Uses of transcription factors in copper regulation and / or rice growth regulation

Abstract

This application relates to the field of rice technology, specifically to the use of transcription factors in copper regulation and / or rice growth regulation. The nucleotide sequence of this transcription factor is located on chromosome 5 of the rice genome, 21655380-21654182 nt. The gRNA, gene knockout vector, and gene knockout kit provided in the examples can effectively knock out the OsMYB67 gene in rice plants, causing the host plant to lose the relevant function of this transcription factor, thereby promoting copper uptake, copper transport, and rice growth.

Description

Technical Field

[0001] This application relates to the field of rice technology, specifically to the use of transcription factors in copper regulation and / or rice growth regulation. Background Technology

[0002] Rice (Oryzasativa Linn.) is a major food crop in my country, and ensuring high-yield and high-quality rice is of great significance to my country's food security and the health of its residents. Copper (Cu) is an essential micronutrient for plant growth and development; Cu deficiency affects the development of flower organs and yield formation in crops. Furthermore, Cu deficiency may also reduce the copper content of grains, leading to micronutrient deficiencies in the population. The absorption, translocation, and distribution of Cu ions in rice are important factors affecting rice grain yield and the micronutrient quality of rice. However, the molecular mechanisms and factors regulating Cu absorption, translocation, and distribution in rice are currently unclear. Summary of the Invention

[0003] Therefore, embodiments of this application provide the use of transcription factors in copper regulation and / or rice growth regulation. The transcription factor is OsMYB67 (RiceGenomeAnnotationProject, MUS-RGAP, http: / / rice.uga.edu / , gene location number LOC_Os05g37060, chromosome 5 21655380-21654182nt, full-length gene 1199bp, coding region 549bp, encoding 182 amino acids; the OsMYB67 gene contains one 5' UTR, one 3' UTR, two exons, and one intron).

[0004] Therefore, the embodiments of this application disclose at least the following technical solutions:

[0005] In one aspect, the embodiments disclose a gRNA, as shown in SEQ ID NO:1 and / or 2. This gRNA targets the coding gene of transcription factor OsMYB67 to guide Cas9 to target and cleave the coding gene of transcription factor OsMYB67.

[0006] Secondly, the vector combination for knocking out the OsMYB67 gene includes a gRNA expression vector and a Cas9 expression vector. The gRNA expression vector carries a guide sequence targeting the OsMYB67 gene, including gRNA1 as shown in SEQ ID NO:1 and gRNA2 as shown in SEQ ID NO:2. The Cas9 expression vector carries the coding sequence for the Cas9 nuclease as shown in SEQ ID NO:3. This vector combination can be introduced into rice to transcribe gRNA and translate it to form the Cas9 nuclease. The gRNA can guide Cas9 to target and cleave the coding gene of the transcription factor OsMYB67, thereby causing the coding gene to lose its protein expression function and thus causing the host rice to lose the related function of the transcription factor.

[0007] In some embodiments, the gRNA expression vector comprises a pGTR vector with the nucleotide sequence shown in SEQ ID NO:1 inserted and a pGTR vector with the nucleotide sequence shown in SEQ ID NO:2 inserted. In some embodiments, the Cas9 expression vector is a pRGEB32 vector with the nucleotide sequence shown in SEQ ID NO:3 inserted.

[0008] Thirdly, the embodiments disclose a gene knockout kit. This kit comprises the gRNA described in the first aspect and / or the vector combination described in the second aspect.

[0009] Fourthly, the embodiments disclose a method for constructing rice mutants. This method includes: introducing the vector combination from the second aspect into wild-type rice lines; and screening positive lines and mutants from their transformants.

[0010] In some embodiments, the screening step includes: using Cas9-F (SEQ ID NO:4) and Cas9-R (SEQ ID NO:5) as primer pairs, PCR amplification of the genomic DNA of the T0 generation line, and if the target band as shown in SEQ ID NO:3 is amplified, it is identified as a positive line.

[0011] In some embodiments, the screening step further includes: using MYB67-Test-F (SEQ ID NO:6) and MYB67-Test-R (SEQ ID NO:7) as primer pairs to amplify the genomic DNA of the line to be screened by PCR, sequencing the obtained PCR amplification product, and comparing the sequencing results of the positive line with the sequencing results of the wild line to determine whether the positive line is a mutant line.

[0012] In some embodiments, the screening step further includes: screening T2 generation lines from T1 generation lines that have the OsMYB67 mutation but do not have the target band as shown in SEQ ID NO:3, performing sequencing verification, and screening from these lines to obtain homozygous lines with stable OsMYB67 mutations.

[0013] Fifthly, the embodiments disclose the use of the gRNA described in the first aspect, the vector combination described in the second aspect, or the gene knockout kit described in the third aspect, the use of which includes promoting at least one of promoting copper absorption in rice, promoting copper translocation in rice, and promoting rice growth.

[0014] This application reveals that the OsMYB67 gene negatively regulates the expression of the OsCOPT7 gene by targeting the OsCOPT7 gene promoter, thereby affecting copper uptake in rice plants. Conversely, the OsMYB67 gene positively regulates the expression of the OsHMA9 gene by targeting the OsHMA9 gene promoter, thus influencing the allocation of copper to the panicle and grains in rice plants, thereby promoting a shorter heading period and increasing rice yield.

[0015] The gRNA, gene knockout vector, and gene knockout kit provided in this application can be effectively introduced into rice plants to knock out the OsMYB67 gene, causing the host plant to lose the relevant function of this transcription factor, thereby promoting the absorption of copper by rice, promoting the translocation of copper by rice, and promoting rice growth. Attached Figure Description

[0016] Figure 1 The target sequence alignments of the wild-type strain and mutant strains (C67-10, C67-11) provided in the examples are shown. Red indicates PAM sequences, and purple * indicates deletions.

[0017] Figure 2 The results of copper absorption and translocation in wild-type strains (WT) and mutant strains (C67-10, C67-11) under different copper application rates provided in the examples. Figure 2 A represents the copper content in the roots and shoots of WT, C67-10, and C67-11 in the -Cu group. Figure 2 B represents the copper content in the roots and shoots of WT, C67-10, and C67-11 in the +Cu group. Figure 2 C represents the copper transport amount in the -Cu and +Cu groups (WT, C67-10, and C67-11).

[0018] Figure 3 The phenotypic comparison results of wild-type strains (WT) and mutant strains (C67-10, C67-11) under different copper application rates provided in the examples. Figure 3a represents the growth phenotypes of WT, C67-10, and C67-11 in the -Cu group (0 mg / kg). Figure 3 b represents the growth phenotypes of WT, C67-10, and C67-11 in the +Cu group (0.5 mg / kg). Figure 3 c represents the ear length distribution ratio of each line in the -Cu group (0 mg / kg) and the +Cu group (0.5 mg / kg). Figure 3 Dry weight of each strain in the d-Cu group (0 mg / kg) and the +Cu group (0.5 mg / kg). Data are expressed as mean ± standard deviation (n = 4). * indicates significant difference compared with wild-type strains (T-test, *P < 0.05).

[0019] Figure 4 Copper content (a) and copper accumulation (b) of wild-type strains (WT) and mutant strains (C67-10, C67-11) under different copper application rates (-Cu group (0 mg / kg) and +Cu group (0.5 mg / kg)) provided in the examples. Data are expressed as mean ± standard deviation (n = 4). * indicates a significant difference compared to wild-type strains (T-test, *P < 0.05).

[0020] Figure 5 The copper distribution ratio in different parts of the plant in wild-type strains (WT) and mutant strains (C67-10, C67-11) under -Cu (0.2 mg / kg) and +Cu (0.6 mg / kg) treatments provided in the examples.

[0021] Figure 6 Phenotypic diagrams of the whole plant, spike, and berries of the wild-type strain (WT) and mutant strains (C67-10, C67-11) provided in the examples.

[0022] Figure 7 Statistical results for plant height (A), effective panicle number (B), panicle length (C), seed setting rate (D), thousand-grain weight (E), yield (F), grain length (G), and grain width (H) of the wild-type strain (WT) and mutant strains (C67-10, C67-11) provided in the examples are shown in the figure. Data are expressed as mean ± standard deviation (n=4). * indicates a significant difference compared with the wild-type strain (T-test, *P<0.05).

[0023] Figure 8 Dry weight (A), node copper content (B), copper accumulation (C), aboveground copper accumulation (D), and polymetallic concentration in grains (E) of wild-type strains (WT) and mutant strains (C67-10, C67-11) provided for the examples. Data are expressed as mean ± standard deviation (n=4). * indicates significant difference compared to wild-type strains (T-test, *P<0.05).

[0024] Figure 9 The expression levels of copper transport-related genes (OsATX1,a; OsYSL16,b; OsHMA5,c; OsHMA9,d; OsCOPT1,e; OsCOPT5,f) in wild-type strains (WT) and mutant strains (C67-10, C67-11) under -Cu (0 mg / kg) and +Cu (0.5 mg / kg) treatments provided in the examples are shown.

[0025] Figure 10 The expression levels of flowering-related genes (OsHd1F,a; OsHd3a,b) in spikes of wild-type lines (WT) and mutant lines (C67-10, C67-11) under -Cu (0 mg / kg) and +Cu (0.5 mg / kg) treatments provided in the examples.

[0026] Figure 11 This is a schematic diagram of the cis-acting element of the OsMYB67 promoter of copper-related genes provided in the example.

[0027] Figure 12 The results of a yeast one-hybrid assay for OsMYB67 and cis-acting elements of copper-related gene promoters provided in this example are shown. Yeast cells containing pGADT7-Rec2-OsMYB67 and pHIS2-cis-element (OsCOPT2, OsCOPT3, OsCOPT4, OsCOPT7, OsHMA5, and OsHMA9) were co-cultured and then grown on SD / -Leu-Trp-His selective medium containing 0 or 60 mM 3-AT.

[0028] Figure 13 The transient expression test results of tobacco interaction between OsMYB67 and the OsCOPT7 and OsHMA9 promoters provided in the examples. Figure 13 a and Figure 13 b,35S:OsMYB67 and proOsHMA9:GUS GUS activity in tobacco, injected alone or co-injected. Figure 13 c and Figure 13 d. The relative LUC activity of tobacco plants injected alone or in combination with pGreenII-62-SK-OsMYB67 and proOsCOPT7:LUC, and proOsHMA9:LUC. Data are expressed as mean ± standard deviation (n = 4). * indicates a significant difference compared to wild-type plants (T-test, *P < 0.05). Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0030] Construction of OsMYB67 knockout vector

[0031] The embodiments provide a combination of vectors for knocking out the OsMYB67 gene, including a gRNA expression vector and a Cas9 expression vector. The gRNA expression vector carries a guide sequence targeting the OsMYB67 gene. The guide sequence includes gRNA1 as shown in SEQ ID NO:1 and gRNA2 as shown in SEQ ID NO:2. The Cas9 expression vector carries a coding sequence for expressing the Cas9 nuclease as shown in SEQ ID NO:3.

[0032] In some embodiments, using pGTR vector (#63143, addgene) as the base vector, the nucleotide sequence shown in SEQ ID NO:1 was inserted into the pGTR vector through steps such as PCR amplification, enzyme digestion, T4 ligase, transformation into E. coli, and screening to obtain a pGTR vector expressing gRNA1. Using pGTR vector as the base vector, the nucleotide sequence shown in SEQ ID NO:2 was inserted into the pGTR vector through steps such as PCR amplification, enzyme digestion, T4 ligase, transformation into E. coli, and screening to obtain a pGTR vector expressing gRNA2.

[0033] In some embodiments, the pRGEB32 vector (LM-8140, LMAIBio) carries a coding sequence as shown in SEQ ID NO:3.

[0034] Constructing OsMYB67 mutant rice plants

[0035] 1. Knockout process

[0036] Using Agrobacterium-mediated transformation, vector combinations for knocking out the OsMYB67 gene were transformed into wild-type rice plants (Jingdao Zhonghua 11, Oryza sativa L. sspjaponicacv Zhonghua 11, which was developed by the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences in 1979 using Jingfeng No. 5 / Tetepu / Fujin for flower cultivation, and bred in 1984, and approved by the Tianjin Variety Approval Committee in 1989), and the transformants were screened.

[0037] The screening steps include: using Cas9-F (SEQ ID NO:4) and Cas9-R (SEQ ID NO:5) as primer pairs, PCR amplification of the genomic DNA of the T0 generation line is performed. If the target band shown in SEQ ID NO:3 is amplified, it is identified as a positive line.

[0038] The screening steps also include: using MYB67-Test-F (SEQ ID NO:6) and MYB67-Test-R (SEQ ID NO:7) as primer pairs, PCR amplification of the genomic DNA of the lines to be screened, sequencing of the obtained PCR amplification products, and comparison of the sequencing results of positive lines with those of wild-type lines to determine whether the positive line is a mutant line.

[0039] The screening steps also include: screening T2 generation lines from T1 generation lines that have the OsMYB67 mutation but do not have the target band as shown in SEQ ID NO:3, performing sequencing verification again, and screening out homozygous lines with stable OsMYB67 mutations, C67-10 and C67-11.

[0040] 2. Results

[0041] like Figure 1 As shown, strain C67-10 has a 1nt nucleotide deletion at the first target site and a 4nt nucleotide deletion at the second target site. Strain C67-11 has a 1nt nucleotide deletion at both the first and second target sites.

[0042] The absence of OsMYB67 promotes Cu uptake and transport.

[0043] 1. Potted plant test

[0044] Pot culture tests were conducted at the Micronutrient Center of Huazhong Agricultural University. Mutant lines (C67-10 and C67-11) and wild-type plants were cultured in normal nutrient solution for 14 days before being transferred to 18 kg (20 L black square container) soil pots for further cultivation. The potting soil contained 5.03 g / kg organic matter, 0.32 g / kg total nitrogen, 2.1 mg / kg available phosphorus, 0.2 mg / kg available copper, and had a pH of 4.3. Simultaneously, nitrogen fertilizer urea (containing 46.4% N) was applied at 0.43 g / kg; phosphorus fertilizer superphosphate (containing 12% P2O5) at 1.25 g / kg; and potassium fertilizer potassium chloride (containing 60% K2O) at 0.33 g / kg.

[0045] The treatment groups included the -Cu group and the +Cu group. Plants in the -Cu group were treated with the above-mentioned basic fertilizer only. Plants in the +Cu group were additionally treated with 0.4 mg / kg Cu. Plants in each group were cultured to the flowering stage, and the growth phenotype at the flowering stage was examined. (1) One tiller of the plant was taken, and the stem, leaves and spike were separated and dried to constant weight to determine the copper content; (2) Fresh samples of the flowering spikes were taken, stored at -80℃, and the expression level of Cu-related genes was determined. This test included 4 biological replicates.

[0046] Referring to the method for determining the metal content in plants and soil in "Soil Agrochemical Testing" (Bao Shidan 2000), the plant sample was ground into powder, and 0.10xxg was weighed and placed in a digestion tube. Then, 2mL of mixed acid (perchloric acid: nitric acid = 1:4) was added. After cold digestion overnight, the sample was digested in a low-temperature digestion oven at 120℃ until it turned colorless or pale yellow (about 24 hours). After cooling, the volume was adjusted to 20mL with deionized pure water, shaken well and filtered. The copper content was determined using a WFX-ID atomic absorption spectrophotometer.

[0047] 2. Results

[0048] like Figure 2 As shown, in the -Cu group, the root copper content of the mutant line (93.5%) was significantly higher than that of the wild plant (83%), the aboveground copper content of the mutant line (68.7%) was significantly higher than that of the wild plant (66.7%), and the copper uptake of the mutant line (114%) was significantly higher than that of the wild plant (62.8%).

[0049] like Figure 2 As shown, in the +Cu group, the root copper content of the mutant strain (213%) was significantly higher than that of the wild-type strain (200%), the aboveground copper content of the mutant strain (90.9%) was significantly higher than that of the wild-type strain (86.3%), and the copper uptake of the mutant strain (63.4%) was significantly higher than that of the wild-type strain (48%).

[0050] It is evident that during the vegetative growth stage, the absence of OsMYB67 can significantly increase the copper content in rice roots and aboveground parts, as well as the copper absorption of the plant, but does not affect the transport of Cu from the roots to the aboveground parts.

[0051] The absence of OsMYB67 shortens the flowering time of rice.

[0052] Because OsMYB67 was expressed at high levels in the panicle and nodes during the flowering stage of rice, it is speculated that OsMYB67 may play an important role during flowering. Therefore, pot tests were also conducted, including a -Cu group and a +Cu group. The -Cu group received 0.2 mg / kg Cu ​​as part of the basal fertilizer, while the +Cu group received an additional 0.4 mg / kg Cu. The soil used had an available copper content of 0.2 mg / kg, classifying it as copper-deficient. Phenotypic differences and copper content were observed between the mutant and wild-type plants at the flowering stage.

[0053] like Figure 3 As shown in ab, under different Cu levels, the mutant lines exhibited an earlier heading period, 5 days earlier than the wild-type lines. For example... Figure 3 According to the CD analysis, in the -Cu group, the proportions of wild plants with ear lengths greater than 10cm, between 5-10cm, and less than 5cm were all 33.30%, while the ear lengths of the mutant lines were all greater than 10cm. In the +Cu group, the proportions of wild plants with ear lengths greater than 10cm, between 5-10cm, and less than 5cm were 33.3%, 50%, and 16.7%, respectively; the ear lengths of the mutant line C67-10 were all greater than 10cm, and in C67-11, 87.5% of the ear lengths were greater than 10cm, and 12.5% ​​were between 5-10cm. Therefore, Figure 3 The results showed that the mutant lines flowered earlier, indicating that OsMYB67 affects the flowering time.

[0054] like Figure 4 As shown, the copper content and cumulative copper amount in the shoots of the mutant strain were both lower than those of the wild strain.

[0055] like Figure 5 As shown, the allocation ratio of Cu in the ears of the mutant lines was 16% and 11%. The allocation ratio of copper in the ears of wild-type lines was 12%, while that in the ears of mutant lines was 15% and 14%, representing increases of 3% and 2%, respectively.

[0056] These results indicate that mutant lines can increase the distribution of Cu to panicle tissues in rice.

[0057] Effects of OsMYB67 on Rice Yield and Copper Content

[0058] like Figure 6 As shown, compared with the wild plant, the number of effective spikes in the mutant line (approximately 11 spikes / plant) was significantly higher than that in the wild plant (approximately 7 spikes / plant).

[0059] like Figure 7 As shown, the thousand-grain weight of the mutant line (25.70262g) was significantly higher than that of the wild line (21.50797g).

[0060] like Figure 8As shown, the copper concentration in the grains of the mutant line (4.151729 μg / g) was significantly higher than that in the wild-type plant (3.072811 μg / g), indicating an increase. The copper concentration in the rachis of the mutant line (2.025472 μg / g) was significantly higher than that in the wild-type plant (1.200539 μg / g). The copper concentration in the nodes of the mutant line (5.575697 μg / g) was significantly higher than that in the wild-type plant (2.896691 μg / g). The cumulative copper content in the grains of the mutant line (157.8232 μg / plant) was significantly higher than that in the wild-type plant (96.20034 μg / plant). Therefore, the cumulative copper content in the aboveground parts of the mutant line was significantly higher than that in the wild-type plant.

[0061] These results indicate that the OsMYB67 mutation can increase the number of effective panicles rather than alter the grain shape, thereby increasing rice yield. The OsMYB67 mutation can also significantly increase the number of effective panicles in rice, further increasing grain yield.

[0062] Effects of OsMYB67 on copper transport and flowering-related gene expression

[0063] 1. Quantitative Real-Time PCR

[0064] The expression levels of copper transport genes in OsMYB67 overexpression and mutant spikelets were tested using quantitative real-time PCR.

[0065] Total RNA was extracted from tissues using the TriZol method. The specific steps are as follows: Plant samples (approximately 100 mg) were cut and placed in a 2 mL centrifuge tube in liquid nitrogen. Two small steel balls were added to the centrifuge tube, and the sample was ground in a chilled grinder for 1-3 minutes. 1 mL of pre-chilled TriZol (4°C) was added at a ratio of 1 mL / 100 mg, and the mixture was vortexed and incubated at room temperature for 10 minutes. 0.2 mL of chloroform was added per mL of TriZol. The centrifuge tube was capped, vortexed for 15 seconds, and then incubated at room temperature for 5 minutes. The mixture was then centrifuged at 12,000 × g for 15 minutes at 4°C. The upper aqueous phase was transferred to a 1.5 mL RNase-free centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 10 minutes and then centrifuged at 12,000 × g for 10 minutes at 4°C. Remove the supernatant, wash the RNA precipitate 3-5 times with 75% ethanol (prepared with 0.1% DEPC water after high-temperature sterilization), and centrifuge at 7,500×g for 5 min at 4°C. Dry the RNA precipitate in a laminar flow hood, and dissolve the RNA in 0.1% DEPC water after high-temperature sterilization.

[0066] RNA concentration and absorbance were measured using a NanoDrop 2000c UV spectrophotometer. High-quality RNA typically has an OD260 / OD280 ratio between 2.0 and 2.2. If OD260 / OD280 is greater than 2.2, the RNA may be degraded; if OD260 / OD280 is less than 2.0, the RNA may be contaminated with genomic DNA, protein, or phenolic compounds. RNA quality was assessed using 1% (w / v) agarose gel electrophoresis. High-quality RNA, under 1% agarose gel electrophoresis, will show three rRNA bands sequentially: 28S, 18S, and 5S, with decreasing brightness in that order. It is crucial that all centrifuge tubes, pipette tips, and related solutions be free of RNase contamination.

[0067] Referencing Yisheng Biotechnology Co., Ltd. The 1stStrand cDNA first-strand synthesis kit reverses RNA into cDNA; refer to the kit instructions for specific methods. Real-time PCR (RT-qPCR) is used for testing. The qPCR SYBR Green Master Mix kit (Shanghai Yisheng Biotechnology Co., Ltd.) and the QuantStudio™ 6 Flex System (Applied Biosystems, Foster City, CA) quantitative PCR instrument used the rice OsUbiquitin gene as an internal control. Quantitative expression of the gene was determined using 2... -ΔΔCT The relative expression level of the target gene was calculated using the geometric mean of the formula (Pattynetal2002). The RT-qPCR reaction system was as follows: 2.0 μL cDNA template, 0.2 μL each of forward and reverse primers, 5.0 μL SYBR Green I (2×) dye, and 2.6 mL ddH2O added to a 10 μL system. The RT-qPCR reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 20 s, for 40 cycles. The melting curve was set to the instrument's default settings: 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s.

[0068] The primer sequences for the corresponding genes are shown in Table 1.

[0069] Table 1

[0070]

[0071]

[0072] 2. Effects on genes related to copper transport

[0073] like Figure 9As shown, under Cu treatment, the expression level of OsCOPT7 in the mutant line was significantly higher than that in the wild-type line, while the expression levels of OsCOPT5, OsHMA5, OsHMA9 and OsYSL16 were significantly lower than those in the wild-type line.

[0074] like Figure 9 As shown, under the +Cu treatment condition, the expression levels of OsCOPT7 and OsYSL16 in the mutant line were significantly higher than those in the wild line, while the expression levels of OsCOPT1, OsCOPT2, OsCOPT5, OsHMA5 and OsHMA9 were significantly lower than those in the wild line.

[0075] The above results indicate that the OsMYB67 mutation significantly affects the expression of copper transport-related genes.

[0076] 3. Effects on the expression levels of flowering genes

[0077] like Figure 10 As shown, under the +Cu treatment condition, the expression level of flowering-related gene OsHd1F in the mutant line was significantly higher than that in the wild line, while the expression level of OsHd3a was significantly lower than that in the wild line (except for C67-11).

[0078] The above results indicate that OsMYB67 does not affect rice flowering time by regulating the expression of flowering genes OsHd1F and OsHd3a. Discovery of MYB Functioning Elements in Rice Copper Transport Gene Promoters

[0079] MYB transcription factors function by binding to the MYB cis-acting element in the promoter region of downstream target genes; therefore, genes whose promoter regions contain MYB cis-acting elements are likely to be their downstream target genes. For example... Figure 11 As shown, the promoter sequences of copper transport genes were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and the promoter regions of copper transport genes were tested using the PlantCARE website (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). It was found that the promoter regions of 8 genes contained abundant MYB functional elements.

[0080] OsMYB67 interacts with the promoter sequence of Cu-related genes.

[0081] 1. Yeast single-hybrid test

[0082] The AD vector is a recombinant whole obtained by ligating the cDNA sequence (SEQ ID NO:28) of the OsMYB67 gene into the pGADT7-Rec2 plasmid (catalog number QCP0234; Shanghai Qincheng Biotechnology Co., Ltd.) via homologous recombination.

[0083] The BD vector is a recombinant vector obtained by inserting the candidate promoter of MYB into the pHIS2 plasmid (Shanghai Yaji Biotechnology Co., Ltd.; catalog number YS-1432F). The candidate promoters are the promoters of the OsCOPT2 gene (SEQ ID NO:29), the OsCOPT3 gene (SEQ ID NO:30), the OsCOPT4 gene (SEQ ID NO:31), the OsCOPT7 gene (SEQ ID NO:32), the OsHMA5 gene (SEQ ID NO:33), and the OsHMA9 gene (SEQ ID NO:34).

[0084] The AD and BD vectors were co-transformed into competent Y187 yeast cells, and the transformed yeast was subjected to a one-hybrid test to verify whether the transcriptional reporter gene was activated when the two vectors were co-transformed with their respective empty vectors.

[0085] In the positive group, Y187 yeast containing AD and BD vectors could grow on SD-3+3-AT medium plates.

[0086] In the negative group, the AD vector containing both the AD vector and the empty pHIS2 vector could not grow on SD-3+3-AT medium plates.

[0087] 2. Results

[0088] like Figure 12 As shown, all yeasts can grow normally on SD / -Leu-Trp-His triple-deficient medium without 3-AT. When 60 mM 3-AT is added to the triple-deficient medium, the positive controls (pGADT7-53 and pHIS2-53) can grow normally, but the negative controls (pGADT7-53 and pHIS2) cannot grow normally. Yeasts transformed with both the AD vector and any one of the BD vectors can grow on the selective medium containing 60 mM 3-AT, indicating that OsMYB67 can interact with OsCOPT2, OsCOPT3, OsCOPT4, OsCOPT7, OsHMA5, and OsHMA9 in the yeast system.

[0089] OsMYB67 activates the expression of Cu-related genes.

[0090] 1. Tobacco transient expression test

[0091] The 35S:OsMYB67 vector is a recombinant vector in which the cDNA sequence of the OsMYB67 gene is cloned into the pCAMBIA1301S vector.

[0092] The proOsCOPT2:GUS vector is a recombinant vector in which the OsCOPT2 promoter (SEQ ID NO:29) is cloned into the DX2181b vector (BioVectorNTCC).

[0093] The proOsCOPT3:GUS vector is a recombinant vector in which the OsCOPT3 promoter (SEQ ID NO:30) is cloned into the DX2181b vector (BioVectorNTCC).

[0094] The proOsCOPT4:GUS vector is a recombinant vector in which the OsCOPT4 promoter (SEQ ID NO:31) is cloned into the DX2181b vector (BioVectorNTCC).

[0095] The proOsCOPT7:GUS vector is a recombinant vector in which the OsCOPT7 promoter (SEQ ID NO:32) is cloned into the DX2181b vector (BioVectorNTCC).

[0096] The proOsHMA5:GUS vector is a recombinant vector in which the OsHMA5 promoter (SEQ ID NO:33) is cloned into the DX2181b vector (BioVector NTCC).

[0097] The proOsHMA9:GUS vector is a recombinant vector in which the OsHMA9 promoter (SEQ ID NO:34) is cloned into the DX2181b vector (BioVector NTCC).

[0098] The proOsCOPT7:LUC vector is a recombinant vector in which the OsCOPT7 promoter (SEQ ID NO:32) is cloned into the pGreenII0800-LUC vector (V010545, Newpro Biotechnology).

[0099] The proOsHMA9:LUC vector is a recombinant vector in which the OsHMA9 promoter (SEQ ID NO:34) is cloned into the pGreenII0800-LUC vector (V010545, Newpro Biotechnology).

[0100] Agrobacterium containing these recombinant vectors was monoclonal in 1 mL of LB broth containing the corresponding antibiotic and incubated at 28°C and 250 rpm for approximately 18 hours. Solution A was prepared by adding 15 mL of LB broth containing the corresponding antibiotic, 300 μL of 0.5 MME, and 6 mL of 100 mAs. 50 μL of Agrobacterium culture was added to every 5 mL of LB broth and incubated at 28°C and 250 rpm until OD600 = 1.0 (approximately 12-18 hours). The cells were centrifuged at 4000 rpm for 10 minutes at room temperature, and the supernatant was discarded. The cells were resuspended in approximately an equal volume of Solution A until OD600 = 1.0 (adjusted to 0 using the above Solution A, and OD600 was measured). The bacterial cultures to be co-injected were premixed in sterile 50 mL centrifuge tubes (using Solution A to ensure uniform concentration of each culture) and incubated at room temperature for at least 3 hours. Tobacco Injection: Inject the bacterial solution into the vigorous growth stage of Nicotiana benthamiana from the back (inject an equal volume of bacterial solution into an equal volume of tobacco leaf) and attach a label. After 24-48 hours, sample with liquid nitrogen and measure GUS enzyme activity or perform GUS staining. Detect luciferase activity according to the instructions of the Dual-Luciferase Reporter Gene Detection Kit (VazymE).

[0101] 2. Results

[0102] The 35S:OsMYB67 vector and the promoter sequences of six downstream candidate genes were fused with a GUS reporter vector and transformed into tobacco leaves individually and co-transformed. Samples were taken 48 hours later for GUS staining and GUS activity detection.

[0103] like Figure 13 a and Figure 13 As shown in b, almost no staining was observed when 35S:OsMYB67 or the vector fused with the downstream GUS gene was injected alone. When co-injected, only when 35S:OsMYB67 and proOsHMA9:GUS were co-injected into tobacco did significant staining be observed, consistent with the GUS activity assay results. No staining was observed when 35S:OsMYB67 was injected with proOsCOPT4:GUS, proOsCOPT7:GUS, or proOsHMA5:GUS. This suggests that OsMYB67 may activate the expression of OsHMA9 by binding to its promoter, while OsMYB67 cannot activate the expression of OsCOPT4, OsCOPT7, and OsHMA5, suggesting that OsMYB67 may inhibit the expression of these genes.

[0104] like Figure 13 c and Figure 13As shown in d, co-expression of OsMYB67 and OsHMA9 promoters enhanced LUC activity, while co-expression of OsMYB67 and OsCOPT7 promoters reduced LUC activity, indicating that OsMYB67 can activate OsHMA9 gene expression but inhibit OsCOPT7 gene expression.

[0105] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. Use of gRNA or vector combination of OsMYB67 gene knockout, the use including at least one of promoting copper uptake and promoting copper translocation in rice. in, The nucleotide sequence of the gRNA is shown in SEQ ID NO:1 and / or 2; The vector combination includes a gRNA expression vector and a Cas9 expression vector. The gRNA expression vector carries a guide sequence targeting the OsMYB67 gene. The guide sequence includes gRNA1 as shown in SEQ ID NO:1 and gRNA2 as shown in SEQ ID NO:

2. The Cas9 expression vector carries a coding sequence for expressing the Cas9 nuclease as shown in SEQ ID NO:

3.

2. The use of the gRNA or vector combination for knocking out the OsMYB67 gene according to claim 1, wherein the gRNA expression vector comprises a pGTR vector having the nucleotide sequence shown in SEQ ID NO:1 inserted therein and a pGTR vector having the nucleotide sequence shown in SEQ ID NO:2 inserted therein.

3. The use of the gRNA or vector combination for knocking out the OsMYB67 gene according to claim 1, wherein the Cas9 expression vector is a pRGEB32 vector with the nucleotide sequence shown in SEQ ID NO:3 inserted.

4. The use of the combination of gRNA for knocking out the OsMYB67 gene or vector for knocking out the OsMYB67 gene according to claim 1, comprising: The vector combination was introduced into wild-type rice lines; as well as Positive lines and mutants were screened from its transformants.

5. The use of the combination of gRNA knockout OsMYB67 gene knockout or vector knockout OsMYB67 gene knockout according to claim 4, wherein the screening step includes: Using Cas9-F and Cas9-R as primer pairs, PCR was used to amplify the genomic DNA of the T0 generation line. If the target band was amplified, the line was identified as a positive line. The nucleotide sequence of Cas9-F is shown in SEQ ID NO:4, and the nucleotide sequence of Cas9-R is shown in SEQ ID NO:

5.

6. The use of the combination of gRNA knockout OsMYB67 gene or vector knockout OsMYB67 gene knockout according to claim 5, wherein the screening step further comprises: Using MYB67-Test-F and MYB67-Test-R as primer pairs, genomic DNA of the lines to be screened was amplified by PCR. The PCR amplification products were sequenced, and the sequencing results of positive lines were compared with those of wild-type lines to determine whether the positive lines were mutant lines. The nucleotide sequence of MYB67-Test-F is shown in SEQ ID NO:6, and the nucleotide sequence of MYB67-Test-R is shown in SEQ ID NO:

7.

7. The use of the combination of gRNA knockout OsMYB67 gene knockout or vector knockout OsMYB67 gene knockout according to claim 6, wherein the screening step further comprises: T2 generation lines with OsMYB67 mutation but without the target band shown in SEQ ID NO:3 were screened from T1 generation lines and sequenced again for verification. Homozygous lines with stable OsMYB67 mutation were obtained from these lines.

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