Rice gene OsANK7 and its use

Abstract

The present invention provides a rice gene OsANK7 and its use. The present invention clones a new gene OsANK7 for controlling plant height in rice for the first time. After the gene mutates, the plant becomes shorter than the wild type, while the plant height of the gene OsANK7 overexpression strain increases significantly, indicating that OsANK7 has a positive regulatory effect on rice plant height. The present invention provides a new gene resource for in-depth understanding of the plant height regulation mechanism and rice plant type breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant gene engineering, in particular to a rice gene OsANK7 and a use thereof. Background Art

[0002] Rice is one of the main food crops in the world. Plant height, as one of the important agronomic traits of rice, affects rice yield by directly affecting the lodging resistance of the plant. Too high plant height and poor lodging resistance will lead to a decrease in rice yield; although too short rice plants have strong lodging resistance, their growth will be insufficient, which will also lead to a decrease in rice yield. The green revolution that emerged in the 1950s based on dwarfing breeding based on the semi-dwarf gene sd1 was an important leap in rice yield (Peng et al., 1999). However, the semi-dwarf gene gene resources currently used are still very narrow, mainly sd1 and its alleles. Therefore, exploring new dwarf or semi-dwarf gene resources is still an important direction in rice genetic breeding research and has important practical application value.

[0003] The measurement of rice plant height is carried out during the maturity period of rice, when the rice grains are full and the plant height has stabilized. When measuring, the height from the ground to the top of the main panicle is generally taken as the plant height, and the measured height does not include the awn of the grain. Gu Fulin et al. (2003) classified rice according to its plant height, and divided it into three types: tall, short and semi-dwarf. The dwarfism of rice refers to a genetic characteristic that the plant height is shorter than normal plants during the maturity period of rice, and does not necessarily refer to short stalks. Mutant lines whose plant height is equal to or less than half of their normal plant height during the maturity period are short stalks, and those with a plant height between normal height and short stalks are semi-dwarfs. Lu Yonggen (1979) believed that tall and semi-dwarf types can be distinguished based on whether they have dwarf genes, while semi-dwarf and dwarf types are distinguished by plant height. Lines with plant heights less than 50 cm are classified as dwarf, lines with plant heights between 50-70 cm can be classified as dwarf or semi-dwarf according to actual conditions, and lines with plant heights greater than 70 cm are classified as semi-dwarf. It is generally believed that the plant height of the semi-dwarf type is the most suitable and has been widely used. However, according to the current situation of rice breeding, in order to achieve high or even super-high yields, it is necessary to use appropriate methods to increase plant height while ensuring a certain yield and harvest index, thereby increasing rice yield. For example, the super rice varieties such as Yuyouhao, Xieyou and Liangyoupeijiu have high plant heights at maturity, and the highest Yuyouhao plant height even exceeds the height of the tall pole. This also shows that in the current rice breeding process, the requirement for plant height is no longer just to be short, but the plant height can also be appropriately increased while ensuring lodging resistance.

[0004] The location and cloning of plant height-related genes are the basis for in-depth research on their molecular mechanisms and genetic mechanisms. Through the study of plant height-related genes, a rice breeding system can be established. By studying, analyzing, and aggregating different plant height genes, a regulatory network of plant height genes can be established, which can effectively regulate plant height and lay the foundation for ideal plant type breeding. Rice plant height is mostly regulated by endogenous hormones, and growth and development are regulated by a variety of plant hormones such as gibberellin (GA), brassinolide (BR), strigolactone (SL), and auxin (IAA). The plant height-related genes cloned so far are also mostly related to hormone pathways (Table 1).

[0005] Table 1 Cloned plant height genes

[0006]

[0007] Note: Reference website https: / / archive.gramene.org /

[0008] However, only a few major plant height QTL loci have been isolated, such as the "Green Revolution" genes sd1 / OsGA20ox2 (PENG et al., 1999; SASAKI et al., 2002), OsGA20ox1 and IPA1 (OIKAWA et al., 2004; JIAO et al., 2010). On the other hand, pleiotropic genes such as Ghd7, Ghd7.1, Ghd8 and Ghd2 that control grain number, plant height and heading date have been successfully cloned (XUE et al., 2008; YAN et al., 2013; WEI et al., 2010; LIU et al., 2016). It has been recently reported that qSBM1 ​​can increase plant height and aboveground biomass, thereby increasing the number of grains per panicle, yield per plant and nitrogen utilization efficiency (XU et al., 2021). IPA1 changes the rice plant structure and increases rice yield by regulating OsmiR156 (SPRINGER, 2010).

[0009] In terms of plant height, the rice breeding project has gone through three stages: dwarf (about 70 cm), semi-dwarf (90-100 cm) and semi-tall (110 cm). When the plant height exceeds a certain range, it is easy to cause the rice stems to lodging, resulting in reduced yields, while too short a plant height will lead to insufficient rice growth, affecting rice yields. Typical dwarf varieties with a plant height of about 70 cm have low biological yields, and semi-dwarf is an important agricultural trait that can reduce lodging and increase the harvest index. In order to increase biological yield, Chinese breeders took the lead in proposing to use dwarf rice as the main breeding object, and first bred three semi-dwarf rice varieties: Dwarf Nante, Taichung Zaila No. 1 (TN1) and Guangchang Ai, which influenced the development trend of global rice breeding (Cheng Shihua, 2021). In 1959, Huang Yaoxiang and others used "Aizaizhan" as a dwarfing source and hybridized it with the tall variety "Guangchang 13" he cultivated, and successfully cultivated my country's first high-yield semi-dwarf new variety "Guangchang Ai", which pioneered rice dwarfing breeding and laid the foundation for the rise of the first green revolution. Influenced by this, in 1966, the International Rice Research Institute (IRRI) used the local variety of Taiwan Province, Dijiao Wujian (DGWG), and hybridized it with "Peta", and finally obtained a high-yield new rice variety IR8 carrying the semi-dwarf gene sd1, which was hailed as the "miracle rice" (PENG et al., 1999). The cultivation of "Square Dwarf" and the introduction of IR8 brought the first "Green Revolution" to my country's rice breeding work, solving the problem of rice fertilizer tolerance and the difficulty of enhancing lodging resistance, and making the rice harvest index in my country undergo a qualitative change for the first time (Gu Fulin et al., 2003). By 2002, the mutation mechanism of DGWG dwarfing was found to be caused by a 383-bp base deletion in SD1 (OsGA20ox2, a key enzyme in gibberellin synthesis) that led to plant dwarfing (SPIELMEYER et al., 2002), and it was named sd1 (AQUINO et al., 1966). So far, all semi-dwarf rice varieties are caused by a specific invalid mutation in the gibberellin (GA) biosynthesis gene sd1 (MONNA et al., 2003). al., 2002). For example, Guiyang Dwarf 1 (sdg) is regulated by a recessive dwarf gene that is not allelic to sdl (Gu Minghong et al., 1988). The semi-dwarf gene sdt2 that is not allelic to sd-1 (Zhao Xiangqiang et al., 2005); a new rice semi-dwarf gene sdt3 was isolated and identified from the multi-tillering dwarf stalks of indica rice (Sui Jiongming et al., 2006); the dominant semi-dwarf gene sdt97 of japonica rice (Tong et al., 2007). However, the promotion of dwarfing breeding controlled by the SD1 single gene will face the risk of reducing rice genetic diversity. At the same time, poor drought resistance and low photosynthesis efficiency are the defects of materials carrying the semi-dwarf gene sd1 (Huang Yaoxiang, 2001), making the discovery of new dwarfing genes particularly important.Subsequently, on the basis of dwarfing, the study of hybrid vigor and the selection of varieties were carried out, which promoted the rise and development of the "Second Green Revolution" and super rice breeding. In order to further cultivate high-yield and high-quality rice varieties under the appropriate plant height background, relevant data show (https: / / ricedata.cn / variety / ) that many varieties have plant heights exceeding 110cm, such as Zhunliangyou 527, Nei 2you 6, Guangliangyou 272, Yongyou 12, Yixiang 2292, Zhongyou 141, Ganwanxian 37 and Tyou 6135. my country's rice has developed from conventional high-stalked rice to dwarfed rice, and then to semi-dwarf hybrid rice, and is now developing towards semi-tall rice. In the future, tall and super-tall rice may appear. Under the premise of maintaining a harvest index of about 0.5, the biological yield increases with the increase of plant height (YUAN, 2012). Therefore, continuing to explore rice plant height genes is of great significance to rice breeding.

[0010] At present, most dwarfing genes have the genetic effect of "one cause with multiple effects", and their application value in breeding is low. Due to the large-scale use of the sd1 gene, the rice gene diversity has been lost, and the materials carrying the sd1 gene also have defects such as poor drought tolerance and poor photosynthetic efficiency. Therefore, it is necessary to continue to explore new dwarfing rice germplasm resources, as well as those gene resources that increase yield by increasing plant height under the premise of unchanged rice harvest index. Therefore, it is of great significance to explore, identify and utilize new germplasm resources. Technically, by using modern cloning technology, plant height genes are obtained, and the molecular mechanism of gene regulation of plant height formation is explored, so as to achieve effective combination of multiple plant height genes, and provide new gene resources for the selection and breeding of ideal rice plant types. In rice, the relevant research on the OsANK7 gene has not been reported so far. The isolation and cloning of the OsANK7 gene is of great significance to the improvement of rice plant type and the molecular biological mechanism of plant growth and development, and plays a vital role in clarifying the regulatory mechanism of plant growth and development. At the same time, applying this semi-dwarf gene to the improvement of rice varieties has important practical significance for breeders. Summary of the invention

[0011] The purpose of the present invention is to provide a rice gene OsANK7 and its application, especially its application in regulating rice plant height.

[0012] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a rice gene OsANK7, which is a gene encoding the following protein (a) or (b):

[0013] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3; or

[0014] (b) A protein derived from (a) in which one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO: 3 and which has the same function.

[0015] The OsANK7 gene (accession number LOC_Os07g33210 or Os07g0515900) is a new gene with unknown function in rice. The nucleotide sequence of the gene OsANK7 contains 8442 bases (SEQ ID NO: 1), the CDS is 1632bp long (SEQ ID NO: 2), and encodes a protein composed of 543 amino acids. OsANK7 is mainly expressed in roots and stems, and the protein is subcellularly localized in cytoplasmic vesicles and may also be associated with the endoplasmic reticulum.

[0016] In a second aspect, the present invention provides an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line containing the gene OsANK7.

[0017] In a third aspect, the present invention provides any of the following applications of gene OsANK7:

[0018] 1) Used to positively regulate plant height;

[0019] 2) Used for rice variety improvement;

[0020] 3) Used to prepare transgenic plants.

[0021] In a fourth aspect, the present invention provides the use of the gene OsANK7 in regulating the height of rice plants, wherein the regulation is positive regulation, that is, the expression of the gene is enhanced in rice, so that the plant becomes taller.

[0022] In a fifth aspect, the present invention provides a method for reducing the height of rice plants, wherein the rice gene OsANK7 is modified by genetic engineering means to render the gene function defective, thereby reducing the height of rice plants.

[0023] The method of gene modification can be selected from at least one of mutagenesis, site-directed mutagenesis, homologous recombination, etc.

[0024] Furthermore, the method includes: taking the rice gene OsANK7 as a target, designing a sgRNA sequence based on CRISPR / Cas9, connecting a DNA fragment containing the encoding sgRNA sequence to a vector carrying CRISPR / Cas9, transforming rice, and thereby obtaining transgenic rice with a loss of gene function.

[0025] Preferably, the nucleotide sequence of the sgRNA action site is 5'-CCGCTTCCCATCCATCTCCCGCG AGG -3'.

[0026] Furthermore, when the 58th to 59th bases of the 5'UTR region of the rice gene OsANK7 are deleted as shown in SEQ ID NO: 4, the function of the gene is lost.

[0027] In a sixth aspect, the present invention provides the use of the transgenic rice obtained according to the method in plant breeding.

[0028] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, selfing, or asexual reproduction.

[0029] The present invention cloned a new gene OsANK7 that controls plant height in rice for the first time. After the gene mutated, the plant became shorter than the wild type, while the plant height of the strain with overexpression of gene OsANK7 increased significantly, indicating that OsANK7 has a positive regulatory effect on rice plant height. The present invention provides a new gene resource for in-depth understanding of the plant height regulation mechanism and rice plant type breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The phenotypic identification results of knockout transgenic plants in the preferred embodiment of the present invention; wherein, (a) knockout site. (b) mutation type. (c) filling stage phenotype; scale: 20 cm. (d) plant height. ** indicates significant at the p<0.01 level, and the data are represented by mean ± standard error.

[0031] Figure 2 The phenotypes of wild-type Zhonghui 8015 (WT) and osank7 in the preferred embodiments of the present invention are shown in the figure below. (a) Seedling stage; scale bar: 1 cm. (b) 30 DAT; scale bar: 15 cm. (c) Mature stage; scale bar: 20 cm. (d) Stem and ear at mature stage; scale bar: 2 cm. (e) Ear; scale bar: 5 cm. (f) Grain width; scale bar: 7 mm. (g) Grain length; scale bar: 20 mm.

[0032] Figure 3 The results of the character investigation of the wild type Zhonghui 8015 (WT) and the knockout transgenic backcross family osank7 in the preferred embodiment of the present invention; wherein, (a) plant height. (b) sword leaf width. (c) sword leaf length. (d) heading date. (e) effective panicle number. (f) grain length. (g) grain width. (h) length-to-width ratio. (i) fruiting rate. The data were analyzed by t-test, * and ** indicate significance at p<0.05 and p<0.01 levels, respectively, and the data are represented by mean ± standard error.

[0033] Figure 4 The results of OsANK7 transcript analysis in a preferred embodiment of the present invention are as follows: (a) the location of the amplification primers of the transcript sequence on the genome; (b) an agarose gel electrophoresis diagram; and (c) a transcript structure diagram.

[0034] Figure 5 The results of OsANK7 transcript sequence analysis in a preferred embodiment of the present invention; wherein, LOC_Os07g33210.1 is XM_015791994.2, LOC_Os07g33210.2 is XM_026026851.1, LOC_Os07g33210.5 is XM_015791989.2 (https: / / www.ncbi.nlm.nih.gov / ); LOC_Os07g33210.4 is Os0 7t0515900-00(https: / / rapdb.dna.affrc.go.jp / ); LOC_Os07g33210.3 is OsR498G0714424710.01.T03(h ttp: / / www.mbkbase.org / R498 / ); LOC_Os07g33210.6 is BGIOSGA025801-TA (https: / / www.gramene.org / ).

[0035] Figure 6 The phenotypes of the overexpressed transgenic plants in the preferred embodiments of the present invention are shown in Figure 1. (a) OE-OsANK7.6 phenotype. (b) OE-OsANK7.3 phenotype. Scale bar: 20 cm. (c) Plant height. Lowercase letters in multiple comparisons indicate significance at the 0.05 level. Data are presented as mean ± standard error.

[0036] Figure 7 This is the tissue-specific expression of the gene OsANK7 in a preferred embodiment of the present invention.

[0037] Figure 8 The results of GUS tissue staining test in a preferred embodiment of the present invention; (ab) base stem nodes and internodes. (c) internodes below the ear. (d) root. (e) leaf. (f) leaf sheath. Scale bar: 1 cm.

[0038] Fig. 9 The subcellular localization of OsANK7 in the preferred embodiment of the present invention is shown in the figure below: (a) subcellular localization of OsANK7 in rice protoplast cells. (b) subcellular localization of OsANK7 in tobacco leaf cells. (c) colocalization of OsANK7 and SYB52. (d) colocalization of OsANK7 and endoplasmic reticulum. DETAILED DESCRIPTION

[0039] The OsANK7 gene (accession number is LOC_Os07g33210 or Os07g0515900) is a new gene in rice whose function has not been reported. After the wild type Zhonghui 8015 is transformed by constructing a CRISPR / Cas9 gene knockout vector, the transgenic plant becomes shorter, and after the wild type Zhonghui 8015 is transformed by a pCAMBIA2300-OsANK7 overexpression vector, the transgenic plant becomes taller, thereby verifying the function of the OsANK7 gene in regulating the height of rice plants, that is, the loss-of-function mutation causes the rice plants to become shorter, and overexpression of the gene can cause the rice plant height to become taller. The present invention provides a new gene resource for rice plant type breeding.

[0040] The present invention adopts the following technical solution:

[0041] 1. Clone a new gene that controls plant height: Clone a new gene that controls plant height in rice, OsANK7. After the mutant gene mutates, the plant becomes shorter than the wild type.

[0042] 2. Verify gene function by gene knockout: After constructing a CRISPR / Cas9 gene knockout vector and transforming the wild-type Zhonghui 8015, the transgenic plants became shorter.

[0043] 3. Verify gene function by overexpression: After the pCAMBIA2300-OsANK7 overexpression vector was used to transform the wild-type Zhonghui 8015 transgenic plants, they became taller.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0045] The primers used for PCR in the following examples are shown in Table 2, and the primers used for constructing the vector are shown in Table 3.

[0046] Table 2 Primers used for PCR

[0047]

[0048]

[0049] Table 3 Primers related to vector construction

[0050]

[0051] Example 1 Identification and analysis of gene knockout of OsANK7 and transgenic plants

[0052] In previous studies, the inventors cloned a dwarf premature senescence gene SUI2 (also known as PLS5), and screened a total of 30 possible interacting proteins through yeast two-hybrid experiments. After consulting the NCBI website (https: / / www.ncbi.nlm.nih.gov), it was found that the LOC_Os07g33210 gene has an ANK domain that may regulate plant growth and development, and may have the function of regulating plant height. Because it is located on chromosome 7 of rice, it is temporarily named OsANK7. In order to study the function of OsANK7, the CRISPR / Cas9 system was used to knock out the OsANK7 gene in the indica rice variety Zhonghui 8015. The specific process is as follows: Use the website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) to design and select a suitable target sequence (the nucleotide sequence of the sgRNA action site is 5'-CCGCTTCCCATCCATCTCCCGCG AGG -3'), and was successfully constructed into a CRISPR / Cas9 vector (BGK03, purchased from BioGene Technology (Jiangsu) Co., Ltd.) through homologous recombination, and introduced into Escherichia coli Trelief5α. The positive monoclonal bacterial solution and plasmid were sent to BioGene Technology (Jiangsu) Co., Ltd. for transformation. After sequencing the target site of the transgenic plants that tested positive for the T0 generation hygromycin gene, a homozygous mutant named osank7C was obtained. PCR amplification and sequencing were performed using primers 7994-F / R, and it was found that the mutation site of the knockout plant was located at the 5'UTR (the -69th and 70th bases before the start codon ATG, Figure 1 a) has a CC deletion ( Figure 1 b). The phenotype of the knockout transgenic plant osank7C was identified and it was found that the plant height of osank7C was reduced by about 50 cm compared with the wild type, and the reduction in plant height was extremely significant ( Figure 1 c, Figure 1 d).

[0053] Considering that the gene editing process undergoes tissue culture, other non-specific mutations may occur, resulting in reduced plant height. In the present invention, the T3 generation knockout transgenic plant osank7C was hybridized with Zhonghui 8015 and then self-pollinated to obtain the F2 generation, and the homozygous knockout genotype plant (named osank7) was screened out. Its plant height was significantly lower than that of the wild type at the seedling stage, before heading and after heading ( Figure 2 , ac). Subsequent studies were conducted on osank7 knockout mutants. First, the phenotype of osank7 in seedlings was significantly shorter than that of Zhonghui 8015, with shorter plant height and root system, and significantly shorter stems, panicles, grain length and width ( Figure 2, dg). Secondly, the agronomic traits of wild-type and knockout transgenic backcross plant osank7 were investigated. The data showed that compared with the wild type, the plant height of osank7 was about 22 cm lower. In addition, the sword leaf length, sword leaf width, grain length, grain width and fruit setting rate of osank7 plants were significantly reduced, the heading period was significantly advanced, the number of effective ears was significantly increased, and there was no significant difference in the grain length-width ratio. It can be concluded that compared with the wild type, in addition to the significant reduction in plant height, osank7 plants will have phenotypes such as shorter sword leaves, fewer tillers, earlier heading period, more effective ears, and reduced fruit setting rate ( Figure 3 , ai).

[0054] Example 2 Genetic Analysis and Transcript Analysis of Gene OsANK7

[0055] 1. Genetic analysis of OsANK7

[0056] The F1 offspring obtained by hybridization of osank7 and Zhonghui 8015 were similar to the wild type phenotype, and there was a segregation phenomenon between the F2 populations. A random selection of 342 strains from the F2 population was performed for trait segregation statistical analysis, and it was found that 75 of them had a similar phenotype to osank7, and the remaining 267 strains had a phenotype similar to the wild type. The chi-square test analysis showed that the statistical results were consistent with the segregation ratio of 3:1, that is, χ 2 =1.559<χ 2 0.05 =3.84 (Table 4), indicating that the dwarf phenotype of osank7 is controlled by a pair of recessive genes.

[0057] Table 4 Statistics of F2 population segregation

[0058]

[0059] 2. Transcript analysis

[0060] According to the predicted sequences of LOC_Os07g33210 gene published by bioinformatics websites such as Gramene, MSU, NCBI, and Rap-DB, it was found that OsANK7 has 6 possible transcripts, which are tentatively named LOC_Os07g33210.1 to LOC_Os07g33210.6. The corresponding primers were designed for these 6 transcripts to perform PCR amplification of their full-length CDS ( Figure 4a represents the corresponding primer sequence and position, respectively). After the PCR product was recovered, it was ligated to the pCAMBIA2300 vector by homologous recombination and transformed into Escherichia coli. Single clones were selected for sequencing and 6 different transcripts were obtained, which were named LOC_Os07g33210.1, LOC_Os07g33210.2, LOC_Os07g33210.3, LOC_Os07g33210.4, LOC_Os07g33210.5 and LOC_Os07g33210.6. Sequence analysis revealed that LOC_Os07g33210.2, LOC_Os07g33210.3 and LOC_Os07g33210.4 all lacked 14 bp of bases at bases 176-189, resulting in premature termination of translation at base 210. Comparison of LOC_Os07g33210.5 with the OsANK7 genome sequence revealed a mutation from base C to T at base 479, resulting in a change in amino acids. In comparison, the transcript of LOC_Os07g33210.1 was shorter, and the transcript of LOC_Os07g33210.6 was the longest ( Figure 4 c, Figure 5 In addition, semi-quantitative experiments found that under the same conditions, the band of LOC_Os07g33210.6-3′UTR (5′UTR-3′UTR) was the brightest ( Figure 4 b). Based on the above sequence comparison analysis and semi-quantitative results, it was inferred that LOC_Os07g33210.6 was likely to be a candidate transcript of OsANK7 regulating plant height (the six transcript amplification sequences LOC_Os07g33210.1, LOC_Os07g33210.2, LOC_Os07g33210.3, LOC_Os07g33210.4, LOC_Os07g33210.5 and LOC_Os07g33210.6 were shown in SEQ ID NOs: 5 to 10, respectively).

[0061] Example 3 Overexpression verification of the function of gene OsANK7

[0062] In order to further verify whether LOC_Os07g33210.6 is a candidate gene of OsANK7, the present invention further constructed an overexpression vector of LOC_Os07g33210.3 and LOC_Os07g33210.6 transcripts for functional verification. Specifically, the LOC_Os07g33210.3 and LOC_Os07g33210.6 CDS obtained by PCR amplification were connected into the overexpression vector pCAMBIA2300 (purchased from CAMBIA) using the Sma I restriction site to successfully construct pCAMBIA2300-LOC_Os07g33210.3 and pCAMBIA2300-LOC_Os07g3310.6 fusion vectors, which were then introduced into Escherichia coli Trelief5α and transformed into wild-type Zhonghui 8015 by Wuhan Aidijing Biotechnology Co., Ltd. PCR reaction was performed using cdsJC-F / R detection primers to identify transgenic plants. Overexpression transgenic positive plants were determined in the T0 generation. The overexpression transgenic plants from LOC_Os07g33210.3 and LOC_Os07g33210.6 were named OE 8015 -OsANK7.3 and OE 8015 -OsANK7.6.

[0063] For T1 generation OE 8015 -OsANK7.6 and OE 8015 -OsANK7.3 transgenic positive plants were identified and analyzed, and the plant height phenotype data were statistically analyzed. It was found that OE 8015 -OsANK7.6 plants were significantly taller than Zhonghui 8015, while OE 8015 -OsANK7.3 plants were significantly taller than OE plants 8015 -OsANK7.6, no significant difference from wild type phenotype ( Figure 6 , ac), indicating that overexpression of LOC_Os0Zg33210.6 caused the plant height to be higher than that of wild-type Zhonghui 8015, but LOC_Os07g33210.3 did not cause the change in plant height. The above experimental results confirmed that LOC_Os07g33210.6 is the candidate transcript of OsANK7 that positively regulates plant height.

[0064] Example 4 Analysis of the expression pattern and subcellular localization of gene OsANK7

[0065] The expression of OsANK7 in roots, stem nodes, internodes, leaves and leaf sheaths of wild type at the jointing stage was detected by qRT-PCR. The results showed that OsANK7 was expressed in all the above tissues, and its expression in stem nodes and roots was more dominant ( Figure 7). By constructing GUS transgenic positive plants, the above tissue-specific results can be further verified. GUS staining results showed that OsANK7 was specifically expressed in roots, stem nodes, internodes, leaves and leaf sheaths, with the highest expression in roots and stem nodes ( Figure 8 , af), which was consistent with the qRT-PCR results, indicating that OsANK7 may control rice plant height traits by affecting stem nodes and internodes.

[0066] By constructing a GFP-OsANK7 vector, transient expression experiments were performed in tobacco and Zhonghui 8015 protoplasts. The experimental results showed that the GFP-OsANK7 fusion protein was expressed in the cytoplasm of tobacco and Zhonghui 8015 protoplasts ( Fig. 9 , ab). In order to further determine the specific location of its expression, GFP-OsANK7 fusion protein was co-expressed with vesicle marker (SYB52-mCherry) in Zhonghui 8015 protoplasts. The results showed that most of the fluorescence signals overlapped. When co-expressed with endoplasmic reticulum marker (mCherry-HDEL), the fluorescence signal of GFP-OsANK7 fusion protein partially overlapped with the fluorescence signal of endoplasmic reticulum marker, indicating that OsANK7 protein is likely to be mainly expressed in cytoplasmic vesicles and may also be related to the endoplasmic reticulum ( Fig. 9 , cd).

[0067] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

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China National Rice Research Institute

Claims

1. Application of rice gene OsANK7 and its biomaterials in positively regulating rice plant height, including: The amino acid sequence of the protein (a) encoded by the rice gene OsANK7 is shown in SEQ ID NO: 3; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineered bacteria.

2. A method for reducing rice plant height, characterized in that: The rice gene OsANK7 is modified by genetic engineering to render the gene functionally ineffective, thereby reducing the height of rice plants. The amino acid sequence of the protein (a) encoded by the rice gene OsANK7 is shown in SEQ ID NO:

3.

3. The method according to claim 2, characterized in that The gene modification method is selected from at least one of site-directed mutagenesis and homologous recombination.

4. The method according to claim 2, characterized in that: The method comprises: taking the rice gene OsANK7 as a target, designing a sgRNA sequence based on CRISPR / Cas9, connecting a DNA fragment containing the encoding sgRNA sequence to a vector carrying CRISPR / Cas9, transforming rice, and thereby obtaining transgenic rice with the gene function missing.

5. The method according to claim 4, characterized in that The nucleotide sequence of the sgRNA action site is 5'-CCGCTTCCCATCCATCTCCCGCGAGG-3'.

6. The method according to claim 2, characterized in that When the 58th to 59th bases of the 5'UTR region of the rice gene OsANK7 are deleted as shown in SEQ ID NO: 4, the function of the gene is lost.

7. Use of the transgenic rice obtained by the method according to any one of claims 2 to 6 in plant breeding.

8. The use according to claim 7, characterized in that: Breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

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