777A1 (en) * 2007-06-15 2008-12-18 Pioneer Hi-Bred International, Inc. Secondary Wall Forming Genes From Maize and Uses Thereof
CN110964733A (zh) * 2020-01-03 2020-04-07 中国科学院合肥物质科学研究院 一种水稻半显性脆秆控制基因、分子标记及应用
CN113862280A (zh) * 2021-08-31 2021-12-31 中国科学院合肥物质科学研究院 一种水稻理想脆秆突变体ibc的突变位点、控制基因IBC及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE Genbank 3 April 2018 (2018-04-03), ANONYMOUS: "Oryza sativa Japonica Group cultivar japonica xylan O-acetyltransferase 13 (XOAT13) mRNA, complete cds", XP055770973, Database accession no. MH037020.1 *
DATABASE Genbank 7 August 2018 (2018-08-07), ANONYMOUS : "PREDICTED: Oryza sativa Japonica Group protein trichome birefringence-like 28 (LOC4332508), mRNA", XP093041611, Database accession no. XM_015776685 *
HU WEI-CHEN, ZHANG TONG; HU ZHEN; WANG LING-QIANG: "Progress on cell wall biosynthesis and regulation in grasses", JIANGSU JOURNAL OF AGRICULTURAL SCIENCES, vol. 34, no. 2, 1 January 2018 (2018-01-01), pages 472 - 480, XP093041636, ISSN: 1000-4440, DOI: 10.3969/j.issn.1000-4440.2018.02.036 *
JIANG HONG-RUI, YE YA-FENG, HE DAN, REN YAN, YANG YANG, XIE JIAN, CHENG WEI-MIN, TAO LIANG-ZHI, ZHOU LI-BIN, WU YUE-JIN, LIU BIN-M: "Identification and gene localization of a novel rice brittle culm mutant bc17", ACTA AGRONOMICA SINICA, vol. 47, no. 1, 1 January 2021 (2021-01-01), pages 71 - 79, XP093041632, ISSN: 0496-3490, DOI: 10.3724/SP.J.1006.2021.02025 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116676284A (zh) * 2023-06-30 2023-09-01 安徽昇谷农业科技有限公司 一种调控作物矮化、抗倒、产量的基因及其应用
CN116676284B (zh) * 2023-06-30 2024-05-07 安徽昇谷农业科技有限公司 一种调控作物矮化、抗倒、产量的基因及其应用

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