Application of transcription factor in copper regulation and / or rice growth regulation

Abstract

The invention relates to the technical field of rice, in particular to application of a transcription factor in copper regulation and / or rice growth regulation. The nucleotide sequence of the transcription factor is located in the No. 5 chromosome 21655380-21654182nt of a rice genome. According to the gRNA, the gene knockout vector and the gene knockout kit provided by the embodiment of the invention, the OsMYB67 gene of a rice plant can be effectively knocked out, so that the host plant loses related functions of a transcription factor, and then copper absorption, copper transfer and rice growth are promoted.

Description

Technical Field

[0001] The present application relates to the field of rice technology, and in particular to the use of transcription factors in copper regulation and / or rice growth regulation. Background Art

[0002] Rice (Oryza sativa Linn.) is a major food crop in my country. Ensuring high yield and high quality of rice is of great significance to my country's food security and residents' health. Copper (Cu) is a trace element necessary for plant growth and development. Cu deficiency affects the development of floral organs and yield formation of crops. At the same time, Cu deficiency may also reduce the copper content in grains and cause trace element deficiency in residents. The absorption, transport and distribution of Cu ions in rice is an important factor affecting rice grain yield and rice trace element quality. However, the molecular mechanism and factors regulating Cu absorption, transport and distribution in rice are still unclear. Summary of the invention

[0003] To this end, the present application provides the use of a transcription factor in copper regulation and / or rice growth regulation. The transcription factor is OsMYB67 (Rice Genome Annotation Project, MUS-RGAP, http: / / rice.uga.edu / , gene location number LOC_Os05g37060, chromosome 5 21655380-21654182nt, gene length 1199bp, coding region length 549bp, encoding 182 amino acids; OsMYB67 gene contains 1 5' UTR, 1 3' UTR, 2 exons and 1 intron).

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

[0005] In a first aspect, the embodiment discloses a gRNA, as shown in SEQ ID NO: 1 and / or 2. The gRNA targets the gene encoding the transcription factor OsMYB67 to guide Cas9 to perform targeted cleavage on the gene encoding the transcription factor OsMYB67.

[0006] In the second aspect, a vector combination for knocking out the OsMYB67 gene includes a gRNA expression vector and a Cas9 expression vector, wherein the gRNA expression vector carries a guide sequence targeting the OsMYB67 gene, wherein the guide sequence includes gRNA1 as shown in SEQ ID NO: 1 and gRNA2 as shown in SEQ ID NO: 2, and the Cas9 expression vector carries a coding sequence as shown in SEQ ID NO: 3 for expressing the Cas9 nuclease. The vector combination can be used to be introduced into rice to transcribe to form gRNA and translate to express the nuclease Cas9, wherein the gRNA can guide Cas9 to perform targeted cutting on the coding gene of the transcription factor OsMYB67, so as to cause the coding gene to lose the protein expression function, thereby causing its host rice to lose the related functions of the transcription factor.

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

[0008] In the third aspect, the embodiment discloses a gene knockout kit. The kit includes the gRNA described in the first aspect and / or the vector combination described in the second aspect.

[0009] In a fourth aspect, the embodiment discloses a method for constructing a rice mutant, which comprises: introducing the vector combination of the second aspect into a wild rice strain; and screening positive strains and mutants from the transformants thereof.

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

[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 a primer pair, PCR amplifying the genomic DNA of the strain to be screened, sequencing the obtained PCR amplification products, and comparing the sequencing results of the positive strain with the sequencing results of the wild strain to determine whether the positive strain is a mutant strain.

[0012] In some embodiments, the screening step further comprises: screening T2 generation strains with OsMYB67 mutation but without the target band shown in SEQ ID NO: 3 from the T1 generation strains, performing sequencing verification, and screening out homozygous strains with stable OsMYB67 mutations.

[0013] In the fifth aspect, 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, and the uses include at least one of promoting the absorption of copper by rice, promoting the transport of copper by rice, and promoting the growth of rice.

[0014] The present application found that the OsMYB67 gene negatively regulates the expression of the OsCOPT7 gene by targeting the OsCOPT7 gene promoter to affect the copper absorption of rice plants. The OsMYB67 gene positively regulates the expression of the OsHMA9 gene by targeting the OsHMA9 gene promoter to affect the distribution of copper to the panicles and grains of rice plants, thereby shortening the heading period of rice and increasing rice yield.

[0015] The gRNA, gene knockout vector and gene knockout kit provided in the embodiments of the present application can be effectively introduced into rice plants to knock out their OsMYB67 gene, causing the host plant to lose the relevant functions of the transcription factor, thereby promoting the absorption of copper by rice, promoting the transport of copper by rice and promoting the growth of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The target sequence alignment of the wild strain and mutant strain (C67-10, C67-11) provided in the examples. The red color represents the PAM sequence, and the purple * represents a deletion.

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

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

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

[0020] Figure 5 The copper distribution ratios in various parts of the wild strain (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 are shown.

[0021] Figure 6 The phenotypes of the whole plant, ear and fruit of the wild strain (WT) and mutant strains (C67-10, C67-11) provided in the examples are shown.

[0022] Figure 7 Statistical results of plant height (A), effective ear number (B), ear length (C), fruit set rate (D), 1000-grain weight (E), yield (F), grain length (G) and grain width (H) of the wild strain (WT) and mutant strains (C67-10, C67-11) provided in the examples. Data are expressed as mean ± standard deviation (n = 4). * indicates significant difference compared with the wild strain (T test, *P < 0.05).

[0023] Figure 8 The dry weight (A), copper content (B), copper accumulation (C), aboveground copper accumulation (D) and multi-metal concentration in grain (E) of the wild strain (WT) and mutant strains (C67-10, C67-11) provided in the examples. The data are expressed as mean ± standard deviation (n = 4). * indicates significant difference compared with the wild strain (T test, *P < 0.05).

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

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

[0026] Fig.11 This is a schematic diagram of the structure of OsMYB67 and the copper-related gene promoter cis-acting elements provided in the examples.

[0027] Fig.12 The yeast one-hybrid test results of OsMYB67 and copper-related gene promoter cis-acting elements provided in the examples. pGADT7-Rec2-OsMYB67 and pHIS2-cis-element (OsCOPT2, OsCOPT3, OsCOPT4, OsCOPT7, OsHMA5 and OsHMA9) were co-cultured with yeast cells and then grown on SD / -Leu-Trp-His selection medium containing 0 or 60 mM 3-AT.

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

[0029] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0030] Construction of OsMYB67 knockout vector

[0031] The embodiment provides a vector combination 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 as shown in SEQ ID NO:3 for expressing the Cas9 nuclease.

[0032] In some embodiments, the pGTR vector (#63143, addgene) is used as the base vector, and the nucleotide sequence shown in SEQ ID NO: 1 is inserted into the pGTR vector by PCR amplification, restriction digestion, T4 ligase, transformation of E. coli and screening to obtain a pGTR vector expressing gRNA 1. The pGTR vector is used as the base vector, and the nucleotide sequence shown in SEQ ID NO: 2 is inserted into the pGTR vector by PCR amplification, restriction digestion, T4 ligase, transformation of E. coli and screening to obtain a pGTR vector expressing gRNA 2.

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

[0034] Construction of OsMYB67 mutant rice plants

[0035] 1. Knockout process

[0036] The vector combination for knocking out the OsMYB67 gene was transformed into a wild rice strain (Oryza sativa L.sspjaponicacvZhonghua 11, which was cultivated by the Crop Research Institute of the Chinese Academy of Agricultural Sciences in 1979 using Jingfeng No. 5 / Tetepu / Fujin, bred in 1984, and approved by the Tianjin Variety Approval Committee in 1989) through Agrobacterium-mediated transformation, and the transformants were screened.

[0037] The screening step includes: using Cas9-F (SEQ ID NO: 4) and Cas9-R (SEQ ID NO: 5) as a primer pair, PCR amplifying the genomic DNA of the T0 generation strain, and if the target band shown in SEQ ID NO: 3 is amplified, it is identified as a positive strain.

[0038] The screening step also includes: using MYB67-Test-F (SEQ ID NO: 6) and MYB67-Test-R (SEQ ID NO: 7) as a primer pair, PCR amplifying the genomic DNA of the strain to be screened, sequencing the obtained PCR amplification products, and comparing the sequencing results of the positive strain with the sequencing results of the wild strain to determine whether the positive strain is a mutant strain.

[0039] The screening step also includes: screening T2 generation strains with OsMYB67 mutation but without the target band shown in SEQ ID NO: 3 from the T1 generation strains, and performing sequencing verification again to screen out homozygous strains with stable OsMYB67 mutations, C67-10 and C67-11.

[0040] 2. Results

[0041] like Figure 1 As shown, the first target site of the C67-10 strain lacks 1 nt nucleotide, and the second target site lacks 4 nt nucleotides. The first target site of the C67-11 strain lacks 1 nt nucleotide, and the second target site lacks 1 nt nucleotides.

[0042] Deletion of OsMYB67 promotes Cu absorption and transport

[0043] 1. Potted plant test

[0044] The pot test was carried out at the test base of the Trace Elements Center of Huazhong Agricultural University. The mutant strains (C67-10 and C67-11) and the wild strain were cultured in normal nutrient solution for 14 days and then transferred to 18kg (20L black square barrel) soil pots for pot culture. The pot soil contained 5.03g / kg organic matter, 0.32g / kg total nitrogen, 2.1mg / kg available phosphorus, 0.2mg / kg effective copper, pH: 4.3. At the same time, nitrogen fertilizer urea (containing N46.4%): 0.43g / kg; phosphorus fertilizer was superphosphate (containing P2O512%): 1.25g / kg; potassium fertilizer was potassium chloride (containing K2O 60%): 0.33g / kg.

[0045] The treatment groups included -Cu and +Cu. The plants in the -Cu group were treated with the above-mentioned basic fertilizer only. The plants in the +Cu group were additionally treated with 0.4 mg / kg of Cu. The plants in each group were cultured until the flowering stage, and the growth phenotype during the flowering stage was examined. (1) A tiller of the plant was taken, and the stem, leaves and ear parts were separated and dried to constant weight to determine the copper content; (2) A fresh sample of the flowering ear was taken, stored at -80°C, 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 Test" (Bao Shidan 2000), the plant sample was ground into powder and 0.10xxg was weighed and put into a digestion tube, and then 2mL of mixed acid (perchloric acid: nitric acid = 1:4) was added. After cold digestion overnight, it was boiled in a low-temperature digestion furnace at a temperature of 120°C until it turned colorless or light yellow (about 24 hours). After cooling, it was diluted to 20mL with deionized pure water, shaken and filtered, and 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 strain (93.5%) was significantly higher than that of the wild-type plants (83%), the aboveground copper content of the mutant strain (68.7%) was significantly higher than that of the wild-type plants (66.7%), and the copper absorption of the mutant strain (114%) was significantly higher than that of the wild-type plants (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 plants (200%), the aboveground copper content of the mutant strain (90.9%) was significantly higher than that of the wild-type plants (86.3%), and the copper absorption of the mutant strain (63.4%) was significantly higher than that of the wild-type plants (48%).

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

[0051] Deletion of OsMYB67 shortens flowering time in rice

[0052] Since OsMYB67 is highly expressed in panicles and nodes during the flowering period of rice, it is speculated that OsMYB67 may play an important role during the flowering period. For this reason, the test case also conducted a potted test including a -Cu group and a +Cu group. Among them, the plants in the -Cu group were applied with 0.2 mg / kgCu on top of the above-mentioned basic fertilizer, and the plants in the +Cu group were additionally applied with 0.4 mg / kgCu. The effective copper content in the soil used was 0.2 mg / kg, which was a copper-deficient soil. The phenotype and copper content of the mutant strains and wild strains were examined during the flowering period.

[0053] like Figure 3 As shown in ab, under different Cu levels, the mutant strain showed an early heading date, which was 5 days earlier than the wild-type strain. Figure 3 cd shows that in the -Cu group, the wild strains with ear lengths greater than 10 cm, between 5-10 cm and less than 5 cm accounted for 33.30% each, and the mutant strains had ear lengths greater than 10 cm. In the +Cu group, the wild strains had ear lengths greater than 10 cm, between 5-10 cm and less than 5 cm accounted for 33.3%, 50% and 16.7% respectively; the mutant strain C67-10 had ear lengths greater than 10 cm, 87.5% of the C67-11 ear lengths were greater than 10 cm, and 12.5% ​​were between 5-10 cm. Therefore, Figure 3 The results showed that the mutant lines flowered earlier, indicating that OsMYB67 affects flowering time.

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

[0055] like Figure 5 As shown, the distribution ratio of Cu in the ears of the mutant strains was 16% and 11%. The distribution ratio of copper in the ears of the wild strains was 12%, and the distribution ratio in the ears of the mutant strains was 15% and 14%, respectively, which were increased by 3% and 2%.

[0056] These results indicate that the mutant lines can increase the allocation 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-type strain, the effective ear number of the mutant strain (about 11 / plant) was significantly higher than that of the wild-type strain (about 7 / plant).

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

[0060] like Figure 8As shown, the copper concentration in the mutant strain grains (4.151729μg / g) was significantly higher than that in the wild strain grains (3.072811μg / g). The copper concentration in the mutant strain cob (2.025472μg / g) was significantly higher than that in the wild strain cob (1.200539μg / g). The copper concentration of the mutant strain (5.575697μg / g) was significantly higher than that of the wild strain (2.896691μg / g). The cumulative amount of copper in the mutant strain grains (157.8232μg / plant) was significantly higher than that of the wild strain (96.20034μg / plant). It can be seen that the cumulative amount of copper in the aboveground part of the mutant strain is significantly higher than that in the aboveground part of the wild strain.

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

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

[0063] 1. Fluorescence quantitative PCR

[0064] The expression levels of copper transport genes in OsMYB67 overexpression and mutant panicles were tested by quantitative PCR.

[0065] The total RNA from tissues was extracted using the TriZol method. The specific steps are as follows: Cut the plant sample (~100 mg), put it into a 2 mL centrifuge tube in liquid nitrogen, add two small steel balls to the centrifuge tube, and grind the sample in a cold sample grinder for 1-3 minutes. Add 1 mL of 4°C pre-cooled Trizol at a ratio of 1 mL / 100 mg, shake and mix, and incubate at room temperature for 10 minutes. Add 0.2 mL of chloroform per mL of Trizol. Fasten the centrifuge tube cap, shake for 15 seconds, and incubate at room temperature for 5 minutes. Centrifuge at 12,000×g for 15 minutes at 4°C. Transfer the upper aqueous phase to a 1.5 mL RNase-free centrifuge tube and add an equal volume of isopropanol. Incubate the mixed sample at room temperature for 10 minutes and centrifuge at 12,000×g for 10 minutes at 4°C. Remove the upper suspension, 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 minutes at 4°C. Blow dry the RNA precipitate in a clean bench, and add 0.1% DEPC water after high temperature sterilization to dissolve the RNA.

[0066] The concentration and absorbance of RNA are measured using a UV spectrophotometer NanoDrop2000c. The OD260 / OD280 of RNA of good quality is generally between 2.0-2.2. 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. The quality of RNA is tested using 1% (w / v) agarose gel electrophoresis. Under 1% agarose gel electrophoresis, RNA of good quality shows three rRNA bands of 28S, 18S and 5S, respectively, and the brightness decreases in sequence. Note that all centrifuge tubes, gun tips and related solutions must be free of RNase contamination.

[0067] Refer to Yisheng Biotechnology Co., Ltd. Ⅱ1stStrandcDNA first strand synthesis kit reverses RNA into cDNA. For specific methods, refer to the instructions for use of the kit. Fluorescence quantitative PCR (RT-qPCR) test use The qPCR SYBR Green Master Mix kit (Shanghai Yisheng Biotechnology Co., Ltd.) and the Quant Studio TM 6 Flex System (Applied Biosystems, Foster City, CA) were used as the internal reference for the reaction. -ΔΔCT The relative expression of the target gene was calculated by the method of (Pattynetal2002). The RT-qPCR reaction system was as follows: 2.0 μL cDNA template, 0.2 μL forward and reverse primers, 5.0 μL SYBRGreenI (2×) dye, 2.6 mL ddH2O to 10 μL system. The RT-qPCR reaction program was as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 10 s, 60°C annealing for 20 s, 72°C extension for 20 s, 40 cycles. The melting curve was set by default at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.

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

[0069] Table 1

[0070]

[0071]

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

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

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

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

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

[0077] like Fig.10 As shown, under +Cu treatment conditions, the expression level of the flowering-related gene OsHd1F in the mutant strain was significantly higher than that in the wild strain, while the expression level of OsHd3a was significantly lower than that in the wild strain (except 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. Mining MYB interaction elements in promoters of rice copper transport genes

[0079] MYB transcription factors function by binding to MYB cis-acting elements in the promoter region of downstream target genes, so genes with MYB cis-acting elements in the promoter region may be their downstream target genes. Fig.11 As shown in the figure, the promoter sequences of copper transport genes were obtained through 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 / ), and it was found that the promoter regions of 8 genes contained rich MYB action elements.

[0080] OsMYB67 interacts with promoter sequences of Cu-related genes

[0081] 1. Yeast single hybrid test

[0082] The AD vector is a recombinant vector obtained by connecting the cDNA sequence of the OsMYB67 gene (SEQ ID NO: 28) to the pGADT7-Rec2 plasmid (Cat. No. QCP0234; Shanghai Qincheng Biotechnology Co., Ltd.) by 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.; Product No. YS-1432F). Among them, the candidate promoters are the promoter of the OsCOPT2 gene (SEQ ID NO: 29), the promoter of the OsCOPT3 gene (SEQ ID NO: 30), the promoter of the OsCOPT4 gene (SEQ ID NO: 31), the promoter of the OsCOPT7 gene (SEQ ID NO: 32), the promoter of the OsHMA5 gene (SEQ ID NO: 33) and the promoter of the OsHMA9 gene (SEQ ID NO: 34).

[0084] The AD vector and the BD vector 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 the corresponding empty vector.

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

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

[0087] 2. Results

[0088] like Fig.12 As shown, all yeasts can grow normally on SD / -Leu-Trp-His triple-deficient medium without 3-AT. When 60mM 3-AT was added to the triple-deficient medium, the positive controls (pGADT7-53 and pHIS2-53) could grow normally, but the negative controls (pGADT7-53 and pHIS2) could not grow normally. Yeasts that were simultaneously transformed with the AD vector and any of the BD vectors could grow in the 60mM 3-AT selection medium, 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 (BioVector NTCC).

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

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

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

[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, NewPu 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, NewPu Bio).

[0100] Agrobacterium monoclonal clones containing these recombinant vectors are placed in 1mL of LB containing the corresponding antibiotics and shaken at 250rpm at 28℃ for about 18h. Prepare A solution: 15mL of LB culture medium containing the corresponding antibiotics, 300μL of 0.5MMES, 6mL of 100mMAS, add 50μL of Agrobacterium bacterial solution to each 5mL A solution, and culture at 250rpm at 28℃ until OD600=1.0 (about 12-18h). Centrifuge at 4000rpm for 10min at room temperature, collect the bacteria, and discard the supernatant. Use an equal volume of A solution to suspend the bacteria to OD600=1.0 (adjust to 0 with the above A solution, and measure OD600) Premix the bacterial solutions to be injected together in a sterilized 50mL centrifuge tube (use A solution to adjust the concentration of each bacterial solution to the same), and let it stand at room temperature for more than 3h. Injection of tobacco: Inject from the back into Nicotiana benthamiana in the vigorous growth period (equal volumes of bacterial solution are injected into equal volumes of tobacco leaves) and hang labels. Take samples with liquid nitrogen after 24-48 hours and measure GUS enzyme activity or 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 the six downstream candidate genes were fused with the GUS reporter vector and transformed into tobacco leaves individually or together. Samples were taken 48 hours later for GUS staining and GUS activity detection.

[0103] like Fig.13 a and Fig.13 As shown in Figure b, almost no staining was observed when 35S:OsMYB67 or a vector fused with the downstream GUS reporter gene was injected alone. When they were co-injected, only when 35S:OsMYB67 and proOsHMA9:GUS were co-injected into tobacco, obvious staining was observed, which was consistent with the results of the GUS activity assay. No staining was observed when 35S:OsMYB67 was combined with proOsCOPT4:GUS, proOsCOPT7:GUS, or proOsHMA5:GUS. This means 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 the above genes.

[0104] like Fig.13 c and Fig.13As shown in (d), co-expression of OsMYB67 with the promoter of OsHMA9 enhanced the activity of LUC, while co-expression of OsMYB67 protein with the promoter of OsCOPT7 reduced the activity of LUC, indicating that OsMYB67 can activate the expression of OsHMA9 gene but inhibit the expression of OsCOPT7 gene.

[0105] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A gRNA as shown in SEQ ID NO: 1 and / or 2.

2. A vector combination for knocking out the OsMYB67 gene, comprising a gRNA expression vector and a Cas9 expression vector, wherein the gRNA expression vector carries a guide sequence targeting the OsMYB67 gene, wherein the guide sequence comprises gRNA1 as shown in SEQ ID NO: 1 and gRNA2 as shown in SEQ ID NO: 2, and the Cas9 expression vector carries a coding sequence as shown in SEQ ID NO: 3 for expressing the Cas9 nuclease.

3. The vector combination according to claim 2, wherein the gRNA expression vector comprises a pGTR vector inserted with the nucleotide sequence shown in SEQ ID NO: 1 and a pGTR vector inserted with the nucleotide sequence shown in SEQ ID NO:

2.

4. The vector combination according to claim 2, wherein the Cas9 expression vector is a pRGEB32 vector inserted with the nucleotide sequence shown in SEQ ID NO:

3.

5. A gene knockout kit, comprising the gRNA according to claim 1 and / or the vector combination according to any one of claims 2 to 4.

6. A method for constructing a rice mutant, comprising: Introducing any one of the vector combinations described in 2 to 4 into a wild rice strain; as well as Positive strains and mutants were selected from the transformants.

7. The construction method according to claim 6, wherein the screening step comprises: Cas9-F (SEQ ID NO: 4) and Cas9-R (SEQ ID NO: 5) were used as primer pairs to PCR amplify the genomic DNA of the T0 generation strain. If the target band shown in SEQ ID NO: 3 was amplified, it was identified as a positive strain.

8. The construction method according to claim 6, wherein the screening step further comprises: MYB67-Test-F (SEQ ID NO: 6) and MYB67-Test-R (SEQ ID NO: 7) were used as primer pairs to PCR amplify the genomic DNA of the strain to be screened, and the obtained PCR amplification products were sequenced. The sequencing results of the positive strains were compared with those of the wild strains to determine whether the positive strains were mutant strains.

9. The construction method according to claim 6, wherein the screening step further comprises: T2 generation strains with OsMYB67 mutations but without the target band shown in SEQ ID NO: 3 were screened from the T1 generation strains, and sequencing verification was performed again to obtain homozygous strains with stable OsMYB67 mutations.

10. Use of the gRNA according to claim 1 or the vector combination according to any one of claims 2 to 4, wherein the use comprises at least one of promoting copper absorption by rice, promoting copper transport by rice, and promoting rice growth.

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