Application of an amino acid transporter gene

Abstract

The present invention relates to the field of plant genetic engineering, and specifically to the application of an amino acid transporter gene. This solution reduces the expression level of the OsAAP7 gene in wild-type rice or knocks out the OsAAP7 gene in wild-type rice; the amino acid sequence of the OsAAP7 protein encoded by the OsAAP7 gene is shown in SEQ ID NO.1, or the OsAAP7 protein is a protein with equivalent activity obtained by substituting, replacing, and / or adding several amino acids to the amino acid sequence shown in SEQ ID NO.1. This solution reveals the function of the OsAAP7 gene. By knocking out the expression of the OsAAP7 gene, the elongation of the tiller buds of a single rice plant can be accelerated, the number of tillers per rice plant can be increased, and the number and weight of grains per rice plant can be increased. The OsAAP7 gene can be used in rice breeding to increase rice yield.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to the application of an amino acid transport gene. Background Art

[0002] Crops that efficiently utilize nitrogen are crucial for global food supply and environmental sustainability. Amino acids in nitrogen are an important form of nitrogen nutrient absorption, transport, storage, and utilization in rice, and can be transported by various amino acid transporters. Studies have shown that nitrogen transporters are effective targets for improving crop productivity and nitrogen utilization efficiency (Tegeder M, Masclaux-Daubresse C. Source and sink mechanisms of nitrogen transport and use. New Phytologist. 2018, 217(1): 35-53.). There are 85 members of amino acid transporters (AATs) in rice (Zhao H, Ma H, Yu L, Wang X, Zhao J. Genome-wide survey and expression analysis of amino acid transporter gene family in rice (Oryza sativa L.). PLoS One, 2012, 7(11): e49210.), which play different roles in various stages of rice growth and development and in various tissue locations. Different amino acid transporter members have significant effects on rice growth and development. Some may have positive regulatory effects, increasing their gene expression to promote growth and development, while others may have negative regulatory effects, decreasing their gene expression to promote growth and development. Furthermore, altered gene expression in these members may lead to diverse phenotypes, with some affecting rice tillering, others leaf senescence, others yield, and still others quality. Experimental studies are needed to uncover their potential biological functions. Rice has 85 amino acid transporter members, of which 19 belong to the AAPs subfamily (OsAAP1-19). The functions of AAPs orthologs in rice are still limited. Thoroughly understanding the functions of individual AAPs subfamily genes in rice will facilitate the development of rice genetic breeding techniques and pave the way for the cultivation of high-yield, high-quality rice varieties. Summary of the Invention

[0003] The present invention aims to provide an application of an amino acid transporter gene to solve the technical problem that the functions of AAPs homologous genes in rice are limited and AAPs cannot be fully utilized for effective rice genetic breeding.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] An amino acid transporter gene is used in rice breeding to reduce the expression level of the OsAAP7 gene in wild-type rice or knock out the OsAAP7 gene in wild-type rice; the amino acid sequence of the OsAAP7 protein encoded by the OsAAP7 gene is shown in SEQ ID NO. 1, or the OsAAP7 protein is a protein with equivalent activity obtained by substituting, replacing and / or adding several amino acids to the amino acid sequence shown in SEQ ID NO. 1.

[0006] The principle and beneficial effects of the above technical solution are as follows: OsAAP7 gene knockout in rice increases the elongation rate of rice tiller buds and the number of rice tillers, as well as the number and weight of filled grains per plant. Overexpression of this gene, however, has the opposite effect. Therefore, OsAAP7 gene knockout can be applied to various aspects of genetic improvement of rice plant architecture and yield.

[0007] In the prior art, it has been reported that the amino acid permease gene OsAAP6 in the rice amino acid transporter gene family is expressed in seeds, positively correlated with grain protein content, and affects rice quality (Peng B, Kong H, Li Y, Wang L, Zhong M, Sun L, Gao G, Zhang Q, Luo L, Wang G, Xie W, Chen J, Yao W, Peng Y, Lei L, Lian X, Xiao J, Xu C, Li X, He Y. OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice. Nature Communication, 2014, 5(1), 4847.). The amino acid permease OsAAP3 specifically transports alkaline and aromatic amino acids (Taylor MR, Reinders A, Ward JM. Transport function of rice amino acid permeases (AAPs). Plant & Cell Physiology, 2015, 56(7): 1355-1363.). Increasing the expression of the OsAAP3 gene reduces the number of rice tillers and causes rice leaf senescence. However, reducing or knocking out the expression of the OsAAP3 gene can accelerate the elongation of rice tiller buds, thereby forming more tillers and increasing rice yield and nitrogen use efficiency (Lu K, Wu BW, Wang J, Zhu W, Nie HP, Qian JJ, Huang WT, Fang ZM. Blocking amino acid transporter OsAAP3 improves grain yield by promoting outgrowth buds and increasing tiller number in rice. Plant Biotechnology Journal,2018,16(10):1710-1722.).Recent studies have shown that OsAAP5 mainly regulates the transport of basic amino acids (lysine, arginine) and neutral amino acids (valine, alanine). Reducing the expression of OsAAP5 can lead to increased tillering and grain yield, while overexpression leads to the opposite phenotype, indicating that OsAAP5 is a gene that negatively regulates rice growth and development (Wang J, Wu BW, Lu K, Wei Q, Qian JJ, Chen YP, Fang ZM. The aminoacid permease5 (OsAAP5) regulates tiller number and grain yield in rice. Plant Physiology, 2019, 180(2): 1031-1045.). Studies have also shown that OsLHT1 is a substrate-nonspecific, high-affinity amino acid transporter that prefers to transport neutral and acidic amino acids. Functional loss leads to reduced amino acid absorption by rice roots, reduced amino acid distribution to the aboveground parts, and reduced rice biomass and grain yield, indicating that OsLHT1 is a gene that positively regulates rice biomass and yield (Wang X, Yang G, Shi M. Disruption of an amino acid transporter LHT1 leads to growth inhibition and low yields in rice. BMC Plant Biology 2019, 19: 268.; Guo N, Hu J, Yan M, Luo L, Qu H, Tegeder M, Xu G. Oryza sativa Lysine-Histidine-type Transporter 1 functions in root uptake and root-to-shoot allocation of amino acids in rice. Plant Journal, 2020. doi: 10.1111 / tpj.14742.).Recent studies have shown that increasing the expression of the rice OsAAP1 gene can increase rice tillering and yield per plant. The protein expressed by this gene can transport neutral amino acids (Ji YY, Huang WT, Wu BW, Fang ZM, Wang XL. The amino acid transporter OsAAP1 mediates growth and grain yield by regulating neutralamino acids uptake and reallocation in Oryza sativa. Journal of Experimental Botany, 2020. doi:10.1093 / jxb / eraa256). This suggests that different amino acid transporter members have significant effects on rice growth and development. Some may have positive regulatory effects, with increased gene expression promoting growth and development, while others may have negative regulatory effects, with decreased gene expression promoting growth and development. Furthermore, altered gene expression in these members may result in diverse phenotypes, with some affecting rice tillering, others leaf senescence, others yield, and still others quality. Experimental studies are needed to uncover their potential biological functions.

[0008] In this study, the amino acid transporter gene OsAAP7 was targeted and its cDNA sequence was cloned from the rice variety Zhonghua 11. By constructing an OsAAP7 overexpression vector and introducing the overexpression vector into Zhonghua 11, OsAAP7-overexpressing plants were obtained. Furthermore, field-grown rice showed reduced tiller number, grain number per plant, and grain weight. By constructing an OsAAP7 knockout vector and introducing the knockout vector into Zhonghua 11, OsAAP7 knockout plants were obtained. Field-grown rice showed increased tiller number, grain number per plant, and grain weight. These results indicate that knocking out the OsAAP7 gene increases the number and weight of seeds per plant in normal rice, thereby improving rice yield per plant.

[0009] Through extensive research, the inventors discovered that the function of the OsAAP7 gene differs from that of known AAPs. Knockout of the OsAAP7 gene accelerated the growth of rice tiller buds in normal hydroponic culture medium or under 1 mM arginine treatment. This effect differs from the effects of the OsAAP1 gene (affecting rice tillering and yield per plant), the OsLHT1 gene (affecting rice biomass and grain yield), the OsAAP3 gene (affecting rice tiller number), the OsAAP5 gene (affecting tillering and grain yield), and the OsAAP6 gene (affecting grain protein content). In other words, the technical effect achieved by knocking out the OsAAP7 gene (accelerated growth of rice tiller buds) cannot be inferred from the functions of known AAPs, resulting in an unexpected technical effect.

[0010] In summary, the functions of the OsAAP7 gene discovered in this invention can be used in rice breeding to increase tiller bud elongation rate, tiller number, and seed number and weight per plant. Specifically, gene knockout can be used to reduce OsAAP7 gene expression, thereby increasing seed number and weight per plant and thereby improving rice yield. The amino acid sequence of the OsAAP7 protein encoded by the OsAAP7 gene is shown in SEQ ID NO. 1; the cDNA sequence of the OsAAP7 gene is preferably shown in SEQ ID NO. 2.

[0011] It should be understood that, provided that the activity of the OsAAP7 protein is not affected (i.e., the mutation is not in the active site of the protein), those skilled in the art can make various substitutions, additions, and / or deletions of one or more amino acids to the amino acid sequence of SEQ ID NO. 1 to obtain an amino acid sequence with equivalent functionality. Therefore, the OsAAP7 protein also includes proteins with equivalent activity obtained by substituting, replacing, and / or adding one or more amino acids to the amino acid sequence of SEQ ID NO. 1. Furthermore, it should be understood that, given codon degeneracy and codon preferences across species, those skilled in the art can utilize codons suitable for expression in a particular species, as needed.

[0012] The beneficial effects of this program are summarized as follows:

[0013] (1) The present invention can reduce the expression of the OsAAP7 gene through gene knockout technology, thereby increasing the number of rice tillers and effective panicles, and increasing the number and weight of seeds per plant. Therefore, plant variety improvement can be achieved by combining gene editing technology and molecular breeding.

[0014] (2) The successful cloning of the OsAAP7 gene confirmed that amino acid transporter genes not only play a role in the change of plant amino acid content and quality regulation, but also play an important role in tiller bud, tiller, and seed development. This can enrich the understanding of amino acid transporters and greatly promote the genetic improvement of plant type and single plant yield.

[0015] Furthermore, the cDNA sequence of the OsAAP7 gene is shown in SEQ ID NO.2.

[0016] By adopting the above technical solution, the OsAAP7 gene can express the OsAAP7 protein and realize relevant biological functions.

[0017] Furthermore, the OsAAP7 gene of wild-type rice was knocked out to obtain a mutant strain; and the mutant strain was planted in a hydroponic solution containing 0-1 mM arginine.

[0018] Using the above technical solution, within the range of 0-1 mM, the higher the arginine concentration, the stronger the effect of arginine on promoting the growth of tiller buds of plants in which the expression of the OsAAP7 gene is knocked out.

[0019] Furthermore, the OsAAP7 gene was knocked out of wild-type rice to generate mutant lines. These mutant lines were then cultured in a hydroponic solution containing either 0 mM or 1 mM arginine. For both wild-type and OsAAP7-overexpressing plants, tiller bud length initially increased with increasing arginine concentration, but then showed growth inhibition.

[0020] Using the above technical solution, mutant strains were constructed and then treated with varying concentrations of arginine. It was found that the elongation of tiller buds was accelerated when no arginine was added or when high concentrations of arginine were used. Through long-term experimental observation, the inventors discovered that when treated with high concentrations of arginine, tiller bud elongation in OsAAP7 mutant plants was faster than in the control Zhonghua 11 and overexpressing plants, promoting tillering. This suggests that genetic improvement could be used to increase rice tillering and yield in soils rich in organic nitrogen.

[0021] Furthermore, knocking out the OsAAP7 gene is used to increase the elongation rate of rice tiller buds.

[0022] Using the above technical solution, experiments have shown that knocking out the OsAAP7 gene in the wild-type rice Zhonghua 11 can effectively increase the elongation rate of rice tiller buds.

[0023] Furthermore, knocking out the OsAAP7 gene is used to increase the number of tillers per rice plant.

[0024] Using the above technical solution, experiments have shown that knocking out the OsAAP7 gene in the wild-type rice Zhonghua 11 can effectively increase the number of tillers per rice plant.

[0025] Furthermore, knocking out the OsAAP7 gene is used to increase the number of filling grains per rice plant.

[0026] Using the above technical solution, experiments have shown that knocking out the OsAAP7 gene in the wild-type rice Zhonghua 11 can effectively increase the number of tillers per rice plant.

[0027] Furthermore, knocking out the OsAAP7 gene is used to increase the weight of filling grains per rice plant.

[0028] Using the above technical solution, experiments have shown that knocking out the OsAAP7 gene in the wild-type rice Zhonghua 11 can effectively increase the weight of filling grains per rice plant.

[0029] Furthermore, the CRISPR / Cas9 system was used to knock out the OsAAP7 gene in wild-type rice to obtain a mutant strain.

[0030] Using the above technical solution, the CRISPR / Cas9 system is an effective tool for accurately knocking out target genes in the existing technology, and can achieve efficient knockout of target genes.

[0031] Furthermore, the target sequence for knocking out the OsAAP7 gene using the CRISPR / Cas9 system is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0032] Using the above technical solution, the inventors tried to design multiple target sequences and found that using the target sequence represented by SEQ ID NO.3 or SEQ ID NO.4 in the gene knockout operation can achieve accurate and efficient knockout of the target gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a comparison chart of the sequencing results after gene knockout in Experimental Example 1 of the present invention.

[0034] Figure 2 This is a statistical bar graph of the OsAAP7 gene expression level in Example 2 of the present invention.

[0035] Figure 3 This is a diagram of the tiller bud phenotype of the rice hydroponic experiment in Experimental Example 1 of the present invention (Arg 0mM treatment, scale bar represents 5mm).

[0036] Figure 4 This is a diagram of the tiller bud phenotype of the rice hydroponic experiment in Experimental Example 1 of the present invention (Arg 0.2 mM treatment, scale bar represents 5 mm).

[0037] Figure 5This is a diagram of the tiller bud phenotype of the rice hydroponic experiment in Experimental Example 1 of the present invention (Arg 0.5 mM treatment, scale bar represents 5 mm).

[0038] Figure 6 This is a diagram of the tiller bud phenotype of the rice hydroponic experiment in Experimental Example 1 of the present invention (treated with 1 mM Arg, the scale bar represents 5 mm).

[0039] Figure 7 This is a statistical histogram of the length of the second tiller bud of Experimental Example 1 of the present invention.

[0040] Figure 8 This is the phenotype diagram of a single rice plant planted in the field of Experimental Example 2 of the present invention.

[0041] Figure 9 This is a statistical chart of the number of tillers per rice plant under field planting in Experimental Example 2 of the present invention.

[0042] Figure 10 This is the rice phenotype diagram of a single rice plant planted in the field of Experimental Example 2 of the present invention.

[0043] Figure 11 The weight of the filling grains of each rice plant in the field planting of Experimental Example 2 of the present invention is statistically shown.

[0044] Figure 12 This is a laser confocal microscopy image of Experimental Example 3 of the present invention (showing subcellular localization). DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods and can be performed according to the described recombinant technology (see Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Ma X et al, A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015, 8 (8): 1274-1284.); the materials, reagents, etc. used can all be obtained from commercial sources.

[0046] Example 1: Construction of OsAAP7 gene mutant plants

[0047] Prepare target sequence:

[0048] F1: 5'-CGAACTGCTTCCACGTCGAG-3' (SEQ ID NO.3);

[0049] F2: 5'-TCGTGCAGAAGAGGATACCG-3' (SEQ ID NO. 4).

[0050] Using the above single target sequence, a gene knockout vector for the OsAAP7 gene (cDNA sequence see SEQ ID NO.2, protein sequence see SEQ ID NO.1) was constructed (method reference Ma X et al, A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015, 8(8): 1274-1284). The gene knockout expression vector was introduced into the normal japonica rice variety Zhonghua 11 using the Agrobacterium EHA105-mediated genetic transformation method. The mutant plants were sequenced at the T0 generation, and it was confirmed that the gene had been knocked out in two strains (OsAAP7-C1 and OsAAP7-C2, sequence alignment as shown in Figure 2). Figure 1 The sequence of the OsAAP7 gene in OsAAP7-C1 is deleted from 490 bp to 1349 bp (a total of 860 bp of bases are deleted, and the deleted sequence is shown in SEQ ID NO. 11), resulting in the loss of the OsAAP7 gene (i.e., knockout). The sequence of the OsAAP7 gene in OsAAP7-C2 has base A inserted into it (as shown in Figure 1 The first knockout line, OsAAP7-C1, was independently propagated to the T1 generation, yielding the independent mutant plant line osaap7 (for field cultivation).

[0051] Example 2: Construction of OsAAP7 gene overexpression plants

[0052] (1) Preparation of OsAAP7 gene overexpressing plants

[0053] RNA was extracted from rice Zhonghua 11 and reverse transcribed into cDNA. The cDNA of the OsAAP7 gene was amplified by PCR. The primer pairs used for PCR amplification were:

[0054] F3: 5'-ATGGTACCATGGGGGAGAACGGTGTGGTGGCG-3' (SEQ ID NO.5);

[0055] R3: 5'-ATGGATCCGTAGGTGGTGGCGAACGGCTTGTA-3' (SEQ ID NO. 6).

[0056] The OsAAP7 gene was then linked into the pCAMBIA-1306 vector (purchased from Cambia) via Kpn I and BamH I to construct the OsAAP7 overexpression vector OsAAP7-p1306. The overexpression vector was then introduced into the normal rice variety Zhonghua 11 using Agrobacterium tumefaciens EHA105-mediated genetic transformation.

[0057] (2) PCR identification

[0058] All transgenic seedlings were transplanted into baskets with soil, watered and fertilized regularly, and planted in the field when the seedlings grew to about 10 cm. After the seedlings grew up, genomic DNA was extracted and tested for transgenic plants by PCR. The primer pair for detection was:

[0059] F4: 5'-GATGTTGGCGACCTCGTATT-3' (SEQ ID NO.7);

[0060] R4: 5'-TCGTTATGTTTATCGGCACTTT-3' (SEQ ID NO. 8).

[0061] If a 517 bp fragment is amplified, the transgenic plant is positive. Positive plants are harvested and planted until homozygous transgenic plants are identified in the T2 generation, thus obtaining plants overexpressing the OsAAP7 gene.

[0062] The leaves of OsAAP7 gene overexpressing plants were taken, RNA was extracted and reverse transcribed into cDNA, and the expression level of OsAAP7 gene in overexpressing plants was detected by real-time fluorescence quantitative PCR. The results showed that ( Figure 2 , mean±SD, n=20) The expression level of OsAAP7 gene in overexpression plants was higher than that in control. If the expression level of control was set as 1, the expression levels of the three strains of overexpression plants were increased by about 100 times. Figure 2 In the data, ZH11 represents the wild-type control rice Zhonghua 11, and OE1, OE2, and OE3 represent three lines of OsAAP7 overexpressing plants. Data were analyzed using SPSS software for analysis of variance (ANOVA), with Duncan's test for significance at the 0.05 level. Lowercase letters indicate significant differences between groups, indicating significant differences in gene expression between OsAAP7 overexpressing plants and the control rice Zhonghua 11. The primer pairs used for real-time fluorescence quantitative PCR were:

[0063] F5: 5'-TGGCGAGCAAGCTGTGCTACC-3' (SEQ ID NO.9);

[0064] R5: 5'-ACCCAACCCAGCTGCCGAT-3' (SEQ ID NO. 10).

[0065] Experimental Example 1: Hydroponic Test

[0066] After obtaining mutant plants and overexpression plants, different concentrations of arginine were added to the rice hydroponic solution for treatment. The samples tested in the experiment were OsAAP7-C1 and OsAAP7-C2 obtained in Example 1, OE1, OE2 and OE3 obtained in Example 2, and the control group rice Zhonghua 11 (wild type, ZH11).

[0067] For OsAAP7-C1 and OsAAP7-C2, it was found that when the arginine concentration was 0 or 1mM, the elongation of rice tiller buds was accelerated and significantly increased compared with the control, while when arginine was 0.2mM or 0.5mM, the elongation of rice tiller buds was accelerated, but there was no significant difference compared with the control; for OE1, OE2 and OE3, it was found that when the arginine concentration was 0 or 1mM, the elongation of rice tiller buds slowed down, while when arginine was 0.2mM or 0.5mM, the elongation of rice tiller buds was accelerated, but there was no significant difference compared with the elongation of tiller buds in the control ( Figure 3-Figure 6 and Figure 7 ).exist Figure 7 The data were analyzed by ANOVA (mean ± SD, n = 20) using SPSS software. T-test was used for significance analysis. ** indicates that there is a significant difference at the 0.01 level compared with the control group ZH11 in the same group. *** indicates that there is a significant difference at the 0.001 level compared with the control group ZH11 in the same group.

[0068] These results indicate that knocking out OsAAP7 can accelerate tiller bud elongation in rice plants exposed to 0 or high arginine concentrations (1 mM). Increased OsAAP7 expression can also slow down tiller bud elongation in normal rice plants.

[0069] The inventors tested the relationship between the length of the second tiller bud (representing the elongation rate of the tiller bud) and arginine concentration. They found that when the arginine concentration was between 0 mM and 1 mM, the length of the second tiller bud in wild-type and OsAAP7-overexpressing plants first increased with increasing arginine concentration, and then showed growth inhibition with increasing arginine concentration. However, this phenomenon did not occur in plants in which the expression of the OsAAP7 gene was knocked out. Within the arginine concentration range of 0-1 mM, the length of the second tiller bud continued to increase with increasing arginine concentration. That is, within the 0-1 mM range, the higher the arginine concentration, the stronger the effect of arginine on promoting the growth of the tiller buds in plants in which the expression of the OsAAP7 gene was knocked out.

[0070] Experimental Example 2: Field Planting Experiment

[0071] Field planting experiments were conducted using the osaap7 strain obtained in Example 1, OE1, OE2, and OE3 obtained in Example 2, and a control rice plant, Zhonghua 11 (wild type, ZH11). One plant each of the OsAAP7 mutant plants (osaap7 strains, OE1, OE2, and OE3) and the control Zhonghua 11 were randomly selected from the field, placed in a small bucket, and photographed. The results are shown in FIG. Figure 8 As shown. It was found that the tillering of mutant osaap7 plants increased, while the tillering of overexpressing plants decreased ( Figure 8 ), the tillering number of individual rice plants was statistically analyzed and it was found that the tillering number of mutant osaap7 plants was significantly increased compared with the control ZH11, while the tillering number of overexpressing plants was significantly reduced compared with the control ZH11 ( Figure 9 , mean±SD, n=20). Figure 9 Data were analyzed by variance analysis (ANOVA) using SPSS software, and Duncan's test was used for significant differences at the 0.05 level. Lowercase letters indicate significant differences between groups. One plant was randomly selected from the mutant osaap7 plant and the control Zhonghua 11. All the shelled and filled rice seeds were arranged in a circle. It was found that the seeds of the mutant osaap7 plant were larger than the circle of the control Zhonghua 11, indicating that the number of rice grains per plant increased; the seeds of the overexpressing plant were smaller than the circle of the control Zhonghua 11, indicating that the number of rice grains per plant decreased ( Figure 10 The weight of each seed in the mutant plants was statistically analyzed, and it was found that the weight of each seed in the mutant osaap7 plants was significantly increased compared with the control plants. The weight of each seed in the overexpression plants was statistically analyzed, and it was found that the weight of each seed in the overexpression plants was significantly decreased compared with the control plants. Figure 11 , mean±SD, n=20). Figure 11SPSS software was used to perform analysis of variance (ANOVA) and Duncan's test was used to perform significant difference analysis at the 0.05 level. Lowercase letters indicate significant differences between different groups.

[0072] These results indicate that knocking out OsAAP7 gene expression increases the number of rice tillers and the weight of grains per plant in field-grown rice. Increased OsAAP7 expression can slow the elongation of normal rice tiller buds. Increased OsAAP7 expression can also reduce the number of rice tillers, the number of grains per plant, and the weight of grains per plant.

[0073] Experimental Example 3: Subcellular localization study

[0074] This experimental example studied the subcellular localization of the OsAAP7 gene. For the research method, please refer to the inventor's previously published paper on the OsAAP5 gene (The Amino Acid Permease 5 (OsAAP5) Regulates Tiller Number and Grain Yield in Rice, Jie Wang, Plant Physiology, June 2019, Vol. 180, pp. 1031–1045). The experimental results are shown in Figure 2. Figure 12The images shown, from left to right, show: GFP subcellular localization of OsAAP7 protein; subcellular localization of ER protein-mCherry; brightfield observation of protoplasts; and colocalization of OsAAP7 protein with ER-localized proteins. The experimental results show that OsAAP7 protein is concentrated in the ER, demonstrating its subcellular localization to the ER. The inventors have previously conducted extensive research on the subcellular localization of the OsAAP5 and OsAAP1 genes. The OsAAP5 protein was found to be subcellularly localized at the plasma membrane (see Figure 2M in the reference for data and images, "The Amino Acid Permease 5 (OsAAP5) Regulates Tiller Number and Grain Yield in Rice," Jie Wang, Plant Physiology, June 2019, Vol. 180, pp. 1031–1045), while the OsAAP1 protein was localized at the plasma membrane and nucleus (see Figure 2A-E in the reference for data and images, "The amino acid transporter AAP1 mediates growth and grain yield by regulating neutral amino acid uptake and reallocation in Oryza sativa," Yuanyuan Ji, Journal of Experimental Botany, Vol. 71, No. 16, pp. 4763–4777, 2020). The OsAAP7 protein was localized to the endoplasmic reticulum, which differs significantly from the subcellular localization of other AAPs subfamily proteins. Based on the subcellular localization of the OsAAP7 protein, the inventors analyzed the mechanism by which arginine-responsive tiller bud growth is promoted in OsAAP7 knockout plants. The endoplasmic reticulum is where proteins are synthesized. Plants overexpressing the OsAAP7 gene transport arginine to the endoplasmic reticulum for protein synthesis, further accumulating inhibitory substances. This inhibits tiller bud elongation. In contrast, plants knocked out of the OsAAP7 gene do not transport arginine to the endoplasmic reticulum in large quantities, thus preventing the growth of tiller buds. In summary, because the OsAAP7 gene and OsAAP7 protein operate differently from other AAPs subfamily genes, knocking out the OsAAP7 gene results in different phenotypic effects in rice plants. In this study, knocking out the OsAAP7 gene in wild-type rice accelerated tiller bud growth in normal hydroponic culture medium or under 1 mM arginine treatment.

[0075] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

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[0082] SEQUENCE LISTING <110> Guizhou University <120> Application of an amino acid transporter gene <130> 2020 / 09 / 09 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 496 <212> PRT <213> Oryza.sativa L. <400> 1 Met Gly Glu Asn Gly Val Val Ala Ser Lys Leu Cys Tyr Pro Ala Ala 1 5 10 15 Ala Met Glu Val Val Ala Ala Glu Leu Gly His Thr Ala Gly Ser Lys 20 25 30 Leu Tyr Asp Asp Asp Gly Arg Leu Lys Arg Thr Gly Thr Met Trp Thr 35 40 45 Ala Ser Ala His Ile Ile Thr Ala Val Ile Gly Ser Gly Val Leu Ser 50 55 60 Leu Gly Trp Ala Ile Ala Gln Leu Gly Trp Val Ala Gly Pro Ala Val 65 70 75 80 Met Leu Leu Phe Ser Phe Val Thr Tyr Tyr Thr Ser Ala Leu Leu Ala 85 90 95 Asp Cys Tyr Arg Ser Gly Asp Glu Ser Thr Gly Lys Arg Asn Tyr Thr 100 105 110 Tyr Met Asp Ala Val Asn Ala Asn Leu Ser Gly Ile Lys Val Gln Val 115 120 125 Cys Gly Phe Leu Gln Tyr Ala Asn Ile Val Gly Val Ala Ile Gly Tyr 130 135 140 Thr Ile Ala Ala Ser Ile Ser Met Leu Ala Ile Lys Arg Ala Asn Cys 145 150 155 160 Phe His Val Glu Gly His Gly Asp Pro Cys Asn Ile Ser Ser Thr Pro 165 170 175 Tyr Met Ile Ile Phe Gly Val Ala Glu Ile Phe Phe Ser Gln Ile Pro 180 185 190 Asp Phe Asp Gln Ile Ser Trp Leu Ser Ile Leu Ala Ala Val Met Ser 195 200 205 Phe Thr Tyr Ser Thr Ile Gly Leu Gly Leu Gly Val Val Gln Val Val 210 215 220 Ala Asn Gly Gly Val Lys Gly Ser Leu Thr Gly Ile Ser Ile Gly Val 225 230 235 240 Val Thr Pro Met Asp Lys Val Trp Arg Ser Leu Gln Ala Phe Gly Asp 245 250 255 Ile Ala Phe Ala Tyr Ser Tyr Ser Leu Ile Leu Ile Glu Ile Gln Asp 260 265 270 Thr Ile Arg Ala Pro Pro Pro Ser Glu Ser Arg Val Met Arg Arg Ala 275 280 285 Thr Val Val Ser Val Ala Val Thr Thr Leu Phe Tyr Met Leu Cys Gly 290 295 300 Cys Thr Gly Tyr Ala Ala Phe Gly Asp Ala Ala Pro Gly Asn Leu Leu 305 310 315 320 Thr Gly Phe Gly Phe Tyr Glu Pro Phe Trp Leu Leu Asp Val Ala Asn 325 330 335 Ala Ala Ile Val Val His Leu Val Gly Ala Tyr Gln Val Tyr Cys Gln 340 345 350 Pro Leu Phe Ala Phe Val Glu Lys Trp Ala Gln Gln Arg Trp Pro Lys 355 360 365 Ser Trp Tyr Ile Thr Lys Asp Ile Asp Val Pro Leu Ser Leu Ser Gly 370 375 380 Gly Gly Gly Gly Gly Gly Arg Cys Tyr Lys Leu Asn Leu Phe Arg Leu 385 390 395 400 Thr Trp Arg Ser Ala Phe Val Val Ala Thr Thr Val Val Ser Met Leu 405 410 415 Leu Pro Phe Phe Asn Asp Val Val Gly Phe Leu Gly Ala Val Gly Phe 420 425 430 Trp Pro Leu Thr Val Tyr Phe Pro Val Glu Met Tyr Ile Val Gln Lys 435 440 445 Arg Ile Pro Arg Trp Ser Thr Arg Trp Val Cys Leu Gln Leu Leu Ser 450 455 460 Leu Ala Cys Leu Ala Ile Thr Val Ala Ser Ala Ala Gly Ser Ile Ala 465 470 475 480 Gly Ile Leu Ser Asp Leu Lys Val Tyr Lys Pro Phe Ala Thr Thr Tyr 485 490 495 <210> 2 <211> 1491 <212> DNA <213> Oryza.sativa L. <400> 2 atgggggaga acggtgtggt ggcgagcaag ctgtgctacc cggcggcggc catggaggtg 60 gtcgccgccg agctcggcca cacggccggc tccaagctgt acgacgacga cggccgcctc 120 aagcgcaccg ggacgatgtg gacggcgagc gcgcacatca tcacggcggt gatcggctcc 180 ggcgtgctgt cgctggggtg ggcgatcgcg cagctgggtt gggtggccgg cccgccgtc 240 atgctgctct tctcgttcgt cacctact acctccgcgc tgctcgccga ctgctaccgc 300 tccggcgacg agagcaccgg caagcgcaac tacacctaca tggacgccgt gaacgccaac 360 ctgagtggca tcaaggtcca ggtctgcggg ttcctgcagt acgccaacat cgtcggcgtc 420 gccatcggct acaccattgc cgcctccatt agcatgctgg cgatcaagcg ggcgaactgc 480 ttccacgtcg aggggcacgg cgacccgtgc aacatctcga gcacgccgta catgatcatc 540 ttcggcgtgg cggagatctt cttctcgcag atcccggact tcgaccagat ctcgtggctg 600 tccatcctcg ccgccgtcat gtcgttcacc tactccacca tcgggctcgg cctcggcgtc 660 gtgcaggtgg tggccaacgg cggcgtcaag gggagcctca ccgggatcag catcggcgtg 720 gtgacgccca tggacaaggt gtggcggagc ctgcaggcgt tcggcgacat cgccttcgcc 780 tactcctact ccctcatcct catcgagatc caggacacca tccgggcgcc gccgccgtcg 840 gagtcgaggg tgatgcggcg cgccaccgtg gtgagcgtcg ccgtcaccac gctcttctac 900 atgctctgcg gctgcacggg gtacgcggcg ttcggcgacg ccgcgccggg caacctcctc 960 accgggttcg gcttctacga gcccttctgg ctcctcgacg ttgccaacgc cgccatcgtc 1020 gtccacctcg tcggcgccta ccaggtctac tgccagccgc tgttcgcctt cgtcgagaag 1080 tgggcgcagc agcggtggcc gaaatcatgg tacatcacca aggatatcga cgtgccgctc 1140 tccctctccg gcggcggcgg cggcggcgga aggtgctaca agctgaacct gttcaggctg 1200 acatggaggt cggcgttcgt ggtggcgacg acggtggtgt cgatgctgct gccgttcttc 1260 aacgacgtgg tggggttcct cggcgcggtg gggttctggc cgctcaccgt ctacttcccg 1320 gtggagatgt acatcgtgca gaagaggata ccgaggtgga gcacgcggtg ggtgtgcctg 1380 cagctgctca gcctcgcctg cctcgccatc accgtcgcct ccgccgccgg ctccatcgcc 1440 ggaatcctct ccgacctcaa ggtctacaag ccgttcgcca ccacctacta a 1491 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 cgaactgctt ccacgtcgag 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 tcgtgcagaa gaggataccg 20 <210> 5 <211> 32 <212> DNA <213> Artificial sequence <400> 5 atggtaccat gggggagaac ggtgtggtgg cg 32 <210> 6 <211> 32 <212> DNA <213> Artificial sequence <400> 6 atggatccgt aggtggtggc gaacggcttg ta 32 <210> 7 <211> twenty two <212> DNA <213> Artificial sequence <400> 7 tcgttatgtt tatcggcact tt 22 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <400> 8 tcgttatgtt tatcggcact tt 22 <210> 9 <211> 21 <212> DNA <213> Artificial sequence <400> 9 tggcgagcaa gctgtgctac c 21 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 acccaaccca gctgcgcgat 20 <210> 11 <211> 860 <212> DNA <213> Oryza.sativa L. <400> 11 gaggggcacg gcgacccgtg caacatctcg agcacgccgt acatgatcat cttcggcgtg 60 gcggagatct tcttctcgca gatcccggac ttcgaccaga tctcgtggct gtccatcctc 120 gccgccgtca tgtcgttcac ctactccacc atcgggctcg gcctcggcgt cgtgcaggtg 180 gtggccaacg gcggcgtcaa ggggagcctc accgggatca gcatcggcgt ggtgacgccc 240 atggacaagg tgtggcggag cctgcaggcg ttcggcgaca tcgccttcgc ctactcctac 300 tccctcatcc tcatcgagat ccaggacacc atccgggcgc cgccgccgtc ggagtcgagg 360 gtgatgcggc gcgccaccgt ggtgagcgtc gccgtcacca cgctcttcta catgctctgc 420 ggctgcacgg ggtacgcggc gttcggcgac gccgcgccgg gcaacctcct caccgggttc 480 ggcttctacg agcccttctg gctcctcgac gttgccaacg ccgccatcgt cgtccacctc 540 gtcggcgcct accaggtcta ctgccagccg ctgttcgcct tcgtcgagaa gtgggcgcag 600 cagcggtggc cgaaatcatg gtacatcacc aaggatatcg acgtgccgct ctccctctcc 660 ggcggcggcg gcggcggcgg aaggtgctac aagctgaacc tgttcaggct gacatggagg 720 tcggcgttcg tggtggcgac gacggtggtg tcgatgctgc tgccgttctt caacgacgtg 780 gtggggttcc tcggcgcggt ggggttctgg ccgctcaccg tctacttccc ggtggagatg 840 tacatcgtgc agaagaggat 860

Claims

1. Application of an amino acid transporter gene in rice breeding, characterized in that: Knockout of wild-type rice PartAAP7 gene, obtaining a mutant strain, and planting the mutant strain in a hydroponic solution containing 1 mM arginine; PartAAP7 The amino acid sequence of the OsAAP7 protein encoded by the gene is shown in SEQ ID NO. 1; Knockout PartAAP7 The gene is used to increase the elongation rate of rice tiller buds, increase the number of tillers per rice plant, increase the number of filling grains per rice plant, and increase the weight of filling grains per rice plant.

2. Use of an amino acid transporter gene in rice breeding according to claim 1, characterized in that: described PartAAP7 The cDNA sequence of the gene is shown in SEQ ID NO.

2.

3. Use of an amino acid transporter gene according to any one of claims 1 or 2 in rice breeding, characterized in that: Knockout of wild-type rice using the CRISPR / Cas9 system PartAAP7 gene, and obtain mutant strains.

4. Use of an amino acid transporter gene in rice breeding according to claim 3, characterized in that: Knockout using the CRISPR / Cas9 system PartAAP7 The target sequence of the gene is shown as SEQ ID NO.3 or SEQ ID NO.4.

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