A rice promoter regulated by amino acids and hormones, an expression vector containing the promoter, and applications thereof

Abstract

The present invention belongs to the technical field of plant genetic engineering, and relates to the technical field of crop breeding, and specifically relates to a rice promoter regulated by amino acids and hormones, an expression vector containing the promoter, and an application thereof. When studying the regulatory mode of the OsAAP14 gene, the promoter of the gene as shown in SEQ ID NO.1 was cloned. The promoter is a conditionally activated gene expression control element. The promoter is applied to transgenic engineering, and the promoter can be connected to a specific effector gene to construct an expression vector, and then a transgenic rice plant is constructed. The technical scheme overcomes the technical problem that the existing transgenic breeding method lacks a regulatory element for regulating the expression of the target gene. After the transgenic plant is acted upon by exogenous hormones and amino acids, the promoter activates a large amount of expression of the effector gene, which can be used in the practical operation of rice and other crop breeding, and will have significant application value and broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering and relates to the technical field of crop breeding, and specifically relates to a rice promoter regulated by amino acids and hormones, an expression vector containing the promoter and applications thereof. Background Art

[0002] Transgenic plants refer to plants that use genetic engineering (DNA recombinant technology) technology to transfer target genes or specific DNA fragments isolated from animals, plants or microorganisms, plus appropriate regulatory elements, into the plant genome through various methods, so that the gene or DNA sequence can be stably expressed and inherited. Genetically modified breeding (GMB) refers to a breeding method that uses modern molecular biology technology to add one or more genes to the genome of an organism to produce organisms with improved characteristics.

[0003] In recent years, plant transgenic engineering has developed rapidly. Various exogenous genes are transferred into recipient plants through vectors to improve plant traits, cultivate high-quality and high-yield crops, or serve as bioreactors. The construction of efficient expression vectors is an important link in transgenic engineering. As eukaryotic organisms, plants can regulate genes at multiple levels, such as DNA level, transcription level and post-transcriptional modification, translation level and post-translational modification, but gene regulation at the transcription level is the main link. Plant gene promoters are key regulatory elements on efficient expression vectors. Promoters play an important role in determining the transcription efficiency, localization introduction and expression enhancement of exogenous genes. In the practical operation of transgenic breeding, the effect of the transferred target gene may be limited due to low expression of the transferred target gene or because the expression time window of the transferred target gene cannot be effectively controlled, thereby affecting the quality of transgenic plants. Inducible promoters are an important form of plant gene promoters. Inducible promoters usually do not initiate gene transcription or have very low transcriptional activity. Only when external conditions change, including light, temperature, water, soil salinity, hormones, and encountering insect pests, can the transcriptional activity be significantly improved. When constructing transgenic vectors, constitutive promoters induce the continuous expression of exogenous proteins, affecting the normal growth and development of plants. However, the use of inducible promoters for transcriptional regulation can make exogenous genes expressed only under special induction conditions. At present, there are not many inducible promoters that can be used for rice breeding, which affects the research progress of rice genetic breeding. It is urgent to research and develop a new type of promoter that can be used for rice transgenic breeding to improve the quality of transgenic breeding rice. Summary of the invention

[0004] The present invention aims to provide a rice promoter regulated by amino acids and hormones to solve the technical problem that the existing transgenic breeding methods lack regulatory elements that can regulate the expression of target genes.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A rice promoter regulated by amino acids and / or hormones, whose nucleotide sequence is shown in SEQ ID NO.1; or its nucleotide sequence is a nucleotide sequence with equivalent functions obtained by replacing, adding and / or deleting one or several nucleotides in the sequence shown in SEQ ID NO.1.

[0007] The principles and advantages of this solution are:

[0008] The inventor of the present technical solution has been engaged in the research of rice genetic breeding for a long time, including the transport genes related to nitrogen absorption in rice development, including ammonium transport protein (AMT), nitrate transport protein (NRT), amino acid transport protein (AAT), peptide transport protein (PTR), etc. When studying the expression regulation pattern of the OsAAP14 gene, the inventor cloned a promoter of the OsAAP14 gene as shown in SEQ ID NO.1. The promoter was connected to the effector gene GUS (β-glucuronidase gene, β-glucuronidase), and then transgenic rice was constructed to study the promoter regulation pattern. β-glucuronidase can catalyze the substrate reaction to develop color, which is used to indicate whether the promoter can effectively serve as a switch for gene expression. The results of the study showed that the rice promoter with the sequence shown in SEQ ID NO.1 can respond to the stimulation of exogenous hormones and amino acids, thereby starting the expression of the effector gene. Therefore, the promoter is a conditionally activated gene expression control element. The promoter is applied to transgenic engineering, and the promoter can be connected to a specific effector gene to construct an expression vector, and then a transgenic rice plant is constructed. After the rice plant is subjected to the action of exogenous hormones and amino acids, the promoter can initiate a large amount of expression of the effector gene in the rice plant, so that the expression product of the downstream target gene under the promoter accumulates specifically in the rice, increasing the overall or local expression of the effector gene and promoting the development of rice. Therefore, the promoter of the OsAAP14 gene (SEQ ID NO.1) has a good application prospect in transgenic engineering.

[0009] Furthermore, the amino acid is histidine; and the hormone is gibberellin or abscisic acid.

[0010] The results of the study showed that the rice promoter with the sequence shown in SEQ ID NO.1 can respond to the stimulation of exogenous hormones and amino acids, thereby initiating the expression of effector genes. In particular, the promoter can respond to two plant hormones, gibberellin and abscisic acid, and can also respond to histidine, a basic amino acid. After screening a large number of amino acids, it was found that the promoter was only regulated by histidine, and had no significant response to other basic amino acids, acidic amino acids, and neutral amino acids (see Figure 7-Figure 9 The staining experiment results shown in FIG. 4A ). Moreover, the regulatory effect of histidine on the promoter shows a certain time-effect relationship as the action time increases. The longer the histidine action time, the more significant the effect of the promoter on the expression of the effector gene.

[0011] Although the inventors have studied the expression control mode of the OsAAP14 gene in the early stage, they have not found the response of the rice promoter shown in SEQ ID NO.1 to histidine and plant hormones. The promoter sequence reported in the present invention has created a breeding model for rice genetic breeding, which has far-reaching significance. More importantly, there is no report on the promoter sequence regulated by histidine. Histidine is not a plant hormone. Using it to induce the expression of effector genes is safer for plants and will not cause other effects induced by excessive hormones. When gibberellins and abscisic acid are applied to plants, in addition to the effector genes on the vector being regulated, the genes of the plants themselves that can respond to gibberellins and abscisic acid will also be regulated, and there are many genes that can respond to these two plant hormones, which will introduce too many unregulated factors to rice development. The promoter sequence regulated by histidine will have significant application value and broad application prospects in the practical operation of rice and other crop breeding.

[0012] Furthermore, an effector gene is connected downstream of the rice promoter.

[0013] By adopting the above technical solution, the promoter of the OsAAP14 gene is connected to the effector gene, and no other gene is inserted between the two. The significance of the promoter is that it is like a switch, controlling whether the gene connected to it is expressed or the degree of expression. Then, the opening and closing of the promoter switch is controlled by exogenous application of histidine and plant hormones, thereby controlling the expression of the effector gene.

[0014] Further, after the rice plants are treated with histidine or gibberellin or abscisic acid, the rice promoter is used to enhance the expression of the effector gene in the rice roots.

[0015] The inventors have found that transgenic rice can promote the expression of target genes in roots after being provided with amino acids and hormones from the outside. The present invention has found that the promoter activity of the rice OsAAP14 gene can be induced by amino acids and hormones; the promoter is used in transgenic rice to enable its downstream genes to be specifically expressed in the roots of rice. The promoter has good application prospects in transgenic engineering and provides new ideas for future exploration of how to regulate nitrogen absorption and utilization.

[0016] Furthermore, the effector gene is one of the OsAAP14 gene, the GUS gene, the OsNRT2.4 gene and the OsAMT1.2 gene.

[0017] The GUS gene mentioned in the embodiment of this scheme only plays an indicator role, showing whether the promoter is working in a more intuitive way; if the GUS staining result is blue, it means that the promoter gene has been expressed normally, and if there is no blue, it means that the gene controlled by the promoter is not expressed normally. The darker the blue, the greater the expression of the gene controlled by the promoter. This technical scheme reveals the regulation mode of the promoter of this scheme through GUS staining. The GUS gene can be replaced with other indicator genes in the prior art.

[0018] In addition, the promoter can be used to connect any other downstream target gene. For example, the 0sAAP14 gene, although in wild-type rice, the promoter of this scheme is the expression regulatory element of the 0sAAP14 gene. However, if we want to enhance the expression of the 0sAAP14 gene in the plant, we can use the promoter of this scheme to directly connect the 0sAAP14 gene to construct an expression vector, and then construct a transgenic plant that conditionally overexpresses the 0sAAP14 gene. The inventors have demonstrated in their previously published papers that overexpression of the 0sAAP14 gene can promote the growth of tiller buds of rice (Yang G, Wei X, Fang Z. Melatoninmediates axillary bud outgrowth by improving nitrogen assimilation and transport in Rice. Frontiers in Plant Science, 2022, 2370.). For another example, the gene OsNRT2.4 with nitrate transport and the gene OsAMT1.2 with ammonium transport function.

[0019] The technical solution also provides an application of a rice promoter regulated by amino acids and / or hormones in rice breeding. When the effector gene is the 0sAAP14 gene, wild-type rice plants are treated with histidine, gibberellin or abscisic acid.

[0020] Since we found that the promoter shown in SEQ ID NO.1 can be regulated by histidine, gibberellin or abscisic acid, and in wild-type rice, the promoter shown in SEQ ID NO.1 can regulate the expression of the 0sAAP14 gene, and the up-regulation of the 0sAAP14 gene expression is conducive to rice development. Therefore, when we breed wild-type rice, we can apply a certain amount of histidine, gibberellin or abscisic acid to the rice to promote the expression of the 0sAAP14 gene ( Figure 10-13 ), thereby promoting the growth of tillering buds of rice plants.

[0021] The technical solution also provides an application of a rice promoter regulated by amino acids and / or hormones in rice breeding, comprising the following steps performed in sequence:

[0022] S1: constructing an expression vector for expressing the effector gene, wherein the promoter of the effector gene is a rice promoter with a sequence as shown in SEQ ID NO.1;

[0023] S2: The expression vector obtained in S1 is transferred into wild-type rice plants to obtain transgenic rice;

[0024] S3: treating the transgenic rice with histidine, gibberellin or abscisic acid.

[0025] The present invention takes the promoter of the OsAAP14 gene, a member of the AAP gene family of rice, as the object. First, the constructed promoter-GUS expression vector is transformed into the callus induced by the mature embryo of rice through Agrobacterium-mediated transformation, and the transgenic plants are obtained and the positive plants are identified, and then the T1 generation transgenic plants are obtained, and then the T2 generation mature seeds are obtained. Then, at different stages of the growth of the T2 generation seeds, the roots, stems, leaves, panicles and other tissues of the plants are respectively subjected to GUS histochemical staining to determine the GUS expression activity, and the transgenic plants are treated with different nitrogen to detect the GUS expression activity and analyze its spatiotemporal expression specificity. The results show that the promoter activity can be strongly induced by the basic amino acids of the external amino acids, especially histidine, and can also be strongly induced by gibberellins and abscisic acid, promoting the specific expression of the downstream target gene in the root of rice. The promoter is applied to transgenic engineering, which can increase the absorption and transportation of nitrogen in the amino acid-rich soil or after the spraying of plant hormones, promote vegetative growth, and can also make the expression product of the downstream target gene under the promoter specifically accumulate in the root and the aboveground part, and increase the local expression amount. Therefore, the promoter of OsAAP14 gene has good application prospects in transgenic engineering.

[0026] Further, in S3, the working concentrations of histidine, gibberellin and abscisic acid were 0.2 mmol / L, 2 μmol / L and 2 μmol / L, respectively.

[0027] The above-mentioned amino acid or hormone treatment conditions can effectively promote the activity of the rice promoter in this scheme, and then promote the expression of downstream effector genes.

[0028] Further, in S1, rice genomic DNA is extracted, and then PCR amplification is performed on the rice genomic DNA using an upstream primer with a sequence as shown in SEQ ID NO.2 and a downstream primer with a sequence as shown in SEQ ID NO.3 to obtain a rice promoter with a sequence as shown in SEQ ID NO.1; the rice promoter with a sequence as shown in SEQ ID NO.1 is integrated into the multiple cloning site of an empty vector to obtain an expression vector; an effector gene is integrated into the empty vector, and the rice promoter is used to initiate the expression of the effector gene.

[0029] The primers SEQ ID NO.2 and SEQ ID NO.3 can be used to effectively amplify the target promoter fragment, and the promoter fragment only needs to be integrated into the multiple cloning site of the empty vector for plant transgenics. Among them, the pCAMBIA 1391Z empty vector is a commercial vector commonly used in the prior art to study promoter activity. It only needs to integrate the promoter fragment into the multiple cloning site of the pCAMBIA 1391Z empty vector to achieve the control of the promoter on the effector gene. Among them, the multiple cloning site refers to an artificially synthesized DNA fragment contained in the vector, which contains multiple single enzyme cutting sites and is the insertion site of exogenous DNA.

[0030] The technical solution also provides an expression vector containing a rice promoter regulated by amino acids and / or hormones, including an empty vector and a promoter fragment whose sequence is shown in SEQ ID NO.1 integrated into the multiple cloning site of the empty vector, and the promoter fragment is used to promote the expression of the effector gene. By integrating the promoter fragment into the multiple cloning site of the empty vector, an expression vector expressing the effector gene regulated by the promoter of SEQ ID NO.1 can be obtained.

[0031] In summary, the technical solution aims at the deficiencies of the prior art and provides a promoter of a rice gene whose expression is regulated by amino acids and hormones and its application method. The promoter sequence of the OsAAP14 gene can respond to external amino acids, alkaline amino acids and hormones, and start the gene to be highly expressed in the roots; it was further found that histidine in alkaline amino acids, gibberellins in hormones and abscisic acid induced and enhanced the activity of the promoter of SEQ ID NO.1 of the OsAAP14 gene. The present invention can increase the local expression of other target genes in the roots in genetic engineering applications, and enhance the absorption of substances from the external environment or the accumulation in the roots. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1It is the agarose electrophoresis diagram of the PCR product of the promoter fragment of Example 1 (M: DL2000 Plus DNA Marker; 1-3: OsAAP14 gene promoter fragment).

[0033] Figure 2 Schematic diagram of the primer binding site of the promoter fragment of Example 1.

[0034] Figure 3 The figure is the vector map of pCAMBIA 1391Z in the prior art.

[0035] Figure 4 The electrophoretic diagrams of bacterial solution PCR verification and enzyme digestion verification of Example 1 (A: bacterial solution PCR electrophoresis: M: DL2000 Plus DNA Marker; 1: negative control amplified with water; 2: positive control amplified with OsAAP14 promoter fragment; 3-12: OsAAP14-pCAMBIA1391Z ligation product, transformed DH5α challenge bacteria for bacterial solution PCR; B: enzyme digestion verification results: M: DL2000 Plus DNA Marker; 1-2 positive clones double enzyme digestion verification).

[0036] Figure 5 The results of identifying the positive plants (OsAAP14 promoter-GUS transgenic plants) in Example 1 (M: DL2000Plus DNA Marker; 1: negative control amplified with water; 2: positive control amplified with OsAAP14-pCAMBIA1391Z recombinant plasmid; 3-18: positive transgenic plants).

[0037] Figure 6 The results of the staining experiment of OsAAP14 promoter-GUS transgenic plants in Example 2 and the real-time fluorescence quantitative PCR experiment results of the OsAAP14 gene in wild-type Zhonghua 1 in Example 4 (A, B, C, D, E, F: staining of roots, bases, leaf sheaths, stems, leaves, and ears of OsAAP14 promoter-GUS transgenic plants; G, H, I, J, K: sections of stained parts of roots, leaf sheaths, stems, leaves, and ears; L: expression levels of OsAAP14 genes in roots, bases, leaf sheaths, stems, leaves, and ears of Zhonghua 11, with the expression level in the stem being set as 1, and data are expressed as mean±SD, n=4).

[0038] Figure 7The effects of exogenous acidic and basic amino acids on the gene expression of the promoter of the OsAAP14 gene promoter in Example 3 (A: the staining results of the roots of the OsAAP14 promoter-GUS transgenic plants treated with 0.5 mmol / L acidic amino acids (aspartic acid 0.2 mmol / L, glutamate 0.3 mmol / L) for 0 h, 1 h, and 2 h; B: the staining results of the roots of the OsAAP14 promoter-GUS transgenic plants treated with 0.5 mmol / L basic amino acids (arginine 0.2 mmol / L, lysine 0.1 mmol / L, histidine 0.2 mmol / L) for 0 h, 1 h, and 2 h).

[0039] Figure 8 The effect of exogenous neutral amino acid treatment on the gene expression effect of the promoter of the OsAAP14 gene promoter in Example 3 (A: 1mmol / L neutral amino acid 1 (serine 0.1mmol / L, threonine 0.2mmol / L, glycine 0.2mmol / L, alanine 0.3mmol / L, proline 0.2mmol / L) treatment 0h, 1h, 2h OsAAP14 promoter-GUS transgenic plant root staining results; B: 1mmol / L neutral amino acid 2 (valine 0.2mmol / L, cysteine ​​0.1mmol / L A: The staining results of the roots of OsAAP14 promoter-GUS transgenic plants treated with 1mmol / L neutral amino acids 3 (methionine 0.1mmol / L, tryptophan 0.1mmol / L, leucine 0.2mmol / L, isoleucine 0.3mmol / L, phenylalanine 0.3mmol / L) for 0h, 1h, and 2h. B: The staining results of the roots of OsAAP14 promoter-GUS transgenic plants treated with 1mmol / L neutral amino acids 3 (methionine 0.1mmol / L, tryptophan 0.1mmol / L, leucine 0.2mmol / L, isoleucine 0.3mmol / L, phenylalanine 0.3mmol / L) for 0h, 1h, and 2h.

[0040] Fig. 9 This is the effect of exogenous histidine, arginine and lysine treatment on the gene expression effect of the promoter of the OsAAP14 gene promoter in Example 3. A: Staining results of the roots of the OsAAP14 promoter-GUS transgenic plants treated with 0.2 mmol / L histidine for 0h, 1h, and 2h; B: Staining results of the roots of the OsAAP14 promoter-GUS transgenic plants treated with 0.2 mmol / L arginine for 0h, 1h, and 2h; C: Staining results of the roots of the OsAAP14 promoter-GUS transgenic plants treated with 0.2 mmol / L lysine for 0h, 1h, and 2h).

[0041] Fig.10Effects of exogenous hormone treatment in Example 3 on the expression of the OsAAP14 gene (A: Real-time fluorescence quantitative PCR detection results of the roots of Zhonghua 11 treated with 2 μmol / L gibberellin for 0h, 1h, and 2h; B: Real-time fluorescence quantitative PCR detection results of the roots of Zhonghua 11 treated with 2 μmol / L abscisic acid for 0h, 1h, and 2h; the expression level at 0h was set as 1, and the data are expressed as mean±SD, n=4).

[0042] Fig.11 Effects of exogenous acidic and basic amino acids in Example 4 on OsAAP14 gene expression (C: Real-time fluorescence quantitative PCR results of roots of S. cerana 11 treated with 0.5 mmol / L acidic amino acids (aspartic acid 0.2 mmol / L, glutamate 0.3 mmol / L) at 0 h, 1 h, and 2 h; D: Real-time fluorescence quantitative PCR results of roots of S. cerana 11 treated with 0.5 mmol / L basic amino acids (arginine 0.2 mmol / L, lysine 0.1 mmol / L, histidine 0.2 mmol / L) at 0 h, 1 h, and 2 h; the expression level at 0 h was set as 1, and the data are expressed as mean ± SD, n = 4).

[0043] Fig.12 The effect of exogenous neutral amino acid treatment on OsAAP14 gene expression in Example 4 (D: Real-time fluorescence quantitative PCR detection results of Zhonghua 11 roots treated with 1mmol / L neutral amino acid 1 (serine 0.1mmol / L, threonine 0.2mmol / L, glycine 0.2mmol / L, alanine 0.3mmol / L, proline 0.2mmol / L) for 0h, 1h, and 2h; E: Real-time fluorescence quantitative PCR detection results of 1mmol / L neutral amino acid 2 (valine 0.2mmol / L, cysteine ​​0.1mmol / L, asparagine 0.3mmol / L, glutamine 0. A: Real-time fluorescence quantitative PCR detection results of the roots of Zhonghua 11 treated with 1mmol / L neutral amino acids 3 (methionine 0.1mmol / L, tryptophan 0.1mmol / L, leucine 0.2mmol / L, isoleucine 0.3mmol / L, phenylalanine 0.3mmol / L) for 0h, 1h, and 2h. The expression level at 0h was set as 1, and the data are expressed as mean±SD, n=4).

[0044] Fig.13This is the effect of exogenous histidine, arginine and lysine treatments on the OsAAP14 gene expression in Example 4 (D: real-time fluorescence quantitative PCR detection results of Zhonghua 11 roots treated with 0.2 mmol / L histidine for 0h, 1h, and 2h; E: real-time fluorescence quantitative PCR detection results of Zhonghua 11 roots treated with 0.2 mmol / L arginine for 0h, 1h, and 2h; F: real-time fluorescence quantitative PCR detection results of Zhonghua 11 roots treated with 0.2 mmol / L lysine for 0h, 1h, and 2h; the expression level at 0h was set as 1, and the data are expressed as mean±SD, n=4). DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with the embodiments, but the embodiments of the present invention are not limited thereto. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative labor conditions belong to the scope of protection of the present invention.

[0046] In the following specific implementation manners, gene recombination technology can be performed with reference to "Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York", and the materials and methods used can all be obtained from commercial channels.

[0047] Example 1: Construction of rice OsAAP14 gene promoter-GUS transgenic plants

[0048] (1) Acquisition of promoter fragments

[0049] DNA was extracted from rice leaf samples using the CTAB method according to the Molecular Cloning Experiment Guide. Primers were designed using Primer Primer 5.0 based on the OsAAP14 (LOC_Os04g56470) sequence provided by the Phytozome website (https: / / phytozome-next.jgi.doe.gov / ) to amplify the promoter sequence. All relevant primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. High-fidelity PCR amplification was performed using 2×Phanta Mix DNA polymerase using Zhonghua 11 genomic DNA as a template. Reaction system: 2×Phanta Mix DNA polymerase 25.0μL, OsAAP14-GUS-F and OsAAP14-GUS-R 2.0μL each, template DNA 1.0μL, supplemented to 50μL with ddH2O. Amplification procedure: 95℃ pre-denaturation for 3min, 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 2min, 35 cycles, and final extension at 72℃ for 5min. Detection was performed using 1.0% agarose gel electrophoresis to recover the PCR product. The purified and recovered product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the homologous sequences obtained from the Phytozome website to determine whether the obtained fragment was the target fragment (see agarose electrophoresis for details). Figure 1 ).

[0050] The reference sequence is shown in Figure 2 , the underline marks the amplification primer binding site. The OsAAP14 gene promoter fragment of about 2000 bp was cloned and obtained. The fragment is located 2088 bp upstream of the ATG of the OsAAP14 gene, see SEQ ID NO.1 (5'→3'):

[0051]

[0052] The sequence of OsAAP14-GUS-F is (5'→3'): TT GGATCC ATGTTAAGCCATAGTTTA (SEQ ID NO. 2, the underline indicates the restriction site);

[0053] The sequence of OsAAP14-GUS-R is (5'→3'): TT GAATTC CTAGTCCACTAACCAATT (SEQ ID NO. 3, the underline indicates the restriction site).

[0054] (2) Expression vector and transgenic plant construction

[0055] The purified OsAAP14 gene promoter fragment (with restriction sites BamHI and EcoRI at both ends) and pCAMBIA 1391Z (see the schematic diagram of the plasmid structure for details) were used to construct the plasmid. Figure 3 , the plasmid is a plant promoter detection reporter vector in the prior art, and the promoter to be studied can be cloned into the plasmid through MCS; the promoter sequence integrated into the multiple cloning site of the plasmid can be used to regulate the expression of the effector gene GUS) and enzyme digestion is performed at the same time, the reaction conditions are 37°C, 4h, and the two enzyme digestion reaction solutions are recovered and purified respectively; the two are linked using T4 DNA ligase, and the reaction conditions are 16°C, 8h; the recombinant plasmid is transformed into Escherichia coli DH5α, and the grown colonies are selected for PCR verification, and the positive colonies are screened out ( Figure 4 A), use LB liquid medium to culture the colony until OD600 is about 0.6, extract the plasmid and perform enzyme digestion verification ( Figure 4 B). According to conventional means in the prior art, the recombinant positive plasmid was transferred into Agrobacterium and transformed into ZH11 callus to obtain transformed plants. When the seedlings were relatively strong, 2 cm of leaves were cut and their genomic DNA was extracted using the CTAB method. The recombinant part of the recombinant plasmid was amplified (the front primer was located in the OsAAP14 promoter region primer sequence GUS-AAP14JD-F, and the rear primer was located in the pCAMBIA1391Z plasmid GUS gene primer sequence GUS-AAP14JD-R), and PCR amplification was performed to detect positive plants (OsAAP14 promoter-GUS transgenic plants). The seedlings verified as positive by PCR were moved to the greenhouse for planting. Figure 5 Agarose electrophoresis images of PCR amplification detection are shown.

[0056] Among them, the primer information for verification is as follows:

[0057] The sequence of GUS-AAP14JD-F is (5'→3'): TGTCGATACGGCAGAGTT (SEQ ID NO. 4);

[0058] The sequence of GUS-AAP14JD-R is (5'→3'): TTTCTACAGGACGGACGA (SEQ ID NO. 5).

[0059] Example 2: Detection of promoter-GUS expression activity in different parts of OsAAP14 promoter-GUS transgenic plants

[0060] The transgenic rice plants obtained in Example 1 were cross-bred to obtain T2 rice plants. GUS staining was performed on different parts of the T2 homozygous transgenic plants during the tillering and reproductive stages of rice. The specific process is as follows: After harvesting the successfully identified T2 seeds, they were soaked in water and cultured in a constant temperature incubator at 37°C; when the buds of the seeds grew to about 2 cm, they were sown in a 96-well plate and hydroponically cultured with a complete nutrient solution under greenhouse lighting. The formula of the rice nutrient solution adopted the conventional nutrient solution of the International Rice Research Institute (Yoshida S, Fomo DA, Cock JH, Gomez KA. 1976. Routine procedure for growing rice plants in culture solution. In: Laboratory manual for physio-logical studies of rice. Los Banos, Philippines: International Rice Research Institute, 61–66.), as shown in Table 1 below.

[0061] Table 1: Composition of conventional nutrient solution at IRRI

[0062]

[0063] The ingredients required for the complete nutrient solution are prepared from analytically pure drugs purchased from Sun Brand. When the seedlings grow to about 20 cm, they are transplanted into the soil and cultured in the greenhouse under normal lighting. When the tillering stage comes, the leaves, roots, stems and bases of the plants are stained with GUS.

[0064] During the reproductive period, GUS staining was performed on leaves, stems, roots and ears of T2 plants at the booting stage, heading stage, flowering stage, grain filling stage and maturity stage. Figure 6 AF).

[0065] The above materials were soaked in GUS staining solution and kept at 37℃ overnight. Then they were decolorized with 75% alcohol three times and stored at 4℃. The materials decolorized with alcohol were observed under a microscope. The blue part was the GUS active expression site. After staining, it was found that GUS activity was higher in the base and leaves of rice, and also expressed to a certain extent in roots, leaf sheaths and panicles, but the expression level was lowest in stems. 2mm small sections were cut from each part of rice roots, leaf sheaths, stems, leaves and panicles, and fixed in FAA fixative for 1.5h. After fixation, gradient elution was performed with 50%, 65%, 85%, and 100% ethanol, and the elution time was 10min. Then, a mixture of xylene and anhydrous ethanol with a ratio of 1:2, 1:1, and 2:1 was used to make it transparent for 10min in sequence, and finally transferred to pure xylene for 2 times, 5min each time, until each tissue part was transparent. The treated transparent tissue was placed in melted paraffin solution and soaked for 1 hour. After cooling, it was directly sliced ​​to a thickness of 5 μm. The slices were dried at 50°C and dewaxed twice with xylene, each time for 15 minutes. Finally, the slices were sealed with neutral gum and observed and photographed. It was found that the GUS gene was highly expressed in the epidermis and cortex of the roots, the mesophyll cells of the leaves, and the internal cells of the glume ( Figure 6 GK).

[0066] Example 3: Comparison of GUS expression activity in OsAAP14 promoter-GUS transgenic plants under different amino acids and hormones

[0067] The harvested OsAAP14 promoter-GUS transgenic plant T2 seeds were immersed in water and cultured in a constant temperature incubator at 37°C. When the seed buds grew to about 2 cm, they were sown into a 96-well plate and hydroponically cultured with a complete nutrient solution under greenhouse lighting. When the seedlings grew to about 3 weeks old, the nitrogen in the complete nutrient solution was replaced with 5 different exogenous amino acid treatments (i.e., NH4NO3 was not added to the complete nutrient solution, but was replaced by the amino acids in each treatment group). The acidic amino acids added were aspartic acid 0.2mmol / L, glutamate 0.3mmol / L at a final concentration of 0.5mmol / L, basic amino acids (arginine 0.2mmol / L, lysine 0.1mmol / L, histidine 0.2mmol / L) 0.5mmol / L, neutral amino acid 1 (serine 0.1mmol / L, threonine 0.2mmol / L, glycine 0.2mmol / L, alanine 0.3mmol / L, proline 0.2mmol / L) 1mmol / L, neutral amino acid 2 (valine 0.2mmol / L, cysteine ​​0.1mmol / L, asparagine 0.3mmol / L, glutamine 0.3mmol / L, tyrosine 0.1mmol / L), and neutral amino acid 3 (methionine 0.1mmol / L, tryptophan 0.1mmol / L, leucine 0.2mmol / L, isoleucine 0.3mmol / L, phenylalanine 0.3mmol / L). The roots were taken for GUS staining at 0h, 1h, and 2h of cultivation, and the GUS activity of the plants under different types of amino acid treatments was compared. After the alkaline amino acid treatment that strongly induced expression was found, 0.2mmol / L arginine, lysine, and histidine were used to treat 0h, 1h, and 2h, and the roots were taken for GUS staining at each time point to compare the GUS activity of the plants under different amino acid treatments. The results showed that the basic amino acid histidine in rice roots strongly promoted the expression of the effector gene by the promoter of the OsAAP14 gene ( Figure 7-Figure 9 ).

[0068] The above results indicate that in histidine-rich soil or when gibberellins and abscisic acid are sprayed, the expression level of the OsAAP14 gene can be increased by regulating the promoter of the OsAAP14 gene, thereby increasing nitrogen absorption and transport and promoting vegetative growth; the expression products of other downstream target genes can also be accumulated specifically in the roots (using the promoter of the OsAAP14 gene to control the expression of effector genes), thereby increasing local expression levels. Therefore, the promoter of the OsAAP14 gene has good application prospects in transgenic engineering.

[0069] Example 4: Analysis of OsAAP14 gene expression using real-time fluorescence quantitative PCR

[0070] During the reproductive period of 11 flowers in the wild type, roots, bases, leaf sheaths, stems, flag leaves, and spikes were collected, and RNA from each part was extracted using the Trizol plant total RNA extraction method. After obtaining cDNA using the HisScriptⅡQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit, real-time fluorescence PCR was performed using OsAAP14 real-time fluorescence primers 5′-CAACGCACCGGGAATCTA-3′ (SEQ ID NO.6) and 5′-GCAGCAACCCACATAACCA-3′ (SEQ ID NO.7), internal reference primers 5′-CGGTGTCATGGTCGGAAT-3′ (SEQID NO.8) and 5′-GCTCGTTGTAGAAGGTGT-3′ (SEQ ID NO.9). The 10 μL reaction system included: Taq Pro Universal SYBR qPCR Master Mix 5 μL, primers qPCR-AAP14-F and qPCR-AAP14-R 1 μL each, ddH2O 2 μL, cDNA 1 μL, and the expression level of OsAAP14 in different rice tissues was determined ( Figure 6 L). The experimental results show that the amino acid permease gene OsAAP14 is expressed in all tissues of rice, and under normal circumstances, the expression level is higher in the base and leaves of rice.

[0071] The wild-type Zhonghua 11 seedlings were simultaneously treated with the amino acid treatment method of Example 3, and the Trizol plant total RNA extraction method was used to extract root RNA, which was reverse transcribed to obtain cDNA, and the expression level of OsAAP14 under different amino acid induction was determined. The wild-type Zhonghua 11 seedlings were treated with 2 μmol / L gibberellin and abscisic acid for 0 h, 1 h, and 2 h, respectively, and the Trizol plant total RNA extraction method was used to extract root RNA, which was reverse transcribed to obtain cDNA, and the expression level of OsAAP14 under exogenous hormone induction was determined. It was further demonstrated that the basic amino acid histidine in rice roots strongly promoted the expression of the OsAAP14 gene, and it was determined that gibberellin and abscisic acid in rice roots also promoted the expression of the OsAAP14 gene ( Figure 10-13 ). When treated with exogenous amino acids and hormones, the expression level in the roots increased sharply, indicating that the OsAAP14 gene may be mainly involved in the transport of amino acids in the aboveground part of rice under normal circumstances, but when the external amino acid and hormone content increases, it responds to the external environment and further participates in the transport of amino acids in the rice roots.

[0072] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is 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 modifications and improvements can be made, which should also be regarded as the protection scope 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 specification can be used to interpret the content of the claims.

Claims

1. A rice promoter regulated by amino acids, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The use of an amino acid-regulated rice promoter in rice breeding according to claim 1, characterized in that: The method includes the following steps: S1: constructing an expression vector for expressing the effector gene, wherein the promoter of the effector gene is a rice promoter with a sequence as shown in SEQ ID NO.1; S2: The expression vector obtained in S1 is transferred into wild-type rice plants to obtain transgenic rice; S3: treating the transgenic rice with histidine.

3. The use of an amino acid-regulated rice promoter in rice breeding according to claim 2, characterized in that: In S3, the working concentration of histidine is 0.2 mmol / L.

4. The use of an amino acid-regulated rice promoter in rice breeding according to claim 3, characterized in that: In S1, rice genomic DNA is extracted, and then PCR amplification is performed on the rice genomic DNA using an upstream primer with a sequence as shown in SEQ ID NO.2 and a downstream primer with a sequence as shown in SEQ ID NO.3 to obtain a rice promoter with a sequence as shown in SEQ ID NO.1; the rice promoter with a sequence as shown in SEQ ID NO.1 is integrated into the multiple cloning site of an empty vector to obtain an expression vector; an effector gene is integrated into the empty vector, and the rice promoter is used to promote the expression of the effector gene.

5. An expression vector containing a rice promoter regulated by amino acids, characterized in that: It comprises an empty vector and a promoter fragment whose sequence is shown in SEQ ID NO.1 and is integrated into the multiple cloning site of the empty vector. The promoter fragment is used to start the expression of the effector gene. The amino acid is histidine.

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