Genetic engineering application of rice grain length gene osbip112

Abstract

The application belongs to the field of genetic engineering, and discloses cloning of a gene OsBIP112 involved in regulating rice grain length and application thereof in regulating rice grain length. The cDNA sequence of the gene is as shown in SEQ ID NO. 1. Overexpression of the rice gene OsBIP112 can reduce the plant height of rice, and is beneficial to genetic improvement of the grain type of rice. The grain length of a gene editing plant with a function loss of OsBIP112 is lengthened, and the number of tillers is increased. The OsBIP112 gene can be used for genetic improvement of yield traits of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the genetic engineering application of the rice grain length gene OsBIP112. Background Technology

[0002] Rice is one of the world's most important food crops, feeding nearly half the world's population. Its influence extends beyond agriculture, with rice cultivation covering the globe, from its native East and Southeast Asia to tropical and subtropical regions worldwide. Rice cultivation began approximately 10,000 years ago, and with the domestication of Asian rice (Oryza sativa), it became a cornerstone of ancient Asian civilizations. In China, rice paddies not only nurture abundant grains but also shaped a "rice-cultivating culture" lifestyle. Meanwhile, African rice (Oryza glaberrima) has developed another cultivation system adapted to arid conditions in the West African basin. With the explosive growth of the world's population, increasing rice production has become an urgent need for global food security.

[0003] Rice yield is primarily determined by three factors: number of panicles per unit area, number of grains per panicle, and thousand-grain weight. These three factors, influenced by both genetics and environment, shape the yield. Grain shape, as a crucial phenotypic characteristic of thousand-grain weight, is a key factor in yield optimization and an important goal in rice breeding. Rice grain shape traits mainly include grain length, width, thickness, and length-to-width ratio, and are quantitative traits controlled by different genetic systems, including the embryo, endosperm, and maternal plant. Rice grain shape also affects the appearance quality and commercial value of rice. In southern China, the United States, and most Asian countries, long or slender rice varieties are preferred, while in South Korea, Japan, and northern China, short, round rice varieties are more common (Harberd NP, 2015). Furthermore, grain shape is an easily selected phenotype in evolution and an important trait for studying rice evolution (Meyer RS, 2013). Therefore, cloning grain shape-related genes and conducting in-depth research and elucidation of the gene regulatory network for grain shape formation can provide an important theoretical foundation and genetic resources for high-yield and high-quality molecular breeding of rice.

[0004] With the development of rice functional genomics, recent studies have identified several quantitative trait loci (QTLs) and some genes as key grain size regulators (Fan and Li, 2019; Xing and Zhang, 2010). Among them, some QTLs regulating rice grain length were found, such as GS3 (GRAIN SIZE 3) (Fan et al., 2006), GS2 (GRAIN SIZE ON CHROMOSOME 2) (Hu et al., 2015), GL3.1 / qGL3 (GRAIN LENGTH 3) (Zhang et al., 2012), GLW7 (GRAIN LENGTH AND WIEIGHT ON CHROMOSOME 7) (Si et al., 2016), and An-1 (Luo et al., 2013); and QTLs regulating grain width, such as GW5 (GRAIN WIDTH 5) / qSW5 (SEEDWIDTH 5) (Wenge et al., 2008), GW2 (GRAIN WIDTH 2) (Song et al., 2007), GS5 (GRAIN SIZE 5) (Li et al., 2011), and GW7 (GRAIN WIDTH 5). 7)(Wang et al., 2015), GW8(GRAIN WIDTH 8))(Wang et al., 2012); QTLs regulating grain weight, such as GIF1(GRAIN INCOMPLETE FILLING 1))(Wang et al., 2008a), TGW6(THOUSAND-GRAIN WEIGHT 6))(Ishimaru et al., 2013), GE(GIANT EMBRYO))(Nagasawa et al., 2013), GW6a(Grain weight on chromosome 6a))(Songet et al., 2015; These regulators are involved in multiple signaling pathways, including the G protein signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, the ubiquitin-proteasome pathway, the plant hormone signaling pathway, and transcription regulators (Fan and Li, 2019; Xing and Zhang, 2010).

[0005] GS3, which encodes an atypical Gγ protein (Fan et al., 2006; Sun et al., 2018), was the first major-effect QTL identified by map-based cloning to regulate grain length and weight, with minimal effect on grain width and thickness (Fan and Li, 2019; Sun et al., 2018). Loss-of-function GS3 alleles significantly increase grain length but often lead to a decrease in the number of panicle branches and grains (Sun et al., 2018; Mao et al., 2010). DEP1 regulates panicle branching and grain number formation; its mutants typically exhibit a more compact panicle type and more grains, but with a smaller grain size (Qiao et al., 2011). These examples illustrate that, due to source-sink relationships, when rice grain size and number are limited by the allocation of carbohydrates and other nutrients, increasing the nutrient supply to individual grains (larger grain type) often reduces the total number of grains. In other words, an increase in grain size often leads to a decrease in the number of grains per panicle. As key agronomic traits, grain shape and grain number are contradictory, and this contradiction exists in most validated QTLs. However, at appropriate levels, the expression level of SMG11 (SMALL GRAIN 11) can improve grain shape, grain weight, and yield in rice without sacrificing grain number (Fang et al., 2016). SMG11 is an allele of DWARF2 (D2) that encodes cytochrome P450 (CYP), which is involved in the biosynthesis of brassinosteroids (BRs) (Fang et al., 2016). Brassinosteroids (BRs) are a class of sterol hormones widely present in plants, participating in the regulation of many important plant physiological activities, such as cell expansion, cell division, vegetative growth, reproduction, senescence, seed germination, and stress tolerance. They play a crucial role in plant growth and development processes, including seed germination, photomorphogenesis, reproductive development, and tropism. Over the past few decades, a series of genes encoding BR biosynthetic enzymes have been cloned in rice, such as BRD1 (Hong et al., 2002), BRD2 (Huang et al., 2022), D2 (Hong et al., 2003), and D11 (Sakamoto et al., 2005; Tanabe et al., 2005). These genes shorten rice grain length and affect rice yield by inhibiting cell expansion and reducing the synthesis of endogenous BRs.Different alleles of D11, namely CLUSTERED PRIMARY BRANCH 1 (Wue et al., 2016), GNS4 (Zhou et al., 2017), and PMM1 (Panicle Morphology Mutant 1) (Li et al., 2018), control rice grain size by influencing spikelet meristem differentiation and thus regulating grain number. In rice, loss of function of OsBRI1 and OsBAK1 results in a BR insensitivity phenotype and smaller grains (Morinaka et al., 2006). Overexpression of OsBZR1 increases rice grain length, width, and weight (Zhu et al., 2015). The BIN2 homolog OsGSK2 negatively regulates rice grain shape (Tong et al., 2012). In rice, OsGSK2 can phosphorylate the positive regulator OsDLT (Tong et al., 2012). qGL3 is a major-effect QTL controlling grain length, encoding a serine / threonine phosphatase (OsPPKL1) containing two Kelch domains. Rice also has two homologues of OsPPKL1, OsPPKL2 and OsPPKL3. OsPPKL1 and OsPPKL3 inhibit grain length, while OsPPKL2, as a homologue of Arabidopsis thaliana AtBSU1 and AtBSL1, promotes grain elongation.

[0006] Because the synthesis and function of BR are typically confined to the intracellular or pericellular space, this "autocrine" or "paracrine" characteristic allows for precise regulation of cellular behavior in individual tissues. Therefore, the pleiotropic nature of BR can act on specific genes or nodes in its signaling pathways to avoid the trade-off between grain size and number (Li et al., 2019). Investigating how OsPPKLs influence rice yield by regulating grain length through the BR signaling pathway while simultaneously avoiding the trade-off between grain size and number is of great significance. We used qGL3 / OsPPKL1 as bait proteins to screen a cDNA expression library of rice panicles and obtained a gene encoding a rice BR signaling receptor-binding protein, OsBIP112. qGL3 can interact with OsBIP112, and OsBIP112 plays an important role in rice grain development. Summary of the Invention

[0007] The purpose of this invention is to disclose the cloning of the rice grain length gene OsBIP112 and its genetic engineering application in rice grain length traits. By silencing the OsBIP112 gene in the rice genome through gene editing technology, rice grain length can be increased, thereby optimizing grain shape characteristics, increasing plant height, and increasing the number of tillers, which can be used for the genetic improvement of rice yield traits.

[0008] This invention uses qGL3 / OsPPKL1 as a bait protein to screen rice panicle cDNA expression libraries and obtains a gene OsBIP112 encoding a rice BR signal receptor binding protein.

[0009] The first object of the present invention is to provide a rice gene OsBIP112, wherein the nucleotide sequence of the OsBIP112 gene is selected from any one of (1) to (3):

[0010] (1) The cDNA sequence of the OsBIP112 gene shown in SEQ ID NO.1;

[0011] (2) The coding region sequence of the OsBIP112 gene shown in SEQ ID NO.2;

[0012] (3) A nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, with or without deletion and / or base mutation and / or vector sequence ligation / recombination.

[0013] A second objective of this invention is to provide the amino acid sequence encoded by the aforementioned rice gene OsBIP112 as shown in SEQ ID NO.3.

[0014] A third objective of this invention is to provide an overexpression vector containing the aforementioned rice gene OsBIP112.

[0015] Preferably, the overexpression vector contains the coding region sequence of the aforementioned OsBIP112 gene; the coding region of the rice gene OsBIP112 is located at 99bp to 1628bp of the sequence shown in SEQ ID NO.1.

[0016] More preferably, the overexpression vector is obtained by inserting the coding region sequence of the aforementioned OsBIP112 gene between the KpnⅠ and SalⅠ restriction sites of the plant binary expression vector pCAMBIA1300s.

[0017] A fourth objective of this invention is to provide a gene-editing vector for the aforementioned rice gene OsBIP112 in the wild-type genome of rice.

[0018] Preferably, the gene editing vector is obtained by inserting the editing target site of the rice OsBIP112 gene into the plant gene editing vector pHUE411.

[0019] More preferably, the editing target points are target point 1 shown in SEQ ID NO.8 and target point 2 shown in SEQ ID NO.9.

[0020] The gene editing vector is obtained by amplifying the target sequence of the rice OsBIP112 gene into the pCBC-MT1T2 plasmid, digesting the vector pHUE411 with restriction endonuclease, and inserting the sgRNA sequence into the vector pHUE411 using T4 DNA ligase.

[0021] The fifth objective of this invention is to provide the genetic engineering application of the aforementioned rice gene OsBIP112, or the aforementioned rice gene OsBIP112 overexpression vector, or the aforementioned OsBIP112 gene editing vector in regulating rice grain length and / or grain width and / or plant height and / or tiller number.

[0022] Furthermore, overexpression of the aforementioned rice gene OsBIP112 or the introduction of an overexpression vector of the aforementioned rice gene OsBIP112 into rice can reduce rice grain length and / or reduce rice grain width and / or reduce plant height and / or reduce tiller number.

[0023] Furthermore, silencing the aforementioned rice gene OsBIP112 or introducing the aforementioned gene-editing vector of OsBIP112 into rice can increase rice grain length and / or increase rice grain width and / or increase plant height and / or increase tiller number.

[0024] Furthermore, this includes the following steps:

[0025] (1) Total RNA was extracted from the rice variety “ZH11” and reverse transcribed to synthesize the first strand of cDNA;

[0026] (2) Cloning of rice gene OsBIP112: Using the first strand of cDNA synthesized by reverse transcription in step (1) as a template, primer pairs were used:

[0027] OsBIP112F: 5'-ATGGCCTCCGACGCCA-3' (SEQ ID NO.4),

[0028] OsBIP112R: 5'-TTACTTCTCGTCACCATCAGCA-3' (SEQ ID NO.5) was amplified by PCR, the cloned fragment was ligated into the pEasyBluntSimple vector, transformed into Escherichia coli strain DH5α, and sequenced to obtain the recombinant plasmid T-OsBIP112 containing the rice OsBIP112 gene cDNA sequence shown in SEQ ID NO.1;

[0029] (3) Construction of overexpression vector: Using the recombinant plasmid T-OsBIP112 obtained in step (2) as a template, primer pairs were used:

[0030] OsBIP112OEF: 5'-AGCTTTCGCGAGCTCGGTACCATGGCCTCCGACGCCA-3' (SEQ IDNO.6),

[0031] OsBIP112OER: 5'-TTGCATGCCTGCAGGTCGACTTACTTCTCGTCACCATCAGCA-3'

[0032] (SEQ ID NO.7) was subjected to PCR amplification to obtain the coding region of the rice OsBIP112 gene. The amplification product was inserted between the KpnⅠ and SalⅠ restriction sites of the plant binary expression vector pCAMBIA1300s to obtain the overexpression vector pCAMBIA1300s-OsBIP112.

[0033] (4) Obtaining transgenic plants: The obtained overexpression vector pCAMBIA1300s-OsBIP112 was transformed into Agrobacterium strain EHA105. The T-DNA region of the overexpression vector was integrated into the genome of the japonica rice variety Nipponbare using Agrobacterium-mediated rice genetic transformation, thereby obtaining transgenic rice with OsBIP112 overexpression.

[0034] Furthermore, this includes the following steps:

[0035] (1) Design sgRNA (one-way guide RNA) targets. The sequence of target 1 is: 5'-GACGGTCTTCCTCTCCCTGG-3' (SEQ ID NO.8), and the sequence of target 2 is: 5'-GGCGGGGGACTTCTTGGCGG-3' (SEQ ID NO.9). Ensure that it is unique in the target gene OsBIP112 and unique in the genome of rice variety “ZH11”.

[0036] (2) Primers were designed to amplify the sgRNA and rice promoter OsU3 in the pCBC-MT1T2 vector:

[0037] BIP112-MT1T2-F0: 5'-gGACGGTCTTCTCTCCTGGgttttagagctagaaatagc-3' (SEQ ID NO.10)

[0038] BIP112-MT1T2-R0: 5'-CCGCCAAGAAGTCCCCGCCCGCTTTCTTGGTGCC-3' (SEQ IDNO.11)

[0039] The pCBC-MT1T2 plasmid was amplified using the primers described above to obtain product 1.

[0040] BIP112-MT1T2-F:5'-aataatggtctcAGGCgGACGGTCTTCCTCTCCCTGG-3'(SEQIDNO.12)

[0041] BIP112-MT1T2-R: 5'-ATTATTGGTCTCTAAACCCGCCAAGAAGTCCCCCGCC-3'(SEQIDNO.13)

[0042] Using the primers described above, the sequence of product 1 obtained by amplification was added with homologous arms and restriction enzyme sites that could be ligated to the pHUE411 vector by PCR. The restriction endonuclease pHUE411 was digested with Bsa I. The sgRNA sequence was inserted into the vector pHUE411 by T4 DNA ligase to obtain the gene editing vector Crispr-OsBIP112.

[0043] Specifically, the pCBC-MT1T2 plasmid was amplified using the primers BIP112-MT1T2-F0 and BIP112-MT1T2-R0. The product was then amplified using primers BIP112-MT1T2-F and BIP112-MT1T2-R to obtain a combined sequence containing target sequence 1 + gRNA scaffold + OsU3 promoter + target sequence 2. The vector pHUE411 was digested with the restriction endonuclease BsaI, and the sgRNA sequence was inserted into the vector pHUE411 using T4 DNA ligase to obtain the gene editing vector Crispr-OsBIP112.

[0044] (3) Obtaining transgenic plants: The obtained gene editing vector CRISpr-OsBIP112 was transferred into Agrobacterium strain EHA105 by heat shock. Agrobacterium-mediated rice genetic transformation was used to target and edit the OsBIP112 gene in the genome of the japonica rice variety Zhonghua 11, thereby obtaining transgenic rice with OsBIP112 gene editing.

[0045] Beneficial effects

[0046] 1. This invention discloses a gene-engineered application of the rice OsBIP112 gene for grain shape. This gene is derived from rice (Oryza sativa L.), and gene editing to silence this gene can increase rice grain length, which is beneficial for the genetic improvement of rice grain shape.

[0047] 2. The OsBIP112 gene cloned in this invention provides a new resource for high-yield and high-quality rice breeding. Attached Figure Description

[0048] Figure 1 Construction of the recombinant vector pCAMBIA1300s-OsBIP112

[0049] A. Schematic diagram of the restriction enzyme sites of the overexpression vector pCAMBIA1300s;

[0050] Identification of B.OsBIP112 overexpression transgenic lines by qRT-PCR.

[0051] Figure 2 Construction of the OsBIP112 gene-edited line Cr-OsBIP112

[0052] A. Schematic diagram of the restriction sites of the gene editing vector pCAMBIA1300s;

[0053] Schematic diagram of different editing types of B.OsBIP112 gene-editing transgenic lines.

[0054] Figure 3 Plant phenotypes of OsBIP112 overexpression transgenic lines and gene-edited lines

[0055] A comparison of the plant types of A.OsBIP112 overexpressing transgenic lines and gene-edited lines with wild-type;

[0056] Statistical graph of spike length of B.OsBIP112 overexpressing transgenic lines and gene-edited lines;

[0057] Statistical graph of plant height of C.OsBIP112 overexpressing transgenic lines and gene-edited lines;

[0058] A statistical chart of tiller numbers in D.OsBIP112 overexpression transgenic lines and gene-edited lines;

[0059] Figure 4 Granular phenotype of OsBIP112 overexpression transgenic lines and gene-edited lines

[0060] Comparison of grain length between wild-type and A.OsBIP112 overexpression transgenic lines and gene-edited lines;

[0061] Comparison of grain width between B.OsBIP112 overexpression transgenic lines and gene-edited lines and wild type;

[0062] Particle length statistics of C.OsBIP112 overexpression transgenic lines and gene-edited lines;

[0063] Particle width statistics of D.OsBIP112 overexpression transgenic lines and gene-edited lines;

[0064] Grain thickness statistics of E.OsBIP112 overexpression transgenic lines and gene-edited lines. Detailed Implementation

[0065] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0066] Example 1

[0067] Rice variety “ZH11” was hydroponically grown in an artificial climate incubator (16h light / 8h darkness, 30℃ during the day and 26℃ at night) using the standard nutrient solution formula from the International Rice Research Institute. When seedlings reached the 3-4 leaf stage, they were flash-frozen in liquid nitrogen and stored at -80℃ for later use. Total RNA was extracted from the stored rice seedling samples using the Invitrogen Trizol method. The quality and concentration of total RNA were analyzed by 1% agarose gel electrophoresis. Total RNA meeting quality standards was further used for the synthesis of cDNA first strand. The synthesis of cDNA first strand was performed according to the Vazyme reverse transcription system manual.

[0068] Primers were designed: upstream OsBIP112F: 5'-ATGGCCTCCGACGCCA-3' (SEQ ID NO.4); downstream OsBIP112R: 5'-TTACTTCTCGTCACCATCAGCA-3' (SEQ ID NO.5). Using the first strand of cDNA synthesized by reverse transcription as a template, cDNA cloning was performed by PCR.

[0069] PCR amplification was performed using PrimeStar GXL DNA polymerase (Takara, Dalian, China). The PCR program was as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 45 s, 28 cycles, followed by 72℃ for 10 min. The PCR products were subjected to agarose gel electrophoresis, and the target fragment was recovered using a DNA gel recovery kit from Vazyme. The cloned fragment was ligated into the pEasy Blunt Simple vector and transformed into E. coli strain DH5α using a heat shock transformation method to obtain plasmid T-OsBIP112. Sequencing yielded the OsBIP112 cDNA sequence with a complete coding region, SEQ ID NO.1.

[0070] The full-length ORF of OsBIP112 is 1530 bp (SEQ ID NO.2). BioXM software (version 2.6) analysis showed that OsBIP112 encodes 509 amino acids (SEQ ID NO.3), and its isoelectric point pI = 6.14 and molecular weight MW = 56.27 kDa were estimated.

[0071] Example 2: Construction of overexpression vector and overexpression line

[0072] Based on the cDNA sequence of the rice OsBIP112 gene, primers were designed to amplify the complete coding reading frame. The upstream primer OsBIP112OEF: 5'-AGCTTTCGCGAGCTCGGTACCATGGCCTCCGACGCCA-3' (SEQ ID NO. 6) contains the KpnI restriction endonuclease site, and the downstream primer OsBIP112OER: 5'-TTGCATGCCTGCAGGTCGACTTACTTCTCGTCACCATCAGCA-3' (SEQ ID NO. 7) contains the SalI restriction endonuclease site. Using the correctly sequenced recombinant vector T-OsBIP112 obtained in Example 1 as a template, PCR amplification was performed. The resulting product was further cloned into the plant binary expression vector pCAMBIA1300s using the Vazyme CloneExpress kit. Figure 1 A) The overexpression vector pCAMBIA1300s-OsBIP112 was obtained.

[0073] The obtained recombinant expression vector pCAMBIA1300s-OsBIP112 was transformed into Agrobacterium strain EHA105 by heat shock. Using Agrobacterium-mediated rice genetic transformation, the T-DNA region of the recombinant vector was integrated into the genome of the japonica rice variety Zhonghua 11, thereby obtaining transgenic rice OE-OsBIP112-2 and OE-OsBIP112-9 with OsBIP112 overexpression.

[0074] After the wild-type “ZH11” and transgenic rice overexpressing OsBIP112 reached seedling stage, qRT-PCR was performed to identify each line. A small amount of transgenic rice leaves were taken, and total RNA was extracted using the Invitrogen Trizol method, then reverse-engineered into cDNA. OsBIP112 gene qRT-PCR specific primers were designed: qOsBIP112F: 5'-TTGGAGTCCCCTTGCATCAC-3' (SEQ ID NO.14) and qOsBIP112R: 5'-CCTCAGAATCAGCAGGACCC-3' (SEQ ID NO.15). Using rice actin rRNA expression as an internal control, qRT-PCR verification was performed on transgenic lines OE-OsBIP112-2 and OE-OsBIP112-3 according to the Roche Fast Start Universal SYBR Green Master (ROX) instructions. The validation results showed that the expression level of OsBIP112 in the two OsBIP112 overexpression lines, OE-OsBIP112-2 and OE-OsBIP112-9, was increased compared with the wild type "ZH11". Figure 1 B).

[0075] Example 3: Construction of gene editing vectors and gene editing lines

[0076] (1) Design sgRNA targets, with target 1 sequence being: 5'-GACGGTCTTCCTCTCCCTGG-3' (SEQ ID NO.8) and target 2 sequence being: 5'-GGCGGGGGACTTCTTGGCGG-3' (SEQ ID NO.9);

[0077] (2) Primers were designed to amplify the sgRNA and rice promoter OsU3 in the pCBC-MT1T2 vector:

[0078] BIP112-MT1T2-F0: 5'-gGACGGTCTTCTCTCCTGGgttttagagctagaaatagc-3' (SEQ ID NO.10)

[0079] BIP112-MT1T2-R0: 5'-CCGCCAAGAAGTCCCCGCCCGCTTTCTTGGTGCC-3' (SEQ IDNO.11)

[0080] BIP112-MT1T2-F:5'-aataatggtctcAGGCgGACGGTCTTCCTCTCCCTGG-3'(SEQIDNO.12)

[0081] BIP112-MT1T2-R: 5'-ATTATTGGTCTCTAAACCCGCCAAGAAGTCCCCCGCC-3'(SEQIDNO.13)

[0082] The pCBC-MT1T2 plasmid was amplified using primers BIP112-MT1T2-F0 and BIP112-MT1T2-R0. The resulting product was then amplified using primers BIP112-MT1T2-F0 and BIP112-MT1T2-R. Homologous arms and restriction enzyme sites were added to ligate the pHUE411 vector, resulting in a combined sequence containing target sequence 1 + gRNA scaffold + OsU3 promoter + target sequence 2. The pHUE411 vector was digested with the restriction endonuclease BsaI, and the sgRNA sequence was inserted into the pHUE411 vector using T4 DNA ligase, yielding the gene editing vector Crispr-OsBIP112. Figure 2 A).

[0083] Following the method described by Chen Qijun's team, gene-edited materials were constructed, and genomic DNA was extracted from T0 generation plants to verify the gene-editing type. Primers YZ-cr-BIP112-F were designed flanking the OsBIP112 gene target site.

[0084] 5'-AAGGAGGAGGCGAAGGC-3' (SEQ ID NO.16); YZ-cr-BIP112-R: 5'-GGTCAGAAACGAAGCAGCAA-3' (SEQ ID NO.17), amplified by PCR technology, and the target editing results were analyzed by sequencing. Figure 2 B).

[0085] Cas9-free validation was performed using universal primers OsU3-FD3: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' (SEQ ID NO.18); TaU3-RD: 5'-CTCACAAATTATCAGCACGCTAGTC-3' (SEQ ID NO.19) to prevent further gene editing in offspring. Homozygous offspring were then produced from different gene-edited Cr-OsBIP112 lines, Cr-OsBIP112-2 and Cr-OsBIP112-3, for further experimental research.

[0086] Example 4: Verification of traits in overexpression and silenced lines

[0087] This experiment was divided into two groups:

[0088] The first group of transgenic overexpression lines (OE-OsBIP112-2, OE-OsBIP112-9) and wild-type (ZH11) obtained in Example 2;

[0089] The second group consists of the gene-edited lines (Cr-OsBIP112-2, Cr-OsBIP112-3) and the wild type (ZH11) obtained in Example 3.

[0090] After soaking and allowing the seeds to sprout, each group of seeds was sown at the base for agronomic trait assessment during the mature plant stage. Each line was planted in three rows with a row spacing of 20cm, with 10 plants per row and a plant spacing of 15cm, and conventional soil and fertilizer management was applied. After grain filling, the plant height and number of tillers of the middle eight plants in each row of both transgenic and wild-type rice were measured and statistically analyzed.

[0091] After the seeds of the overexpression transgenic lines (OE-OsBIP112-2, OE-OsBIP112-9), gene-edited lines (Cr-OsBIP112-2, Cr-OsBIP112-3), and wild-type line (ZH11) matured, the panicle nodes of each group of rice were cut off, and the seeds were dried at 42℃ for 5 days before grain length analysis.

[0092] Statistical results show:

[0093] The panicle length of the overexpressing transgenic lines (OE-OsBIP112-2, OE-OsBIP112-9) was not significantly different from that of the wild-type ZH11, but the plant height was lower and the number of tillers was significantly less than that of the wild-type ZH11.

[0094] The panicle length of the gene-edited lines (Cr-OsBIP112-2 and Cr-OsBIP112-3) was not significantly different from that of the wild-type ZH11, but their plant height and the number of tillers were significantly higher. Figure 3 This indicates that loss of function of OsBIP112 can increase the number of tillers in rice, which is beneficial to the genetic improvement of rice yield.

[0095] The rice grains of the overexpressing transgenic lines (OE-OsBIP112-2, OE-OsBIP112-9) were significantly shorter and wider than those of the wild-type ZH11, while the grain thickness was not significantly different from that of the wild-type ZH11.

[0096] The rice grains of the gene-edited lines (Cr-OsBIP112-2 and Cr-OsBIP112-3) were significantly longer and wider than those of the wild-type ZH11, while the grain thickness was not significantly different. Figure 4This indicates that loss of function of OsBIP112 can increase rice grain length and width, which is beneficial to the genetic improvement of rice grain shape.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Rice gene OsBIP112, characterized in that, The nucleotide sequence of the OsBIP112 gene is selected from any one of (1) to (3): (1) The cDNA sequence of the OsBIP112 gene shown in SEQ ID NO.1; (2) The coding region sequence of the OsBIP112 gene shown in SEQ ID NO.2; (3) A nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, with or without deletion and / or base mutation and / or vector sequence ligation / recombination.

2. The amino acid sequence encoded by the rice gene OsBIP112 according to claim 1 is shown in SEQ ID NO.

3.

3. An overexpression vector containing the rice gene OsBIP112 as described in claim 1. Preferably, the overexpression vector contains the coding region sequence of the OsBIP112 gene as described in claim 1 (2); more preferably, the overexpression vector is obtained by inserting the coding region sequence of the OsBIP112 gene as described in claim 1 (2) between the KpnⅠ and SalⅠ restriction sites of the plant binary expression vector pCAMBIA1300s.

4. Gene editing vector for the rice gene OsBIP112 described in claim 1 in the wild-type genome of silent rice. Preferably, the gene editing vector is obtained by inserting the editing target site of the rice OsBIP112 gene into the plant gene editing vector pHUE411. More preferably, the editing target is target 1 shown in SEQ ID NO.8 and target 2 shown in SEQ ID NO.

9.

5. The application of the rice gene OsBIP112 of claim 1, the overexpression vector of the rice gene OsBIP112 of claim 3, or the gene editing vector of OsBIP112 of claim 4 in the genetic engineering of regulating rice grain length and / or grain width and / or plant height and / or tiller number.

6. The application according to claim 5, characterized in that, Overexpression of the rice gene OsBIP112 as described in claim 1 or the introduction of the overexpression vector of the rice gene OsBIP112 as described in claim 3 into rice can reduce rice grain length and / or reduce rice grain width and / or reduce plant height and / or reduce tiller number.

7. The application according to claim 5, characterized in that, Silencing the rice gene OsBIP112 of claim 1 or introducing the gene editing vector of OsBIP112 of claim 4 into rice can increase rice grain length and / or increase rice grain width and / or increase plant height and / or increase tiller number.

8. The application according to claim 5 or 6, characterized in that, Includes the following steps: (1) Total RNA was extracted from the rice variety "ZH11" and reverse transcribed to synthesize the first strand of cDNA; (2) Cloning of rice gene OsBIP112: Using the first strand of cDNA synthesized by reverse transcription in step (1) as a template, primer pairs were used: OsBIP112F: 5'-ATGGCCTCCGACGCCA-3' (SEQ ID NO.4), OsBIP112R: 5'-TTACTTCTCGTCACCATCAGCA-3' (SEQ ID NO.5) was amplified by PCR, the cloned fragment was ligated into the pEasyBluntSimple vector, transformed into Escherichia coli strain DH5α, and sequenced to obtain the recombinant plasmid T-OsBIP112 containing the rice OsBIP112 gene cDNA sequence shown in SEQ ID NO.1; (3) Construction of the overexpression vector: Using the recombinant plasmid T-OsBIP112 obtained in step (2) as a template, primer pairs were used: OsBIP112OEF: 5'-AGCTTTCGCGAGCTCGGTACCATGGCCTCCGACGCCA-3' (SEQ ID NO.6), OsBIP112OER: 5'-TTGCATGCCTGCAGGTCGACTTACTTCTCGTCACCATCAGCA-3' (SEQ ID NO.7) was subjected to PCR amplification to obtain the coding region of the rice OsBIP112 gene. The amplification product was inserted between the KpnⅠ and SalⅠ restriction sites of the plant binary expression vector pCAMBIA1300s to obtain the overexpression vector pCAMBIA1300s-OsBIP112. (4) Obtaining transgenic plants: The obtained overexpression vector pCAMBIA1300s-OsBIP112 was transformed into Agrobacterium strain EHA105. The T-DNA region of the overexpression vector was integrated into the genome of the japonica rice variety Nipponbare using Agrobacterium-mediated rice genetic transformation, thereby obtaining transgenic rice with OsBIP112 overexpression.

9. The application according to claim 5 or 7, characterized in that, Includes the following steps: (1) Design sgRNA targets, with target 1 sequence being: 5'-GACGGTCTTCCTCTCCCTGG-3' (SEQ ID NO.8) and target 2 sequence being: 5'-GGCGGGGGACTTCTTGGCGG-3' (SEQ ID NO.9); (2) Primers were designed to amplify the sgRNA and rice promoter OsU3 in the pCBC-MT1T2 vector: BIP112-MT1T2-F0: 5'-gGACGGTCTTCTCTCCTGGgttttagagctagaaatagc-3' (SEQ ID NO.10) BIP112-MT1T2-R0: 5'-CCGCCAAGAAGTCCCCGCCCGCTTTCTTGGTGCC-3' (SEQ ID NO.11) The pCBC-MT1T2 plasmid was amplified using the primers described above to obtain product 1. BIP112-MT1T2-F: 5'-aataatggtctcAGGCgGACGGTCTTCCTCTCCCTGG-3' (SEQ ID NO.12) BIP112-MT1T2-R: 5'-ATTATTGGTCTCTAAACCCGCCAAGAAGTCCCCGCC-3'(SEQID NO.13) Using the primers described above, the sequence of product 1 obtained by amplification was added with homologous arms and restriction enzyme sites that could be ligated to the pHUE411 vector by PCR. The restriction endonuclease pHUE411 was digested with Bsa I. The sgRNA sequence was inserted into the vector pHUE411 by T4 DNA ligase to obtain the gene editing vector Crispr-OsBIP112. (3) Obtaining transgenic plants: The obtained gene editing vector CRISpr-OsBIP112 was transferred into Agrobacterium strain EHA105 by heat shock. Agrobacterium-mediated rice genetic transformation was used to target and edit the OsBIP112 gene in the genome of the japonica rice variety Zhonghua 11, thereby obtaining transgenic rice with OsBIP112 gene editing.

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