Method for increasing carotenoid content in common rice and transgenic golden rice

Abstract

Provided is a method for increasing the carotenoid content in common rice and transgenic golden rice, relating to the technical field of breeding. Black rice is used as a breeding material to obtain a rice variety with a far higher lutein and β-carotene content than that of regular white rice or red rice varieties; using the black rice material as an acceptor, genes controlling carotenoid synthesis are transferred to obtain black golden rice, in which the lutein and β-carotene content is significantly increased. Using the transgenic golden rice line Ky1-4 as a donor and the black rice variety 'Heishuai' (HS) as an acceptor, hybridization, backcrossing, and selfing are carried out to obtain a GR line having a black seed coat, GRB; compared to the donor, the wholegrain rice of GRB has a significantly increased β-carotene and lutein content, and unlike GR, which only preferentially accumulates β-carotene, GRB accumulates more β-carotene and lutein.

Description

A method for increasing the carotenoid content in ordinary rice and genetically modified golden rice Technical Field

[0001] The present invention belongs to the field of seed breeding technology, and in particular relates to a method for increasing the carotenoid content in common rice and genetically modified golden rice (GR). Background Art

[0002] Crops rich in beta-carotene (provitamin A) are considered an effective and sustainable way to address vitamin A deficiency (VAD) worldwide. Golden rice rich in beta-carotene came into being and was called the first generation of golden rice GR1. The second generation of golden rice GR2 that followed had a total carotenoid content of up to 37μg / g, which is 23 times that of GR1 (mainly beta-carotene). The food safety of GR2E, a transformation event of GR2 that meets human food biosafety, has been approved in four countries: the United States, Canada, Australia and New Zealand; this leading GR2 event was approved for commercial cultivation in the Philippines in 2021.

[0003] However, rapid degradation of β-carotene during post-harvest storage is a common problem in rice, severely impacting the efficacy of β-carotene biofortified crops. GR2E degrades by 50% after 25 days of post-harvest storage at room temperature. By 72 days, the β-carotene content in GR2E drops to 4.1 μg / g, representing an 87% loss. This rapid degradation of β-carotene during storage significantly reduces the actual efficacy of GR2 after transportation and storage.

[0004] There is currently no consensus or regulations on how to use non-genetic engineering methods with low regulatory costs and low public attention to increase the accumulation of carotenoids.

[0005] Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a method for increasing the carotenoid content in conventional rice and genetically modified Golden Rice (GR), so that significantly more lutein and β-carotene are accumulated in the offspring black-coated Golden Rice.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides application of black rice materials in creating rice germplasm with high carotenoid content.

[0009] Preferably, the rice germplasm with high carotenoid content includes common rice germplasm, transgenic golden rice germplasm and black seed coat golden rice germplasm.

[0010] The present invention provides a method for increasing the carotenoid content of common rice varieties, comprising using the common rice variety as a receptor, introducing the golden rice gene, and obtaining a transgenic rice variety with increased carotenoid content.

[0011] Preferably, the golden rice genes include the phytoene synthase gene ZmPsy from maize and the phytoene desaturase gene PaCrtI from Pantoea ananas.

[0012] Preferably, the nucleotide sequence of the phytoene synthase gene ZmPsy is shown as SEQ ID No. 25, and the nucleotide sequence of the phytoene desaturase gene PaCrtI is shown as SEQ ID No. 26.

[0013] The present invention also provides a method for cultivating black-skin golden rice with high carotenoid content, using black rice material as a receptor, and transferring golden rice genes to obtain black-skin golden rice with high carotenoid content.

[0014] The present invention also provides a method for cultivating a transgenic golden rice variety with a high carotenoid content, comprising the following steps: using black rice material as a receptor and golden rice as a donor to perform hybridization to obtain a transgenic golden rice variety with a high carotenoid content.

[0015] Preferably, the black rice material includes conventional rice varieties or transgenic rice varieties with high carotenoid content cultivated using black rice varieties as materials.

[0016] The present invention also provides a breeding method for black-skinned golden rice, comprising the following steps: (1) hybridizing a black rice variety as a recipient and a golden rice variety as a donor to obtain a hybrid F1;

[0017] (2) performing four consecutive backcrosses on the hybrid F1 to obtain a backcross BC4F1;

[0018] (3) The backcross variety BC4F1 is self-pollinated once to obtain black seed golden rice.

[0019] Preferably, the black rice variety in step (1) includes a conventional rice variety or a transgenic rice variety with a high carotenoid content cultivated using the black rice variety as a material.

[0020] Preferably, the golden rice variety in step (1) includes golden rice Ky1-4.

[0021] The present invention provides a method for increasing the carotenoid content in golden rice and delaying carotenoid degradation. In an embodiment of the present invention, the GR2 transformation event Ky1-4 (the seed coat is white because it does not contain anthocyanins, and is named GRW in the present invention) is used as a donor, and the black rice variety "Hei Shuai" (HS) is used as a recipient. According to the process shown in Figure 13, hybridization, backcrossing and selfing are carried out to obtain a GR family with a black seed coat - GRB. Compared with the donor GRW, the content of β-carotene and lutein in the brown rice of GRB is significantly increased. Unlike GR, which preferentially accumulates β-carotene, GRB accumulates more lutein and β-carotene. Because anthocyanins have the characteristics of absorbing visible light and ultraviolet light, and carotenoids are sensitive to visible light and ultraviolet light, the presence of anthocyanins avoids the photodecomposition of carotenoids, which is beneficial to promote the accumulation of carotenoids during seed development. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 is a statistical diagram of the natural variation of β-carotene and lutein in black rice (n=51), red rice (n=75), and white rice (n=421);

[0023] Figure 2 is a graph showing the results of HPLC detection of carotenoid content in brown rice, wherein Peak 1: lutein; Peak 2: zeaxanthin; Peak 3: β-cryptoxanthin; Peak 4: β-carotene;

[0024] FIG3 is a graph showing the correlation analysis results (n=51) of β-carotene and anthocyanidins, and lutein and anthocyanidins in black rice;

[0025] FIG4 is a distribution diagram of lutein and β-carotene in brown rice and polished rice of a representative black rice variety;

[0026] Figure 5 shows the results of the light-induced in vitro degradation test of β-carotene and lutein; a) Test apparatus setup, including a xenon lamp simulating sunlight, an outer tube containing 0 mg / L, 1 mg / L, or 10 mg / L anthocyanin solution, and an inner tube containing β-carotene or lutein solution; b) Light-induced in vitro degradation curves of β-carotene and lutein;

[0027] Figure 6 is a schematic diagram of the structure of the pYLgRNA-OsU# plasmid vector;

[0028] Figure 7 is a schematic diagram of the structure of the pYLCRISPR / Cas9-MH expression vector;

[0029] FIG8 is a diagram showing the sequencing verification results of OsMYB3, Kala4 and OsTTG1 gene knockout families;

[0030] Figure 9 shows the phenotypes and anthocyanin contents of OsMYB3, Kala4, and OsTTG1 gene knockout families

[0031] FIG10 is a graph showing changes in β-carotene and lutein content in OsMYB3, Kala4, and OsTTG1 gene knockout families;

[0032] Figure 11 is a schematic diagram of the pC2300 expression vector structure Ky1-4;

[0033] FIG12 is a graph showing the β-carotene and lutein contents in brown rice, polished rice, and rice bran of transgenic rice obtained by introducing the GR gene into black rice B02 and its Kala4 knockout line Kala4_KO8 (n=7);

[0034] FIG13 is a flow chart of the cultivation of GRB according to the present invention;

[0035] Figure 14 shows the color map of GRW and GRB seeds;

[0036] Figure 15 is a graph showing changes in β-carotene and lutein content in GRW and GRB during seed development;

[0037] FIG16 is a graph showing changes in β-carotene and lutein content in GRW and GRB during storage;

[0038] Figure 17 shows the degradation of GRB anthocyanins during storage. DETAILED DESCRIPTION

[0039] The present invention provides application of black rice materials in creating rice germplasm with high carotenoid content.

[0040] The present invention found that lutein and β-carotene are the carotenoids with the highest content in black rice. In the absence of anthocyanin solution, both β-carotene and lutein are rapidly degraded, while anthocyanin solution significantly reduces their degradation rates. Moreover, anthocyanin not only significantly reduces the rate of photodegradation of carotenoids, but also has a dosage effect. The results show that the presence of anthocyanin is beneficial to promoting the accumulation of carotenoids during seed development, and the seed coat of black rice is rich in anthocyanin.

[0041] The present invention is based on the fact that black rice is rich in anthocyanins, which are beneficial to promoting the accumulation of carotenoids in seeds and can be used to increase the carotenoid content of various rice germplasms, such as ordinary rice germplasm, genetically modified golden rice germplasm and black seed coat golden rice germplasm.

[0042] The present invention provides a method for increasing the carotenoid content of common rice varieties, comprising using the common rice variety as a receptor, introducing the golden rice gene, and obtaining a transgenic rice variety with increased carotenoid content.

[0043] The common rice varieties of the present invention preferably include white rice, red rice, white rice and its offspring, red rice and its offspring, or even its offspring, and their seeds contain almost no carotenoids or no carotenoids.

[0044] The present invention preferably utilizes genetic engineering to introduce Golden Rice genes into common rice varieties. The Golden Rice genes preferably include the phytoene synthase gene ZmPsy from maize and the phytoene desaturase gene PaCrtI from Pantoea ananatis. The nucleotide sequence of the phytoene synthase gene ZmPsy is preferably as shown in SEQ ID No. 25, and the nucleotide sequence of the phytoene desaturase gene PaCrtI is preferably as shown in SEQ ID No. 26. The present invention does not specifically limit the genetic engineering method; genetic transformation can be performed using conventional methods in the art.

[0045] The present invention also provides a method for cultivating black-coated golden rice with high carotenoid content. Using black rice as a recipient, the golden rice gene is transferred to produce black-coated golden rice with high carotenoid content. The present invention does not specifically limit the type of black rice material, but is preferably the same as described above. The golden rice gene described herein is preferably the same as described above and will not be further described here.

[0046] The present invention also provides a method for cultivating a transgenic golden rice variety with a high carotenoid content, comprising the following steps: using black rice material as a receptor and golden rice as a donor to perform hybridization to obtain a transgenic golden rice variety with a high carotenoid content.

[0047] The black rice material described in the present invention preferably includes conventional rice varieties or transgenic rice varieties with high carotenoid content, cultivated using black rice varieties. The present invention hybridizes the black rice material as the female parent with transgenic golden rice as the male parent. The transgenic golden rice preferably includes golden rice obtained through genetic engineering. After the hybridization, the present invention preferably also includes screening based on the color of the endosperm. If it is yellow, it is a true hybrid, and if it is white, it is a false hybrid.

[0048] The present invention also provides a breeding method for black-skinned golden rice, comprising the following steps: (1) hybridizing a black rice variety as a recipient and a golden rice variety as a donor to obtain a hybrid F1;

[0049] (2) performing four consecutive backcrosses on the hybrid F1 to obtain a backcross BC4F1;

[0050] (3) The backcross variety BC4F1 is self-pollinated once to obtain black seed golden rice.

[0051] The black rice variety of the present invention preferably includes conventional rice varieties or transgenic rice varieties with high carotenoid content cultivated using black rice varieties as materials. The golden rice variety of the present invention preferably includes golden rice Ky1-4.

[0052] The present invention preferably utilizes the flow chart shown in FIG13 to increase the carotenoid content and slow carotenoid degradation in Golden Rice. The black rice variety described herein preferably includes Heishuai HS (derived from the Shaanxi Academy of Agricultural Sciences and disclosed in Li Yanxia's master's thesis: Li Yanxia, ​​Lin Yongjun, Chen Hao. Functional Study of Three Rice Transcription Factors Regulating Anthocyanin Biosynthesis. Master's Thesis, Huazhong Agricultural University, 2016). The Golden Rice variety described herein preferably includes Golden Rice Ky1-4 (Golden Rice from the japonica rice variety Kongyu 131 (KY131), designated GRW), which has been disclosed in the Journal of Huazhong Agricultural University: Du Lique, Zhao Mingchao, Lin Yongjun, Chen Hao. Cultivation of Transgenic Rice with Enhanced β-Carotene. Journal of Huazhong Agricultural University, Issue 5, 2014. The authors pledge to distribute this material to the public within 20 years of the application date.

[0053] The black-coated golden rice GRB, bred by the breeding method described above, is a new rice variety rich in anthocyanins and carotenoids. Its seed coat is black and rich in anthocyanins; its endosperm is golden yellow and rich in carotenoids ( FIG11 ).

[0054] The black-coated golden rice described in the present invention is the hybrid offspring of golden rice and black rice. The anthocyanins in black rice absorb visible light and ultraviolet light, while carotenoids are sensitive to visible light and ultraviolet light. The presence of anthocyanins delays the light-induced degradation of carotenoids, which is beneficial for promoting the accumulation of carotenoids during seed development.

[0055] The following describes in detail a method for increasing the carotenoid content in conventional rice and genetically modified golden rice (GR) provided by the present invention in conjunction with the following examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] Analysis of carotenoid content in natural rice seed resources

[0058] 1.1 Determination of carotenoid content in rice seeds

[0059] For the extraction and determination of carotenoids, the method of Egesel et al. (Egesel, CO, Wong, JC, Lambert, RJ, & Rocheford, TR: Combining maize inbreds for carotenoids and tocopherols. 2003, Crop Science, 43:818-823) was used with minor modifications: After all samples were collected, they were freeze-dried at -80°C in a vacuum freeze dryer until ready for use. After grinding the sample into rice flour using a grinder, 600 mg of the sample was weighed and placed in a 50-ml centrifuge tube. 6 ml of anhydrous ethanol containing 0.1% (v / v) BHT was added, mixed, and incubated at 85°C for 5 min. Next, 120 μl of 80% (w / v) KOH was added, vortexed for 15 s, and then saponified at 85°C for 10 min, vortexing once during the reaction. Immediately pre-cool the sample on ice, add 3 ml of n-hexane and 3 ml of pre-cooled ultrapure water. Vortex to mix well and centrifuge at 2700 rpm for 5 minutes. Transfer the supernatant to a new 15 ml centrifuge tube. Extract the rice flour twice with 3 ml of n-hexane. Add 3 ml of pre-cooled ultrapure water to the total extract, wash once, and transfer the supernatant to a 10 ml conical bottom glass centrifuge tube. Remove the n-hexane by vacuum centrifugal concentration technology, and then remove the water in a freeze dryer. Add 1 ml of acetonitrile: methanol: dichloromethane (v:v:v = 45:20:35) to fully dissolve the dried material. After mixing the solute, transfer it to a brown sample bottle through a 0.22 μm filter and put it into the machine.

[0060] Carotenoid content in rice seeds was determined using high-performance liquid chromatography (HPLC, Aglient 1260). All reagents used (acetonitrile, methanol, and dichloromethane) were HPLC-grade, and the column used was a YMC-carotenoid column (2.0 × 100 mm, 3 μm). Mobile phase A consisted of acetonitrile; mobile phase B consisted of a mixture of methanol and dichloromethane in a 4:1 volume ratio. The mobile phase contained 0.05% w / v triethylamine and 0.1% BHT. The volume ratio of mobile phases A to B was 3:1. The sample load was 20 μl, the flow rate was 1.8 ml / min, the column temperature was 30°C, and the measurement wavelength was 450 nm. Lutein, zeaxanthin, β-cryptoxanthin, and β-carotene standards were purchased from Sigma-Aldrich.

[0061] 2.2 Determination of anthocyanin content in rice seeds

[0062] Anthocyanin content in rice seeds was determined using HPLC (Zhu F, Yi-Zhong Cai, Jinsong Bao, Harold Corke, Effect of γ-irradiation on phenolic compounds in rice grain, Food Chemistry, 2010, 120:74-77): Rice seeds 30 days after anthesis were freeze-dried in a vacuum freeze dryer and ground into rice flour. 100 mg of rice flour was weighed and placed in a 2.0 ml centrifuge tube. 1 ml of extractant (105 ml extractant: 80 ml HPLC-grade methanol, 20 ml sterile ddH2O, and 5 ml 5 mol / L HCl solution) was added. Extraction was performed on a shaker at 120 rpm at room temperature in the dark for 24 hours. The supernatant was carefully aspirated, filtered through a 0.22 μm filter, and then transferred into a 2.0 ml brown vial for analysis.

[0063] Anthocyanin content in rice seeds was determined using high-performance liquid chromatography (HPLC, Aglient 1260). All reagents used were HPLC-grade, and the chromatographic column used was a C18 column (ZORB-AX SB-Aq 4.6×250 mm, 5 μm). Mobile phase A consisted of ddH2O and pure formic acid (V:V = 100:5), and mobile phase B was pure methanol. The mobile phase settings were: 20-70% B for 0-12 min; 70-78% B for 12-20 min. The flow rate was 0.8 ml / min, the sample load was 10 μl, the column temperature was 25°C, and the measurement wavelength was 520 nm.

[0064] 2.3 Distribution of carotenoids in rice germplasm resources

[0065] This study investigated the natural variation in carotenoid content in 544 rice germplasm accessions, including 51 black rice accessions, 72 red rice accessions (which accumulate proanthocyanidins in the seed coat), and 421 white rice accessions. The results, published in the academic journal New Phytologist, are as follows: Zheng J, Wu H, Zhu H, Huang C, Liu C, Chang Y, Kong Z, Zhou Z, Wang G, Lin Y, Chen H. Determining factors, regulation system, and domestication of anthocyanin biosynthesis in rice leaves. New Phytol. 2019, 223:705-721. The study found that lutein and β-carotene were the most abundant carotenoids in black rice, while other carotenoids were present at very low or even absent levels. The average lutein content in black rice was 2.80±1.14μg / g, and the average β-carotene content was 0.64±0.36μg / g; the average lutein content in red rice was 0.29±0.60μg / g, and the average β-carotene content was 0.04±0.09μg / g; the average lutein content in white rice was 0.04±0.07μg / g, and the average β-carotene content was 0.03±0.09μg / g (Figures 1 and 2). The lutein and β-carotene contents in black rice were significantly higher than those in red rice and white rice (Figure 1). The β-carotene content of black rice germplasm B03 reached 2.05μg / g, which is equivalent to the first-generation Golden Rice GR (1.6μg / g).

[0066] 2.4 Correlation analysis between carotenoids and anthocyanins in black rice

[0067] Black rice seeds accumulate both anthocyanins and carotenoids. The relationship between anthocyanins, lutein and β-carotene in black rice was analyzed. The study found that the correlation coefficient R 2 The correlation coefficient R between anthocyanin and β-carotene is 0.42. 2 The correlation coefficient was 0.39, indicating a positive correlation (Figure 3). Varieties with high lutein and β-carotene contents generally also had high anthocyanin contents.

[0068] 2.5 Distribution of carotenoids in seeds

[0069] We randomly selected five portions of black rice and measured the lutein and β-carotene content in both brown and polished rice. We found that polished rice contained very low or even no lutein (Figure 4). This suggests that carotenoids, like anthocyanins, are synthesized only in the rice bran.

[0070] Example 2

[0071] Anthocyanins have a light absorption effect that delays the degradation of carotenoids

[0072] Carotenoids have the chemical properties of being easily oxidized and decomposed by light, while anthocyanins naturally have the properties of being antioxidant and absorbing sunlight (especially blue-green light and ultraviolet rays). Therefore, in theory, anthocyanins have a protective effect on carotenoids. In plant cells, carotenoids accumulate only in plastids, while anthocyanins accumulate only in vacuoles, and anthocyanins and carotenoids are in a non-contact, isolated state. Therefore, we speculate that it is difficult for anthocyanins in plants to protect carotenoids through antioxidant properties, and their property of absorbing sunlight may provide a protective effect for carotenoids to delay photodegradation. In order to verify this speculation, the present invention designed a light-induced in vitro degradation test of carotenoids. The experimental device uses a xenon lamp (PLS-SXE300 / 300UV, Beijing Perfect Light) to simulate sunlight. Two transparent, nested glass tubes were set up (Figure 5a). The outer tube contained anthocyanin (Shengqing Biotechnology, Xi'an) solutions of varying concentrations (1 mg / mL or 10 mg / mL) (methanol:water:5M HCl = 16:4:1 v / v / v). The inner tube contained β-carotene (Sigma-Aldrich, starting concentration 9.21±0.79 μg / g) or lutein (Sigma-Aldrich, starting concentration 13.40±0.20 μg / g) (acetonitrile:methanol:dichloromethane = 75:20:5 v / v / v). The ends of both tubes were sealed with parafilm to prevent evaporation and then sealed with black plastic bags to prevent simulated sunlight from irradiating the anthocyanin-free solution from above. The outer tube of the control sample contained a colorless solvent without anthocyanin, while the inner tube contained the same β-carotene or lutein solution as the test sample. Set the xenon lamp current to 20A, adjust the angle of the light source and the distance between the light source and the sample to ensure that the light intensity at the position of the sample being measured is 0.14W / cm 2 Sampling was performed at 0 min, 30 min, 60 min, 90 min, 120 min, and 150 min after simulated sunlight stress. The samples were immediately placed in liquid nitrogen to avoid light until analysis.

[0073] In vitro simulated sunlight degradation tests showed that without the protection of anthocyanin solution, both β-carotene and lutein were rapidly degraded, while anthocyanin solution significantly reduced their degradation rates (Figure 5b). Without the protection of anthocyanin solution, β-carotene solution was degraded by 91.4% after 150 minutes of light stress; while under the protection of 1 mg / mL and 10 mg / mL anthocyanin solution, β-carotene solution was degraded by 46.0% (P = 3.1 × 10 -6) and 33.9% (P = 0.0012) (Figure 5(b)). Without the protection of anthocyanin solution, the lutein solution degraded by 37.9% after 150 minutes of light stress. However, with the protection of 1 mg / mL and 10 mg / mL anthocyanin solutions, the lutein solution degraded by 14.1% (P = 0.0063) and 7.2% (P = 0.0018), respectively (Figure 5(b)). These results indicate that anthocyanin solution not only significantly reduces the rate of carotenoid photodegradation but also has a dose-effect, as the 10 mg / mL anthocyanin solution provides better protection against carotenoid photodegradation than the 1 mg / mL anthocyanin solution.

[0074] Example 3

[0075] Golden Rice with Black Rice as Background Increases Carotenoid Accumulation Levels

[0076] 3.1 Determination of carotenoid content in OsMYB3, Kala4, and OsTTG1 knockout materials

[0077] It is known that the synthesis of anthocyanins in black rice is regulated by the activation of a ternary complex consisting of the transcription factors OsMYB3, Kala4, and OsTTG1. The CRISPR / Cas9 system was used to knock out the OsMYB3, Kala4, and OsTTG1 genes in the black rice material B02. The CRISPR / Cas9 knockout expression vector was constructed according to the method of Ma et al. (2015). Two knockout target sites were designed for each knockout gene. PCR and enzyme ligation were used to construct expression vectors for U3-driven gRNA1, U6a-driven gRNA2, and Ubiqutin promoter-driven Cas9 protein. Finally, these two fragments were simultaneously ligated into pYLCRISPR / Cas9-MH digested with Bsa I. The specific process is as follows:

[0078] 1) Based on the gene sequences of OsMYB3, Kala4, and OsTTG1, design two pairs of primers targeting two target sites per gene (see Table 1 for specific sequences). Prepare a 100 μM stock solution of each primer pair in TE. Take 1 μl of each primer and mix them in 98 μl of 0.5× TE to dilute to 1 μM. Denature at 98°C for 30 seconds and then anneal at room temperature to form adapters.

[0079] Table 1 Primer sequence list

[0080] 2) Perform enzymatic digestion and ligation simultaneously. Add ATP to a final concentration of 0.5-1.0 mM in 1× Bsa I digestion buffer, then add ~20 ng of pYLgRNA-OsU# plasmid (Figure 6), 0.5 μL of target linker (final concentration 0.05 μM), ~5 U of Bsa I, ~35 U of T4 DNA ligase, and fill with ddH2O to 10 μL. Incubate in a PCR machine for 5 cycles: 37°C for 5 min, then 20°C for 5 min.

[0081] 3) PCR amplification products.

[0082] First round PCR (2 PCR reactions per gRNA expression cassette):

[0083] Take 1 μl of the above ligation product as a template, primer UF / adapter reverse primer (reaction 1), or adapter forward primer / gRNA-R (reaction 2) 0.2 μM each (specific primer sequences are shown in Table 1), KOD enzyme 0.2 μl, dNTP (10 μM) 0.4 μl, 2× KOD buffer 10 μl, add water to 20 μl, and perform PCR reaction for 25-28 cycles: 95°C for 10 s, 60°C for 15 s, and 68°C for 20 s.

[0084] Second round of PCR:

[0085] Pre-mix the specific primer pairs B1′+B2 and B2+BL (for specific sequences, see Table 1) into a 10× working solution (1.5 μM each). Dilute 1 μL of the products from reactions 1 and 2 in the first round of PCR 10-fold with ddH2O and mix 1 μL each as the template for the second round of PCR. Add 3 μL of the primer pair working solution (final concentration 0.15 μM), 0.3 μL of KOD enzyme, 0.6 μL of 10 μM dNTPs, and 15 μL of 2× KOD buffer to each expression cassette, and finally add water to 30 μL. Incubate the reaction in a PCR machine for 15-20 cycles: 95°C for 10 s, 58°C for 15 s, and 68°C for 20 s. Purify the second-round PCR product using a PCR product purification kit.

[0086] 4) Take 70 ng of PCR product and add 80 ng of uncut pYLCRISPR / Cas9-MH plasmid (Figure 7). In a 15 μl reaction system (1× Bsa I digestion buffer), add 10 U of Bsa I and digest at 37°C for 10 min. Then, add 0.5 μL of 10× NEB T4 DNA ligase buffer and 35 U of T4 ligase. Incubate the reaction on a PCR machine for 10-15 cycles: 37°C for 2 min; 10°C for 3 min; 20°C for 5 min; and finally at 37°C for 2 min.

[0087] 5) Precipitate the ligation product with ethanol, wash with 70% ethanol, air-dry, and dissolve in 5 μL ddH2O. Electroporate 1 μL of the ligation product into E. coli DH10B competent cells. After electroporation, add 1 mL of SOC medium and incubate at 37°C for 1 hour. Plate the cells onto LB medium supplemented with 25 μg / mL karatomycin, 0.3-0.5 mM IPTG, and an appropriate amount of X-gal. Select positive clones that produce blue plaques.

[0088] 6) Confirmation of positive clones: Select positive clones and shake them, extract the plasmids, digest them with Mlu I, and verify them by sequencing.

[0089] 7) The obtained positive clones were transformed into Agrobacterium tumefaciens strain EHA105 and stored at -70°C until use.

[0090] The constructed CRISPR / Cas9 knockout vector was transformed into black rice variety B02 using the Agrobacterium-mediated method. The specific experimental steps are as follows:

[0091] 1) Callus induction:

[0092] Mature ZH11 rice seeds were hulled and then surface-sterilized with a 75% ethanol solution for 1 minute and a 0.15% mercuric chloride solution for 20 minutes. The seeds were washed four to five times with sterile, single-distilled water and evenly distributed on induction medium. The inoculated medium was incubated in a dark room at 28 ± 1°C for 4 to 6 weeks.

[0093] 2) Callus subculture

[0094] Bright yellow, compact and relatively dry embryogenic calli were selected and placed on subculture medium and cultured in a dark room for 3 weeks at a culture temperature of 28±1°C.

[0095] 3) Pre-culture

[0096] Select compact and relatively dry embryogenic calli, place them on pre-culture medium and culture them in the dark for 4 days at a temperature of 28±1°C.

[0097] 4) Agrobacterium culture

[0098] Pre-culture Agrobacterium on LA medium with the corresponding resistance selection for 2 days at 28 ± 1°C. Transfer the Agrobacterium to the suspension culture medium and culture at 28°C, 200 rpm, and shake for 2-3 hours.

[0099] 5) Agrobacterium infection

[0100] Transfer the pre-cultured callus to a sterilized bottle. Adjust the Agrobacterium suspension to an OD of 600= approximately 0.3. Soak the callus in the Agrobacterium suspension for 10 minutes. Transfer the callus to sterilized filter paper, blot dry, and then incubate on co-culture medium for 3 days at 19-20°C.

[0101] 6) Callus washing and selective culture

[0102] Wash the infected callus 7-8 times with sterile water, then soak it in sterile water containing 400 mg / L carbenicillin for 30 minutes. Transfer the callus to sterilized filter paper, blot it dry, and then transfer the callus to selective culture medium for 3 times, each time for 2 weeks.

[0103] 7) Differentiation

[0104] The resistant calli were transferred to pre-differentiation medium and cultured in the dark for 7 days at 26±1°C. The pre-differentiation cultured calli were transferred to differentiation medium and cultured under light at 26±1°C.

[0105] 8) Rooting

[0106] Cut off the roots produced during differentiation; then transfer them to rooting medium and culture them under light for 2 to 3 weeks at a culture temperature of 26±1℃.

[0107] 9) Transplanting

[0108] The residual culture medium on the roots was washed clean, and the seedlings with good root systems were transferred to the greenhouse and kept moist in the first few days (for the culture medium and its preparation method of this embodiment, please refer to patent No. 2011100832269).

[0109] The obtained transgenic regenerated plants were detected by PCR to amplify the region containing the two target sites and sequenced to confirm the sequence. The primers used for target sequencing are shown in Table 1. Two independent homozygous mutant families were selected for each transformation, and the target site sequences that were knocked out were confirmed by sequencing (Figure 8).

[0110] The anthocyanin content and carotenoid content were determined by HPLC. Knocking out Kala4 or OsTTG1 resulted in a complete loss of anthocyanins in the black rice seed coat; after knocking out OsMYB3, the anthocyanin content in the seed coat decreased from 234.14±5.81μg / g to 2.45±0.04μg / g and 2.94±0.00μg / g, respectively, with a loss of 99% of the anthocyanins (Figure 9). In addition, knocking out OsMYB3, Kala4, or OsTTG1 caused a significant decrease in β-carotene and lutein (Figure 10). This result once again verified that anthocyanin synthesis plays an important protective role in the accumulation of carotenoids.

[0111] 3.2 Transgenic Golden Rice with Black Rice as the Receptor Increases Carotenoid Accumulation

[0112] It is known that the birth of black rice is caused by a mutation in the Kala4 gene. The Kala4 gene is not expressed in red rice and white rice, so anthocyanins are not synthesized; however, the promoter region of the Kala4 gene in black rice has changed, causing the originally unexpressed Kala4 gene to be expressed in the seed coat, thereby activating the expression of anthocyanin synthesis genes in the seed coat, leading to the production of black rice. We knocked out the Kala4 gene in the black rice material B02, and the knockout material changed from black rice to white rice. We used wild-type black rice B02 and its Kala4-knocked-out white rice mutant Kala4_KO8 (Figures 8 and 9) as recipients to transfer golden rice genes, namely the phytoene synthase gene ZmPsy (SEQ ID No. 25) from corn and the phytoene desaturase gene PaCrtI (SEQ ID No. 26) from Pantoea ananatis. The specific method is as follows:

[0113] 1) Target gene synthesis. The target genes, ZmPsy and PaCrtI, are 2765 and 2760 bp, respectively, and were commissioned for synthesis by Shanghai Yingjun Biotechnology Co., Ltd. Both genes are driven by an endosperm-specific glutelin promoter, with transcription terminated by the nos terminator. During gene synthesis, HindIII and SalI restriction sites were added to both ends of the ZmPsy gene, and EcoRI and KpnI restriction sites were added to both ends of the PaCrtI gene.

[0114] 2) The expression vector pC2300 and the Zmpsy gene were double-digested with HindIII and SalI, and the exogenous Zmpsy gene fragment was recovered and purified. The ZmPsy gene fragment was then ligated to the vector to form the intermediate vector pC2300-psy (Figure 11). The PaCrtI gene and the pC2300-Psy vector were double-digested with EcoRI and KpnI. Finally, the expression vector pC2300-psy / crtI, which linked the ZmPsy and PacrtI genes, was obtained. The constructed pC2300-psy / crtI expression vector was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation and stored at -70°C.

[0115] 3) The expression vector pC2300-psy / crtI was introduced into the black rice variety B02 and its Kala4 knockout white rice mutant Kala4_KO8 using Agrobacterium-mediated transfection, and seven independent transgenic rice plants were regenerated from each. The transformation method was the same as in Example 3.1, except that the screening agent was replaced with G418 instead of hygromycin B.

[0116] The carotenoid content of T3 transgenic plants was detected. The β-carotene content in brown rice, polished rice and rice bran of transgenic plants with black rice B02 as the recipient was 11.44±2.96μg / g, 8.10±1.27μg / g and 13.64±4.76μg / g (n=7), respectively. The lutein content was 7.25±1.60μg / g, 1.36±0.35μg / g and 13.62±3. 84 (n=7); while the transgenic plants using the white rice mutant as the recipient had β-carotene contents of 3.17±0.92μg / g, 3.15±0.68μg / g, and 5.36±1.80μg / g (n=7), and lutein contents of 0.57±0.41μg / g, 0.24±0.16μg / g, and 0.99±0.47μg / g (n=7) (Figure 12). Comparison of the two sets of data revealed that the transgenic Golden Rice using black rice as the recipient accumulated significantly more carotenoids than the Golden Rice using white rice as the recipient (Figure 12).

[0117] 3.3 Rice hybridization and the cultivation process of Black Golden Rice

[0118] Following the process shown in Figure 13, select a black rice HS plant that is about to flower that day as the female parent. Scald the flowering ears at 45°C for 5 minutes, then remove the stamens and bag the plants. Collect natural pollen from the white rice recipient Golden Rice GRW (male parent) and artificially pollinate the emasculated HS. The hybridization bag is sealed and the pollinated plants are grown in the field for approximately 25 days before harvesting. This represents the F1 generation. Visually inspect the endosperm color of the F1 hybrid seeds; if yellow, it indicates a true hybrid; if white, it indicates a false hybrid. Using the same hybridization method as shown in Figure 13, four backcrosses and one self-pollination are performed to obtain Black Golden Rice (GRB).

[0119] GRB is a new rice variety rich in two nutrients: anthocyanins and carotenoids. The seed coat of GRB is black and rich in anthocyanins; the endosperm is golden yellow and rich in carotenoids (Figure 14).

[0120] 3.4 Changes in carotenoid content during GRW and GRB seed development

[0121] GRW and GRB seeds were sown at the appropriate time to ensure consistent flowering. All rice materials were grown under normal field management. Ears that flowered on the same day were labeled, with approximately 200 ears per variety labeled. The top third of the labeled ears were harvested 10, 15, 20, 25, 30, and 35 days after flowering (typically, a single ear takes three days to fully bloom from the top). The harvested ears were placed in liquid nitrogen and stored at -80°C until all samples were prepared.

[0122] Figure 12 shows the dynamic changes in carotenoid content during seed development in GRW and GRB from 10 to 35 days after flowering. The study found that the β-carotene content of GRB was consistently higher than that of GRW throughout seed development; the β-carotene content of both GRW and GRB peaked at 15 days after flowering and then continued to decline until seed maturity at 35 days after flowering; at 35 days after flowering, the β-carotene contents of GRW and GRB were 7.8 ± 0.3 μg / g and 18.2 ± 0.9 μg / g, respectively, indicating that the β-carotene content of GRB was 2.3 times that of GRW (Figure 15).

[0123] Lutein content in both GRW and GRB reached its highest level 10 days after anthesis and continued to decline as the seeds matured. During seed development, lutein content in both GRW and GRB did not change significantly until 20 days after anthesis, but then decreased dramatically in GRW, while the decline in GRB was relatively gradual. This resulted in the lutein content in GRB (4.21 ± 0.35 μg / g) being 10.5 times higher than that in GRW (0.40 ± 0.02 μg / g) at 35 days after anthesis (Figure 15).

[0124] 3.5 Changes in carotenoid content during storage of GRW and GRB seeds

[0125] After harvesting, GRW and GRB were hulled and stored in mesh bags at room temperature in the dark. At 0, 2, 4, 8, 16, 24, 32, and 40 weeks after storage, equal amounts of seeds were vacuum-packed and stored at -80°C. After all samples were prepared, they were uniformly processed and the carotenoid and anthocyanin contents were determined.

[0126] The changes in carotenoid content in GRW and GRB brown rice during room temperature storage were compared. β-carotene and lutein contents were determined by HPLC at 0, 2, 4, 8, 16, 24, 32, and 40 weeks of storage. After 40 weeks of storage, the β-carotene content in GRW was 0.89 ± 0.01 μg / g, representing an 85.8% degradation compared to the initial content; the β-carotene content in GRB was 2.37 ± 0.07 μg / g, representing an 86.7% degradation compared to the initial content. After 40 weeks of storage, the β-carotene content in GRB was 2.7 times that of GRW. Lutein content after storage was 0.15 ± 0.00 μg / g in GRW, representing a 48.2% degradation compared to the initial content; while the lutein content in GRB was 2.56 ± 0.01 μg / g, representing a 44.9% degradation compared to the initial content (Figure 16). After 40 weeks of storage, the lutein content in GRB was 17.1 times that in GRW. During storage, the degradation rate of anthocyanins in GRB was so slow that it could be ignored (Figure 17). Given that the β-carotene and lutein contents in GRB were much higher than those in GRW at harvest, and that the degradation rates of β-carotene and lutein in GRB were roughly comparable to those in GRW after storage, the carotenoid content in GRB was also much higher than that in GRW after long-term storage.

[0127] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of black rice materials in creating rice germplasm with high carotenoid content.

2. The application according to claim 1, characterized in that: The rice germplasm with high carotenoid content includes common rice germplasm, transgenic golden rice germplasm and black seed coat golden rice germplasm.

3. A method for increasing the carotenoid content of common rice varieties, characterized in that: This includes using ordinary rice varieties as receptors, transferring golden rice genes, and obtaining genetically modified rice varieties with increased carotenoid content.

4. The method according to claim 3, characterized in that: The golden rice genes include the phytoene synthase gene ZmPsy from corn and the phytoene desaturase gene PaCrtI from Pantoea ananas.

5. The method according to claim 4, characterized in that: The nucleotide sequence of the phytoene synthase gene ZmPsy is shown in SEQ ID No.25, and the nucleotide sequence of the phytoene desaturase gene PaCrtI is shown in SEQ ID No.

26.

6. A method for cultivating black-coated golden rice with high carotenoid content, characterized in that: Using black rice material as the receptor, the golden rice gene was transferred to obtain black-skin golden rice with high carotenoid content.

7. The cultivation method according to claim 6, characterized in that: The golden rice genes include the phytoene synthase gene ZmPsy from corn and the phytoene desaturase gene PaCrtI from Pantoea ananas.

8. The cultivation method according to claim 7, characterized in that: The nucleotide sequence of the phytoene synthase gene ZmPsy is shown in SEQ ID No.25, and the nucleotide sequence of the phytoene desaturase gene PaCrtI is shown in SEQ ID No.

26.

9. A method for cultivating a transgenic golden rice variety with high carotenoid content, characterized in that: The following steps are involved: Using black rice material as the recipient and golden rice as the donor, hybridization was carried out to obtain a genetically modified golden rice variety with a high carotenoid content.

10. The cultivation method according to claim 9, characterized in that: The black rice material includes conventional rice varieties or transgenic rice varieties with high carotenoid content cultivated using black rice varieties as materials.

11. The cultivation method according to claim 9, characterized in that: The hybridization is carried out with the golden rice as the male parent, and the golden rice includes genetically modified golden rice.

12. The cultivation method according to claim 9 or 11, characterized in that: The hybridization may be followed by screening, wherein the trait to be screened includes the color of the endosperm.

13. The cultivation method according to claim 12, characterized in that: The screening is as follows: screening is performed based on the color of the endosperm, wherein yellow indicates a true hybrid and white indicates a false hybrid.

14. A method for breeding black seed golden rice, characterized in that: The following steps are involved: (1) hybridizing a black rice variety as a recipient and a golden rice variety as a donor to obtain a hybrid F1; (2) performing four consecutive backcrossings on the hybrid F1 to obtain a backcross variety BC4F1; (3) The backcross variety BC4F1 is self-pollinated once to obtain black-coated golden rice.

15. The breeding method according to claim 14, characterized in that: The black rice variety in step (1) includes a conventional rice variety or a transgenic rice variety with a high carotenoid content cultivated using the black rice variety as a material.

16. According to the breeding method of claim 15, the black rice variety in step (1) comprises Heishui HS.

17. The cultivation method according to claim 14, characterized in that: The golden rice varieties in step (1) include golden rice Ky1-4.

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