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

Abstract

The invention provides a method for increasing the carotenoid content in common rice and transgenic golden rice, and relates to the technical field of breeding. According to the method, the black rice is used as a breeding material, and a rice variety with the content of xanthophyll and beta-carotene far higher than that of common white rice or red rice varieties can be obtained. According to the black golden rice obtained after the gene for controlling synthesis of carotenoid is transferred by using a black rice material as a receptor, the content of xanthophyll and beta-carotene is remarkably increased. A transgenic golden rice family Ky1-4 is used as a donor, a black rice variety HS is used as a receptor, hybridization, backcross and selfing are carried out, a GR family-GRB with black seed coats is obtained, compared with the donor, the content of beta-carotene and the content of xanthophyll in brown rice of the GRB are remarkably increased, and compared with GR which only accumulates beta-carotene preferentially, the content of the beta-carotene and the content of xanthophyll in brown rice of the GRB are remarkably increased. And more beta-carotene and lutein are accumulated by the GRB.

Description

Technical Field

[0001] The present invention belongs to the technical field of seed breeding, 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 β-carotene (provitamin A) are considered an effective and sustainable way to address vitamin A deficiency (VAD) worldwide. Golden rice rich in β-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 β-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, the rapid degradation of β-carotene during storage after rice harvest is a common problem, which seriously affects the efficacy of β-carotene biofortified crops. After 25 days of storage at room temperature after harvest, the β-carotene in GR2E can be degraded by 50%; after 72 days of storage after harvest, the β-carotene content in GR2E dropped to 4.1μg / g, equivalent to a loss of 87%. Due to the rapid degradation of β-carotene during storage, the actual efficacy of GR2 after transportation and storage is very low.

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

[0005] In view of this, the object of the present invention is to provide a method for increasing the carotenoid content in common rice and transgenic golden rice (GR), and significantly more lutein and β-carotene are accumulated in the offspring black-coated golden rice cultivated.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of black rice material in creating rice germplasm with high carotenoid content.

[0008] Preferably, the rice germplasm with high carotenoid content includes common rice germplasm, transgenic golden rice germplasm and black seed coat golden rice germplasm.

[0009] The present invention provides a method for increasing the carotenoid content of common rice varieties, comprising using the common rice varieties as receptors, transferring golden rice genes into the common rice varieties, and obtaining transgenic rice varieties with increased carotenoid content.

[0010] Preferably, the golden rice genes include the phytoene synthase gene ZmPsy from corn and the phytoene desaturase gene PaCrtI from Pantoea ananas.

[0011] 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.

[0012] The present invention also provides a method for cultivating black-coated golden rice with high carotenoid content, wherein black rice material is used as a receptor, golden rice genes are introduced, and black-coated golden rice with high carotenoid content is obtained.

[0013] The present invention also provides a method for breeding 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.

[0014] Preferably, the black rice material includes conventional rice varieties or transgenic rice varieties with high carotenoid content cultivated using black rice varieties as materials.

[0015] The present invention also provides a method for breeding 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;

[0016] (2) performing four consecutive backcrossings on the hybrid F1 to obtain a backcross variety BC4F1;

[0017] (3) The backcross variety BC4F1 is self-pollinated once to obtain black-coated golden rice.

[0018] 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.

[0019] Preferably, the golden rice variety in step (1) includes golden rice Ky1-4.

[0020] The present invention provides a method for increasing the carotenoid content in golden rice and delaying the degradation of carotenoids. In the 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 receptor. Fig.13 The process shown in the figure was followed by hybridization, backcrossing and selfing to obtain a GR lineage with black seed coat, GRB. Compared with the donor GRW, the contents of β-carotene and lutein in the brown rice of GRB were significantly increased. Unlike GR, which preferentially accumulated β-carotene, GRB accumulated more lutein and β-carotene. Since anthocyanins have the characteristics of absorbing visible and ultraviolet light, and carotenoids are sensitive to visible and ultraviolet light, the presence of anthocyanins is conducive to promoting the accumulation of carotenoids during seed development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The natural variation statistics of β-carotene and lutein in black rice (n=51), red rice (n=75) and white rice (n=421) are shown;

[0022] Figure 2 This is the result of HPLC detection of carotenoid content in brown rice, where Peak 1: Lutein; Peak 2: Zeaxanthin; Peak 3: β-cryptoxanthin; Peak 4: β-carotene;

[0023] Figure 3 The result diagram of the correlation analysis between β-carotene and anthocyanidin and between lutein and anthocyanidin in black rice (n=51);

[0024] Figure 4 The distribution diagram of lutein and β-carotene in brown rice and polished rice of representative black rice varieties;

[0025] Figure 5 The results of the light-induced in vitro degradation test of β-carotene and lutein are shown in the figure; a) The test device settings include 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) The light-induced in vitro degradation curves of β-carotene and lutein;

[0026] Figure 6 This is a schematic diagram of the structure of the pYLgRNA-OsU# plasmid vector;

[0027] Figure 7 Schematic diagram of the structure of the pYLCRISPR / Cas9-MH expression vector;

[0028] Figure 8 This is the sequencing verification result of OsMYB3, Kala4 and OsTTG1 gene knockout families;

[0029] Fig. 9 Phenotypes and anthocyanin contents of OsMYB3, Kala4, and OsTTG1 knockout families

[0030] Fig.10 The graph shows the changes in β-carotene and lutein content in OsMYB3, Kala4 and OsTTG1 gene knockout families;

[0031] Fig.11 Schematic diagram of pC2300 expression vector structure Ky1-4;

[0032] Fig.12 To obtain the contents of β-carotene and lutein in brown rice, polished rice, and rice bran of transgenic rice by introducing GR gene into black rice B02 and its Kala4 knockout line Kala4_KO8 (n=7) Fig.;

[0033] Fig.13 is a flow chart of the cultivation of GRB described in the present invention;

[0034] Fig.14 Color map for GRW and GRB seeds;

[0035] Fig.15 The graphs showing the changes in β-carotene and lutein content in GRW and GRB during seed development;

[0036] Fig.16 The graphs showing the changes in β-carotene and lutein contents of GRW and GRB during storage;

[0037] Fig.17 This is the degradation of GRB anthocyanins during storage. DETAILED DESCRIPTION

[0038] The invention provides application of black rice material in creating rice germplasm with high carotenoid content.

[0039] The present invention has found that lutein and β-carotene are the carotenoids with the highest content in black rice, and without the protection of anthocyanin solution, both β-carotene and lutein are rapidly degraded, while anthocyanin solution significantly reduces their degradation rates, and anthocyanin not only significantly reduces the rate of photodegradation of carotenoids, but also has a dose 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.

[0040] 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 common rice germplasm, genetically modified golden rice germplasm and black seed coat golden rice germplasm.

[0041] The present invention provides a method for increasing the carotenoid content of common rice varieties, comprising using the common rice varieties as receptors, transferring golden rice genes into the common rice varieties, and obtaining transgenic rice varieties with increased carotenoid content.

[0042] The common rice varieties of the present invention preferably include white rice, red rice, white rice and the offspring thereof, red rice and the offspring thereof, and even the offspring thereof, and their seeds contain almost no carotenoids or no carotenoids.

[0043] The present invention preferably utilizes transgenic means to transfer golden rice genes into common rice varieties, and the golden rice genes preferably include the phytoene synthase gene ZmPsy from corn and the phytoene desaturase gene PaCrtI from Pantoea ananas. 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 transgenic method, and genetic transformation can be performed using conventional methods in the art.

[0044] The present invention also provides a method for cultivating black seed golden rice with high carotenoid content, using black rice material as a receptor, transferring golden rice genes, and obtaining black seed golden rice with high carotenoid content. The present invention does not specifically limit the type of black rice material, which is preferably the same as above. The golden rice gene of the present invention is preferably the same as above, and will not be repeated here.

[0045] The present invention also provides a method for breeding 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.

[0046] The black rice material 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 present invention uses black rice materials as female parents and transgenic golden rice as male parents for hybridization, and the transgenic golden rice preferably includes golden rice obtained by transgenic means. The present invention preferably further includes screening after the hybridization, that is, screening according to the color of the endosperm, if it is yellow, it is a true hybrid, and if it is white, it is a false hybrid.

[0047] The present invention also provides a method for breeding 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;

[0048] (2) performing four consecutive backcrossings on the hybrid F1 to obtain a backcross variety BC4F1;

[0049] (3) The backcross variety BC4F1 is self-pollinated once to obtain black-coated golden rice.

[0050] 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.

[0051] The present invention is preferably based on Fig.13 The flow chart shown in the figure is used to increase the carotenoid content in golden rice and delay the degradation of carotenoids. The black rice variety of the present invention preferably includes Heishuai HS (derived from Shaanxi Academy of Agricultural Sciences and disclosed in Li Yanxia's master's degree thesis: Li Yanxia, ​​Lin Yongjun, Chen Hao. Functional study of three rice anthocyanin synthesis regulatory transcription factors. Master's degree thesis of Huazhong Agricultural University. 2016). The golden rice variety of the present invention preferably includes golden rice Ky1-4 (golden rice in the background of japonica rice variety Kongyu 131 (KY131), named 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 05, 2014, and promises to distribute the material to the public within 20 years from the date of application.

[0052] The black seed coat golden rice GRB bred by the breeding method of the present invention is a new rice variety rich in two nutrients, anthocyanins and carotenoids. Its seed coat is black and rich in anthocyanins; its endosperm is golden yellow and rich in carotenoids ( Fig.11 ).

[0053] The black-coated golden rice of the present invention is the hybrid offspring of golden rice and black rice. The anthocyanins in the 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 to promoting the accumulation of carotenoids during seed development.

[0054] The following is a detailed description of a method for increasing the carotenoid content in common rice and genetically modified golden rice (GR) provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] Analysis of carotenoid content in natural rice seed resources

[0057] 1.1 Determination of carotenoid content in rice seeds

[0058] For the extraction and determination of carotenoids, the method of Egesel et al. (Egesel, CO, Wong, JC, Lambert, RJ, & Rocheford, TR Combining ability of maize inbreds for carotenoids and tocopherols. 2003, Crop Science, 43: 818-823) was referred to, with a slight improvement: After all samples were taken, they were all freeze-dried in a vacuum freeze dryer at -80°C for later use. After the sample to be tested was ground into rice flour using a grinder, 600 mg was weighed into a 50 ml centrifuge tube. 6 ml of anhydrous ethanol containing 0.1% (v / v) BHT was added, mixed and then bathed at 85°C for 5 min. Then 120 μl of 80% (w / v) KOH was added, vortexed for 15 s, and then continued to bathe at 85°C for 10 min for saponification reaction, vortexing once during the period. Immediately pre-cool the sample on ice, add 3 ml of n-hexane and 3 ml of pre-cooled ultrapure water. Vortex to mix, 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 on the machine.

[0059] Carotenoids in rice seeds were determined by high performance liquid chromatography (HPLC, Aglient 1260). The reagents used (acetonitrile, methanol, dichloromethane) were all of HPLC grade, and the chromatographic column was a YMC-carotenoid column (YMC-carotenoid, 2.0×100mm, 3μm). Mobile phase A was acetonitrile; mobile phase B was a mixture of methanol and dichloromethane, with a volume ratio of 4:1. The total mobile phase contained 0.05% w / v triethylamine and 0.1% BHT. The volume ratio of mobile phase A and B was 3:1, the sample volume was 20μl, the flow rate was 1.8ml / min, the column temperature was 30℃, and the measurement wavelength was 450nm. Four standard samples of lutein, zeaxanthin, β-cryptoxanthin and β-carotene were purchased from Sigma-Aldrich.

[0060] 2.2 Determination of anthocyanin content in rice seeds

[0061] The content of anthocyanin in rice seeds was determined by 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): Take rice seeds 30 days after flowering, freeze-dry them in a vacuum freeze dryer, grind them into rice powder, weigh 100 mg and put them into a 2.0 ml centrifuge tube. Add 1 ml of extractant (105 ml of extractant: 80 ml of HPLC grade pure methanol, 20 ml of sterilized ddH2O and 5 ml of 5 mol / L HCl solution), extract on a shaker at 120 r / min at room temperature and avoid light for 24 hours, then carefully aspirate the supernatant, filter it through a 0.22 μm filter membrane, and inject it into a 2.0 ml brown injection bottle for detection.

[0062] Anthocyanins in rice seeds were determined by high performance liquid chromatography (HPLC, Aglient 1260). All reagents used were of HPLC grade, and the chromatographic column was a C18 column (ZORB-AX SB-Aq4.6×250mm, 5μm). Mobile phase A was ddH2O and pure formic acid (V:V=100:5), and mobile phase B was pure methanol. The mobile phase settings were: 0~12min, 20~70%B; 12~20min, 70~78%B. The flow rate was 0.8ml / min, the sample load was 10μl, the column temperature was 25℃, and the measurement wavelength was 520nm.

[0063] 2.3 Distribution of carotenoids in rice germplasm resources

[0064] This study investigated the natural variation of carotenoid content in 544 rice germplasm seeds, including 51 black rice, 72 red rice (accumulation of proanthocyanidins in the seed coat) and 421 white rice, which has been published in the academic journal New Phytologist: 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 are the highest content carotenoids in black rice, and the content of other types of carotenoids is very low or even absent. 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 ( Figure 1 and Figure 2 The content of lutein and β-carotene in black rice is significantly higher than that in red rice and white rice ( Figure 1 ). Among them, the β-carotene content of B03 in the black rice germplasm reached 2.05μg / g, which is equivalent to the first-generation golden rice GR (1.6μg / g).

[0065] 2.4 Correlation analysis between carotenoids and anthocyanins in black rice

[0066] 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 between anthocyanins and lutein, R 2 The correlation coefficient between anthocyanin and β-carotene is 0.42. 2 is 0.39, both are positively correlated ( Figure 3 ). Varieties high in lutein and beta-carotene are generally high in anthocyanins.

[0067] 2.5 Distribution of carotenoids in seeds

[0068] Five portions of black rice were randomly selected to measure the lutein and β-carotene content in brown rice and polished rice. It was found that the lutein and β-carotene content in polished rice was very low or even absent. Figure 4). This suggests that the synthesis of carotenoids, like anthocyanins, is only in the rice bran layer.

[0069] Example 2

[0070] Anthocyanins have a light-absorbing effect that delays the degradation of carotenoids

[0071] 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 light). 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 ability to absorb 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 ( Figure 5 a), wherein the outer tube is injected with anthocyanin (Shengqing Biotechnology, Xi'an) solution of different concentrations (1 mg / mL or 10 mg / mL) (solvent is methanol: water: 5M HCl = 16:4:1 V / V / V), and the inner tube is β-carotene (Sigma-Aldrich, starting concentration 9.21±0.79 μg / g) or lutein (Sigma-Aldrich, starting concentration 13.40±0.20 μg / g) solution (solvent is acetonitrile: methanol: dichloromethane = 75:20:5 V / V / V). The mouths of the two tubes are sealed with parafilm to prevent water from evaporating, and then sealed with a black plastic bag to prevent simulated sunlight from irradiating from the top without anthocyanin solution. The outer tube of the control sample is a colorless solvent without anthocyanin, and the inner tube is a β-carotene or lutein solution like the sample to be tested. 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 0min, 30min, 60min, 90min, 120min and 150min of simulated sunlight stress. After sampling, it was immediately placed in liquid nitrogen and kept away from light until content analysis.

[0072] 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, the β-carotene solution was degraded by 91.4% after 150 min of light stress; while under the protection of 1 mg / mL and 10 mg / mL anthocyanin solution, the 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 was degraded by 37.9% after 150 min of light stress; while under the protection of 1 mg / mL and 10 mg / mL anthocyanin solution, the lutein solution was degraded by 14.1% (P=0.0063) and 7.2% (P=0.0018), respectively ( Figure 5 In b). The above results indicate that anthocyanin solution not only significantly reduces the rate of photodegradation of carotenoids, but also has a dose effect, because the protective effect of 10 mg / mL anthocyanin solution on photodegradation of carotenoids is better than that of 1 mg / mL anthocyanin solution.

[0073] Example 3

[0074] Golden Rice with Black Rice as Background Increases Carotenoid Accumulation Levels

[0075] 3.1 Determination of carotenoid content in OsMYB3, Kala4 and OsTTG1 knockout materials

[0076] It is known that the synthesis of anthocyanins in black rice is activated and regulated by the ternary complex composed of transcription factors OsMYB3, Kala4 and OsTTG1. The OsMYB3, Kala4 and OsTTG1 genes of B02 in the black rice material were knocked out using the CRISPR / Cas9 system. The CRISPR / Cas9 knockout expression vector was constructed with reference to the method of Ma et al. (2015). Two knockout target sites were designed for each knockout gene, and the expression vectors of U3-driven gRNA1, U6a-driven gRNA2 and Ubiqutin promoter-driven Cas9 protein were constructed by PCR and enzyme ligation. Finally, the two fragments were simultaneously connected to pYLCRISPR / Cas9-MH digested with Bsa I. The specific process is as follows:

[0077] 1) According to the gene sequences of OsMYB3, Kala4 and OsTTG1, two pairs of primers were designed for each gene with two target sites (see Table 1 for specific sequences). Each pair of primers was dissolved in TE to prepare a 100 μM stock solution, and 1 μl of each was mixed and added to 98 μl of 0.5×TE to dilute to 1 μM. Denatured at 98°C for 30 seconds, and then annealed at room temperature to form a linker.

[0078] Table 1 Primer sequence list

[0079]

[0080]

[0081] 2) Digest and ligate at the same time. Add ATP to a final concentration of 0.5-1.0 mM in 1×Bsa I digestion buffer, and add ~20 ng pYLgRNA-OsU# plasmid ( Figure 6 ), 0.5 μL target linker (final concentration 0.05 μM), ~5 U BsaI, ~35 U T4 DNA ligase, supplemented with ddH2O to 10 μL. PCR reaction for 5 cycles: 37℃5min, 20℃5min.

[0082] 3) PCR amplification products.

[0083] First round of PCR (2 PCR reactions per gRNA expression cassette):

[0084] Take 1 μl of the above ligation product as template, primer UF / linker reverse primer (reaction 1), or linker 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×KODbuffer 10 μl, add water to 20 μl, PCR reaction 25-28 cylces: 95 ° C 10 s, 60 ° C 15 s, 68 ° C 20 s.

[0085] Second round of PCR:

[0086] Pre-mix the specific primer pairs B1′+B2, B2+BL (specific sequences are shown in Table 1) into 10× working solution (1.5 μM each): Take 1 μL of the products of reactions 1 and 2 of the first round of PCR and dilute them 10 times with ddH2O, then take 1 μL each and mix them as the template for the second round of PCR. Add 3 μL of primer pair working solution (final concentration 0.15 μM), 0.3 μL of KOD enzyme, 0.6 μL of dNTP (10 μM), 15 μL of 2×KOD buffer to each expression cassette, and finally add water to 30 μL. PCR instrument reaction 15-20 cycles: 95℃10s, 58℃15s, 68℃20s). Purify the second round of PCR products with a PCR product purification kit.

[0087] 4) Take 70 ng of PCR product and add 80 ng of uncut pYLCRISPR / Cas9-MH plasmid ( Figure 7), add 10U Bsa I to 15μl reaction system (1× Bsa I digestion buffer) and digest at 37℃ for 10min, then add 0.5μL 10×NEB T4 DNA ligase buffer and 35U T4 ligase. PCR instrument reaction 10-15 cycles: 37℃ 2min; 10℃ 3min, 20℃ 5min; finally 37℃ 2min.

[0088] 5) The ligation product was precipitated with ethanol, washed with 70% ethanol, air-dried and dissolved in 5 μL ddH2O), and 1 μL of the ligation product was electroporated to transform E. coli DH10B competent cells. After electroporation, 1 ml of SOC medium was added and cultured at 37°C for 1 hour. The plate was spread on LB medium containing 25 μg / mL karanomycin, 0.3-0.5 mM IPTG and an appropriate amount of X-gal, and positive clones that produced blue plaques were selected.

[0089] 6) Confirmation of positive clones. Positive clones were selected and shaken, plasmids were extracted, digested with Mlu I, and sequenced for verification.

[0090] 7) The obtained positive clones were transformed into Agrobacterium tumefaciens strain EHA105 and stored at -70°C until use.

[0091] 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:

[0092] 1) Callus induction:

[0093] Mature ZH11 rice seeds were shelled, then treated with 75% ethanol aqueous solution for 1 minute, and 0.15% mercuric chloride aqueous solution for 20 minutes. The seeds were washed 4 to 5 times with sterilized single distilled water, and the seeds were evenly placed on the induction medium. The inoculated medium was placed in a dark room for 4 to 6 weeks at a culture temperature of 28 ± 1 °C.

[0094] 2) Callus subculture

[0095] Select bright yellow, compact and relatively dry embryonic callus, place it on subculture medium and culture it in a dark room for 3 weeks at a culture temperature of 28±1℃.

[0096] 3) Pre-culture

[0097] Select compact and relatively dry embryonic calli, place them on the preculture medium and culture them in the dark for 4 days at a temperature of 28±1°C.

[0098] 4) Agrobacterium culture

[0099] Pre-culture Agrobacterium on LA medium with corresponding resistance selection for 2 days at 28±1°C. Transfer Agrobacterium to suspension medium and culture at 28°C, 200 rpm in a shaking incubator for 2-3 hours.

[0100] 5) Agrobacterium infection

[0101] Transfer the pre-cultured callus to a sterilized bottle. Adjust the Agrobacterium suspension to an OD of 600 = about 0.3. Soak the callus in the Agrobacterium suspension for 10 min. Transfer the callus to a sterilized filter paper and blot dry, then place it on the co-culture medium and culture it for 3 days at 19-20°C.

[0102] 6) Callus washing and selection culture

[0103] 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 and blot dry, then transfer the callus to selective culture medium for 3 times, each time for 2 weeks.

[0104] 7) Differentiation

[0105] The resistant calli were transferred to the predifferentiation medium and cultured in the dark for 7 days at 26±1°C. The predifferentiation cultured calli were transferred to the differentiation medium and cultured under light at 26±1°C.

[0106] 8) Rooting

[0107] 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℃.

[0108] 9) Transplanting

[0109] The residual culture medium on the roots is washed off, and the seedlings with good root systems are transferred into the greenhouse, while keeping them moist in the first few days (the culture medium and its preparation method of this embodiment are referred to patent No. 2011100832269).

[0110] The obtained transgenic regenerated plants were detected by PCR to amplify the region containing the two target sites and sequenced to determine the sequence. The primers used for target site 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 determined by sequencing ( Figure 8 ).

[0111] The anthocyanin content and carotenoid content were determined by HPLC. Knocking out Kala4 or OsTTG1 resulted in the 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 ( Fig. 9 In addition, knockout of OsMYB3, Kala4, or OsTTG1 caused a significant decrease in β-carotene and lutein ( Fig.10 ). This result once again verifies that anthocyanin synthesis plays an important protective role in the accumulation of carotenoids.

[0112] 3.2 Transgenic Golden Rice with Black Rice as Receptor Increases Carotenoid Accumulation

[0113] 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; while the promoter region of the Kala4 gene in black rice has changed, causing the originally non-expressed 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-knockout white rice mutant Kala4_KO8 ( Figure 8 and Fig. 9 ) as the receptor, and the 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, were transferred into the Golden Rice. The specific method is as follows:

[0114] 1) Target gene synthesis. The sizes of the two target genes, ZmPsy and PaCrtI, were 2765bp and 2760bp respectively, and they were commissioned to be synthesized by Shanghai Yingjun Biotechnology Co., Ltd. Both genes were expressed under the drive of the endosperm-specific glutenin promoter, and the transcription of the genes was terminated by the nos terminator. During gene synthesis, HindⅢ and SalⅠ restriction sites were added at both ends of the ZmPsy gene, and EcoRⅠ and KpnⅠ restriction sites were added at both ends of the PaCrtI gene.

[0115] 2) Double digest the expression vector pC2300 and Zmpsy gene with HindⅢ and SalⅠ, recover the exogenous fragment of Zmpsy gene, purify the vector fragment, connect the exogenous fragment of Zmpsy gene and the vector to form the intermediate vector pC2300-psy( Fig.11 ). The PaCrtI gene and pC2300-Psy vector were double-digested with EcoRⅠ and KpnⅠ. Finally, the expression vector pC2300-psy / crtI connecting the ZmPsy gene and the PacrtI gene was obtained. The constructed pC2300-psy / crtI expression vector was transformed into the Agrobacterium strain EHA105 by electroporation and stored at -70℃.

[0116] 3) The expression vector pC2300-psy / crtI was introduced into the black rice variety B02 and its Kala4 knockout white rice mutant Kala4_KO8 by Agrobacterium-mediated method, and 7 independent regenerated transgenic rice plants were obtained from each. The transformation method was the same as that in Example 3.1 above, except that the screening agent was replaced by G418 instead of hygromycin B.

[0117] The detection of carotenoid content in T3 transgenic plants showed that the contents of β-carotene in brown rice, polished rice and rice bran of transgenic plants with black rice B02 as receptor were 11.44±2.96μg / g, 8.10±1.27μg / g and 13.64±4.76μg / g (n=7), and the contents of lutein were 7.25±1.60μg / g, 1.36±0.35μg / g and 13.62± 3.84 (n=7); while the contents of β-carotene in brown rice, polished rice and rice bran of transgenic plants with white rice mutant as recipient were 3.17±0.92μg / g, 3.15±0.68μg / g and 5.36±1.80μg / g (n=7), and the contents of lutein were 0.57±0.41μg / g, 0.24±0.16μg / g and 0.99±0.47μg / g (n=7) ( Fig.12 ). Comparing the two sets of data, it can be found that the genetically modified golden rice with black rice as the receptor accumulates significantly more carotenoid content than the golden rice with white rice as the receptor ( Fig.12 ).

[0118] 3.3 Rice hybridization and the breeding process of black golden rice

[0119] according to Fig.13 The process shown in the figure is to select the black rice variety HS plant that is about to bloom on the same day as the female parent, scald the ears that are about to bloom with warm water at 45℃ for 5 minutes, then remove the stamens and put them in bags; collect the natural pollen of the golden rice material GRW (male parent) with white rice as the receptor, and then artificially pollinate the emasculated HS, and seal the hybridization bag. The pollinated plants are grown in the field for about 25 days and harvested as the F1 generation. Observe the color of the endosperm of the hybrid F1 seeds with the naked eye. If it is yellow, it is a true hybrid, and if it is white, it is a false hybrid. Use the same hybridization method according to Fig.13 The black golden rice (GRB) was obtained by backcrossing 4 times and selfing once.

[0120] GRB is a new rice variety rich in anthocyanins and carotenoids. The seed coat of GRB is black and rich in anthocyanins; the endosperm is golden yellow and rich in carotenoids ( Fig.14 ).

[0121] 3.4 Changes in carotenoid content during seed development of GRW and GRB

[0122] GRW and GRB seeds were sown at the right time to ensure that their flowering period was consistent. All rice materials were planted under normal field management. The panicles that bloomed on the same day were marked, and about 200 panicles were marked for each variety. The upper third of the marked panicles were collected at 10 days, 15 days, 20 days, 25 days, 30 days and 35 days after flowering (usually one panicle blooms from top to bottom for three days) and placed in liquid nitrogen, and stored at -80℃ until all samples were prepared.

[0123] like Fig.12 As shown in the figure, the dynamic changes of carotenoid content in GRW and GRB during seed development from 10 days to 35 days after flowering. The study found that the β-carotene content of GRB was always higher than that of GRW throughout the seed development process; the β-carotene content of GRW and GRB reached a peak at 15 days after flowering, and then continued to decline until the seeds matured 35 days after flowering; 35 days after flowering, the β-carotene content of GRW and GRB was 7.8±0.3μg / g and 18.2±0.9μg / g, respectively, and the β-carotene content of GRB was 2.3 times that of GRW ( Fig.15 ).

[0124] The lutein content of both GRW and GRB was at its highest level 10 days after flowering, and the content continued to decline as the seeds matured. During seed development, the lutein content of GRW and GRB did not change significantly before 20 days after flowering, but then the lutein content of GRW dropped sharply, while the lutein content of GRB dropped relatively slowly. This resulted in the lutein content of GRB (4.21±0.35μg / g) being 10.5 times that of GRW (0.40±0.02μg / g) at 35 days after flowering. Fig.15 ).

[0125] 3.5 Changes in carotenoid content during storage of GRW and GRB seeds

[0126] After harvesting, the husks of GRW and GRB were removed 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 taken and vacuum-packed and stored at -80°C. After all samples were prepared, they were uniformly processed and the carotenoid and anthocyanin contents were determined.

[0127] The changes of carotenoid contents in brown rice of GRW and GRB under room temperature storage were compared. The contents of β-carotene and lutein were determined by HPLC at 0, 2, 4, 8, 16, 24, 32 and 40 weeks after storage. After 40 weeks of storage, the content of β-carotene in GRW was 0.89±0.01μg / g, which was degraded by 85.8% compared with the initial content; the content of β-carotene in GRB was 2.37±0.07μg / g, which was degraded by 86.7% compared with the initial content. After 40 weeks of storage, the content of β-carotene in GRB was 2.7 times that of GRW. As for the content of lutein after storage, the content of GRW was 0.15±0.00μg / g, which was degraded by 48.2% compared with the initial content; the content of GRB was 2.56±0.01μg / g, which was degraded by 44.9% compared with the initial content. Fig.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 ( Fig.17 ). Given that the contents of β-carotene and lutein in GRB are much higher than those in GRW at harvest, and that the degradation rates of β-carotene and lutein in GRB are roughly the same as those in GRW after storage, the carotenoid content of GRB is also much higher than that of GRW after long-term storage.

[0128] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

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

2. The use 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. 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.

6. 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.

7. The cultivation method according to claim 6, 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.

8. 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.

9. The breeding method according to claim 8, 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.

10. The breeding method according to claim 8, characterized in that: The golden rice varieties in step (1) include golden rice Ky1-4.

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