阅读说明：本技术 一种基于CRISPR/Cas9转变水稻果皮色的基因编辑方法及其应用 (Gene editing method for converting rice peel color based on CRISPR/Cas9 and application thereof ) 是由 戴伟民 强胜 宋小玲 孔梦瑶 孙茜茜 于 2019-11-07 设计创作，主要内容包括：本发明涉及了一种基于CRISPR/Cas9变换水稻果皮色(白至红、红至白)的基因编辑方法,其包括如下步骤：(1)合成转录因子rc基因的特定gRNA-spacer；(2)构建CRISPR/Cas-gRNA-spacer载体；(3)采用农杆菌转化法转化水稻品种的成熟胚、再生植株。T<Sub>0</Sub>代种子即可实现水稻品种果皮色的白至红、红至白的相互转变。红米在世界各国长期用作滋补食品,是水稻育种的重要目标之一。经过人工长期驯化的水稻果皮色为白色,抗性弱,而来自栽培稻脱驯化的杂草稻果皮色为红色,抗性强。通过相互转换果皮色,可能培育出抗性强的白\红果皮水稻品种。本技术具有良好的商业化应用前景。(The invention relates to a gene editing method for transforming the peel color (white to red and red to white) of rice based on CRISPR/Cas9, which comprises the following steps: (1) synthesizing a specific gRNA-spacer of a transcription factor rc gene; (2) constructing a CRISPR/Cas-gRNA-spacer vector; (3) mature embryos and regenerated plants of rice varieties are transformed by an agrobacterium transformation method. T is 0 The mutual transformation of the white to red and red to white of the peel color of the rice variety can be realized by the generation of seeds. Red rice is used as a tonic food in countries of the world for a long time and is one of the important targets of rice breeding. Long-term domesticated riceThe peel color of the weedy rice is white and the resistance is weak, while the peel color of the weedy rice which is removed and domesticated from the cultivated rice is red and the resistance is strong. By mutually converting the color of the peel, the rice variety with the white/red peel and strong resistance can be cultivated. The technology has good commercial application prospect.)

1. A gRNA spacer-Rc1 sequence for changing the color of rice pericarp based on CRISPR/Cas9, characterized in that the nucleotide sequence of the gRNA spacer-Rc1 comprises

gRNA-Spacer-Rc 1F: 5'-GGCAGGGGCGGGAAAGGCGCAAG-3' (SEQ ID NO.1), and

gRNA-Spacer-Rc1R：5’-AAACCTTGCGCCTTTCCCGCCCC-3’(SEQ ID NO.2)。

a construction method of a CRISPR/Cas9-gRNA-spacer expression vector pRGEB32-Rc1, which is characterized by comprising the following steps:

(1) annealing gRNA-Spacer-Rc1F shown in SEQ ID NO.1 and gRNA-Spacer-Rc1R shown in SEQ ID NO.2 to form a double strand;

(2) digesting the plasmid pRGEB32 by BsaI, connecting a double chain formed by gRNA spacer-Rc1F and gRNA spacer-Rc1R, and constructing an expression vector pRGEB32-Rc 1;

(3) the expression vector pRGEB32-Rc1 was PCR amplified using the primer pRGEB32-3 and sent to sequencing company for sequencing verification, and the sequencing result included the sequence shown in SEQ ID NO.1, namely pRGEB32-Rc1 which was indicated as being successfully constructed.

3. The method for constructing the CRISPR/Cas9-gRNA-Spacer expression vector pRGEB32-Rc1 according to claim 2, wherein the annealing system of the gRNA-Spacer-Rc1F and the gRNA-Spacer-Rc1R in the step (1) is a10 μ l system: gRNA-Spacer-Rc1F 1ul (100. mu.M); gRNA-Spacer-Rc1R 1ul (100. mu.M); 10 XT 4 DNA ligase buffer 1. mu.l (50mM Tris-HCl,10mM MgCl)₂,10mM DTT,1mM ATP,pH7.5，25℃)；ddH₂07 mu l of the mixture; placing the PCR tube in a PCR instrument at 37 deg.C for 60min and 95 deg.C for 10min, and standingCooled to 25 ℃ for 1min to form double strands.

4. The method for constructing the expression vector pRGEB32-Rc1 of CRISPR/Cas9-gRNA-spacer according to claim 2, wherein the primer pRGEB32-3 in the step (3) comprises the following steps:

pRGEB32-3-F：5’-CTGGGTACGTTGGAAACCAC-3’(SEQ ID NO.3),

pRGEB32-3-R：5’-CGGCCCAAATTGAAAAGATA-3’(SEQ ID NO.4)；

the PCR amplification system of the expression vector pRGEB32-Rc1 is 2 XPCR Mix 4.0 uL, ddH₂O 4.5μL,pRGEB32-3-F(10μmol/L)0.25μL,pRGEB32-3-R(10μmol/L)0.25μL,DNA(10-20ng/μL)1μL,

The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, and elongation at 72 ℃ for lmin for 30 cycles; extension was carried out at 72 ℃ for 8 min.

5. The CRISPR/Cas9-gRNA-spacer expression vector pRGEB32-Rc1 constructed based on the construction method of any one of claims 2 to 4.

6. A gene editing method for converting the peel color of rice based on CRISPR/Cas9, which is characterized by comprising the following steps:

(1) aiming at the structure of the rice Rc protein, a specific gRNA-spacer-Rc1 for synthesizing a transcription factor Rc gene is designed: the nucleotide sequence of the gRNA spacer comprises:

gRNA-Spacer-Rc 1F: 5'-GGCAGGGGCGGGAAAGGCGCAAG-3' (SEQ ID NO.1), and

gRNA-Spacer-Rc1R：5’-AAACCTTGCGCCTTTCCCGCCCC-3’(SEQ ID NO.2)；

(2) constructing the CRISPR/Cas9-gRNA-spacer vector pRGEB32-Rc1 of claim 5;

(3) and (3) agrobacterium infection regeneration, and obtaining pericarp mutation seeds: the constructed pRGEB32-Rc1 is transferred into Agrobacterium tumefaciens EHA105, the infected Agrobacterium is used for culturing callus with the callus of mature rice and weedy rice, and then the mutant seed for changing the peel color of the rice is obtained by screening and regenerating in a culture medium containing 50mg/L hygromycin.

7. The gene editing method for converting the color of rice pericarp based on CRISPR/Cas9 as claimed in claim 6, wherein the specific step of step (3) is as follows: transferring the constructed pRGEB32-Rc1 into Agrobacterium tumefaciens EHA105, performing genetic transformation test by an Agrobacterium mediated method, respectively shelling rice and weed rice seeds, selecting full and non-speck healthy seeds, sterilizing the seeds by using 75 vol% ethanol and 30 vol% sodium hypochlorite, and inoculating the seeds on a culture medium containing 2,4-D hormone; carrying out subculture after dark culture for two weeks, and selecting light yellow callus with good activity for agrobacterium infection transformation; dark culture is carried out for two days after the callus is infected, and two rounds of screening are carried out by transferring the callus into a culture medium containing 50mg/L hygromycin; obtaining resistant callus after one month after 15 days in one turn, and transferring the resistant callus into a differentiation culture medium; culturing and differentiating at 26 ℃ by illumination to obtain T₀The generation plants are then screened and regenerated in a culture medium containing 50mg/L hygromycin to obtain mutant seeds for changing the peel color of the rice.

8. The gene editing method for converting rice pericarp color based on CRISPR/Cas9 of claim 6, wherein the converting rice pericarp color is converting white pericarp to red pericarp or converting red pericarp to white pericarp.

9. The gRNA spacer-Rc1 sequence for CRISPR/Cas 9-based rice pericarp color conversion of claim 1, or

The CRISPR/Cas9-gRNA-spacer vector pRGEB32-Rc1 of claim 5, or

The application of the CRISPR/Cas 9-based gene editing method for converting the color of rice pericarp in the conversion of the color of rice pericarp in claim 6.

10. The use of claim 9, wherein the color of the rice pericarp is changed to white pericarp or to red pericarp.

Technical Field

The invention belongs to the field of molecular biology, and relates to a gene editing method for converting rice pericarp color (white to red, red to white) based on CRISPR/Cas9 and application thereof.

Background

Colored rice, including red rice, is of great interest because of its rich flavonoid content, which is a dietary polyphenol of plant origin, and is believed to have antioxidant, cardioprotective, hypoglycemic, cancer and cardiovascular functions (Tsudaet al, 2002; Engler et al, 2004; Walter et al, 2011; Jaeger et al, 2017). The long-term artificial selection and domestication enables modern rice varieties to be basically white pericarp without flavonoid bioactive substances, and more residents begin to pursue healthy diet instead of just satisfying the requirements of being full of warmness with the development of economy. Reintroduction of flavonoid-rich substances into modern varieties has become one of the important targets in rice breeding (gunnaratene et al, 2013; shara et al, 2014; Jun et al, 2018). The peel color of the rice domesticated for a long time is white and the resistance is weak, while the peel color of the weedy rice domesticated from the cultivated rice is red and the resistance is strong. By mutually converting the color of the fruit peel, the white/red fruit peel rice variety with strong resistance can be cultured.

The Rc allele has a key role in the formation of red pericarp. Rc (LOC _ Os07g11020) is located on chromosome 7, approximately 6400bp in length, and the translation product is a protein with basic helix-loop-helix (bHLH) elements (Sweeney et al, 2006). The bHLH protein has 4 conserved functional regions, respectively, an interaction region (I), an acidic region (A), a basic helix-loop-helix region (bHLH), and a C-terminal region (C) (Buck and Atchley, 2003; Fan et al, 2014; Hichr et al, 2011).

The Rc allele has a 14bp deletion in its exon 6 compared to the Rc allele. 97.9% of the white caryopsis are due to the 14bp deletion resulting in an incomplete translated bHLH protein structure (Sweeney et al, 2006; Sweeney et al, 2007). Brooks et al (2008) found that the native red pericarp of the American cultivar "Wells" resulted from a single base deletion 20bp upstream of the 14bp deletion, i.e., a total of 15 bases relative to the Rc allele, restoring the bHLH protein structure of the Rc gene. Lee et al (2009) reported that Italian rice "Perla" is a single base deletion at 44bp upstream of 14bp, restoring the bHLH protein structure of the Rc gene, and also leading to red peel recovery. Therefore, it is theoretically possible to restore the function of Rc by mutating the Rc gene.

The CRISPR/Cas9(clustered regularly interspersed short palindromic repeats) gene editing technology newly developed in recent years can accurately edit the functions of genes, and provides a possibility for realizing red/white interconversion of peel colors. The CRISPR-Cas9 system is useful for genome editing in a variety of organisms, including plant and crop species, by creating a Double Strand Break (DSB) at a specific site of chromosomal DNA and introducing an INDEL mutation (Xie and Yang 2013) at the DSB. The system can realize the site-specific mutagenesis and stable inheritance of the rice genome, and can obviously improve the breeding efficiency (Feng et al, 2013; Zhang et al, 2014; Xu et al, 2015). At present, a gene editing method for freely changing the skin color of red ginkgo is not established, and in the process of mutating the Rc \ Rc gene by using the CRISPR/Cas9 technology, the primer is discovered to be mutated unintentionally, so that the free change of the skin color of red ginkgo can be realized.

Disclosure of Invention

In order to solve the technical problems of the mutual conversion of the skin colors of the red ginkgo and the cultivation of the white/red fruit peel rice variety with strong resistance, the invention designs and screens a specific gRNA-spacer on the basis of the previous protein structure research on the rc gene of the rice transcription factor so as to construct a CRISPR/Cas9-gRNA-spacer vector. Introducing the vector by an agrobacterium infection method to obtain T₀Plant generation, T₀Substitute for seedThe fruit can obtain the color mutation of the peel. The research realizes the free conversion of the peel color of the rice variety from white to red and from red to white.

The purpose of the invention can be realized by the following technical scheme:

the first purpose of the invention is to provide a gRNA spacer sequence for converting the color of rice pericarp based on CRISPR/Cas9, wherein the nucleotide sequence of the gRNA spacer-Rc1 comprises

gRNA-Spacer-Rc 1F: 5'-GGCAGGGGCGGGAAAGGCGCAAG-3' (SEQ ID NO.1), and

gRNA-Spacer-Rc1R：5’-AAACCTTGCGCCTTTCCCGCCCC-3’(SEQ ID NO.2)。

the second purpose of the invention is to provide a construction method of CRISPR/Cas9-gRNA-spacer vector, which comprises the following steps:

(1) annealing the gRNA-Spacer-Rc1F shown in SEQ ID NO.1 and the gRNA-Spacer-Rc1R shown in SEQ ID NO.2 to form a double strand.

(2) Adopting BsaI to carry out enzyme digestion on the plasmid pRGEB32, connecting a double chain formed by gRNA spacer-Rc1F shown in SEQ ID NO.1 and gRNA spacer-Rc1R shown in SEQ ID NO.2, and constructing to obtain an expression vector pRGEB32-Rc 1;

(3) the expression vector pRGEB32-Rc1 was PCR amplified using the primer pRGEB32-3 and sent to sequencing company for sequencing verification, and the sequencing result included the sequence shown in SEQ ID NO.1, namely pRGEB32-Rc1 which was indicated as being successfully constructed.

Further, the annealing system of the gRNA-Spacer-Rc1F and the gRNA-Spacer-Rc1R in the step (1) is a10 μ l system: gRNA-Spacer-Rc1F 1ul (100. mu.M); gRNA-Spacer-Rc1R 1ul (100. mu.M); 10 XT 4 DNA ligase buffer, 1. mu.l (50mM Tris-HCl,10mM MgCl)₂,10mM DTT,1mM ATP,pH7.5，25℃；NEBcompany；http://www.neb-china.com/)；ddH₂07 mu l of the mixture; the PCR tube was placed in a PCR apparatus (PCR thermal cycler Dice; Takara company) at 37 ℃ for 60min, at 95 ℃ for 10min, and naturally cooled to 25 ℃ for 1min to form a double strand.

Further, in the step (3), the primer pRGEB32-3 comprises:

pRGEB32-3-F：5’-CTGGGTACGTTGGAAACCAC-3’(SEQ ID NO.3),

pRGEB32-3-R：5’-CGGCCCAAATTGAAAAGATA-3’(SEQ ID NO.4)。

the PCR amplification system of the expression vector pRGEB32-Rc1 is 2 XPCR Mix 4.0 uL, ddH₂O4.5. mu.L, pRGEB32-3-F (10. mu. mol/L) 0.25. mu.L, pRGEB32-3-R (10. mu. mol/L) 0.25. mu.L, DNA (10-20 ng/. mu.L) 1. mu.L, PCR reaction program: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min, 30 cycles; extension was carried out at 72 ℃ for 8 min.

The third purpose of the invention is to provide the CRISPR/Cas9-gRNA-spacer expression vector pRGEB32-Rc1 constructed based on the construction method.

The fourth purpose of the invention is to provide a gene editing method for converting the peel color of rice based on CRISPR/Cas9, which comprises the following steps:

(1) aiming at the structure of the rice rc protein, a specific gRNA-spacer for synthesizing a transcription factor rc gene is designed: the nucleotide sequence of the gRNA spacer comprises:

gRNA-Spacer-Rc 1F: 5'-GGCAGGGGCGGGAAAGGCGCAAG-3' (SEQ ID NO.1), and

gRNA-Spacer-Rc1R：5’-AAACCTTGCGCCTTTCCCGCCCC-3’(SEQ ID NO.2)；

(2) construction of the aforementioned CRISPR/Cas9-gRNA-spacer vector pRGEB32-Rc 1:

preferably, the construction method comprises the following steps: the gRNA-Spacer-Rc1F and gRNA-Spacer-Rc1R were annealed to form a double strand. The annealing system is a10 μ l system: gRNA-Spacer-Rc1F 1ul (100. mu.M); gRNA-Spacer-Rc1R 1ul (100. mu.M); 10 XT 4 DNA ligase buffer 1. mu.l (50mM Tris-HCl,10mM MgCl)₂10mM DTT,1mM ATP, pH7.5, 25 ℃; ) (ii) a ddH 207. mu.l. The PCR tube was placed in a PCR apparatus (PCR Thermal Cycler Dice) at 37 ℃ for 60min and 95 ℃ for 10min, and naturally cooled to 25 ℃ for 1min to form a double strand. BsaI is adopted to cut the plasmid pRGEB32, and a double chain formed by gRNA spacer-Rc1F shown in SEQ ID NO.1 and gRNA spacer-Rc1R shown in SEQ ID NO.2 is connected, so that an expression vector pRGEB32-Rc1 is constructed. PCR amplification detection, the PCR system is 2 XPCR Mix 4.0 uL, ddH₂O4.5. mu.L, pRGEB32-3-F (10. mu. mol/L) 0.25. mu.L, pRGEB32-3-R (10. mu. mol/L) 0.25. mu.L, DNA (10-20 ng/. mu.L) 1. mu.L, PCR reaction program: pre-denaturation at 94 ℃ for 5 min; 94Denaturation at 55 deg.C for 45s, annealing at 55 deg.C for 45s, extension at 72 deg.C for l min, and 30 cycles; extension was carried out at 72 ℃ for 8 min. Sending the DNA sequence to a sequencing company for sequencing verification, wherein the sequencing result comprises a sequence shown in SEQ ID NO.1, namely pRGEB32-Rc1 which shows that the construction is successful. The primer pRGEB32-3 comprises:

pRGEB32-3-F：5’-CTGGGTACGTTGGAAACCAC-3’(SEQ ID NO.3),

pRGEB32-3-R：5’-CGGCCCAAATTGAAAAGATA-3’(SEQ ID NO.4)。

(3) and (3) agrobacterium infection regeneration, and obtaining pericarp mutation seeds: the constructed pRGEB32-Rc1 is transferred into Agrobacterium tumefaciens EHA105, the infected Agrobacterium is used for culturing callus with the callus of mature rice and weedy rice, and then the mutant seed for changing the peel color of the rice is obtained by screening and regenerating in a culture medium containing 50mg/L hygromycin.

Further, the constructed pRGEB32-Rc1 was transferred into Agrobacterium tumefaciens EHA105, and genetic transformation experiments were performed by Agrobacterium-mediated method, rice and weed rice seeds were separately dehulled, and healthy seeds with plump grains and no spots were selected and sterilized with 75 vol% ethanol and 30 vol% sodium hypochlorite, and inoculated on a 2,4-D hormone-containing medium. And (4) carrying out subculture after dark culture for two weeks, and selecting light yellow callus with good activity for agrobacterium infection transformation. After the infection of the callus, the callus is cultured in the dark for two days, and then is transferred into a hygromycin-containing culture medium for two rounds of screening. After 15 days, resistant callus is obtained after one month and transferred into a differentiation culture medium. Culturing and differentiating at 26 ℃ by illumination to obtain T₀And (3) generating plants, and then screening and regenerating in a culture medium containing 50mg/L hygromycin to obtain mutant seeds for changing the peel color of the rice.

Further, the rice pericarp color is changed into white pericarp or red pericarp.

The fifth purpose of the invention is to provide the gRNA spacer-Rc1 sequence shown in the SEQ ID NO.1 and SEQ ID NO.2 and used for converting the color of the rice pericarp based on CRISPR/Cas9, or

The aforementioned CRISPR/Cas9-gRNA-spacer vector pRGEB32-Rc1, or

The application of the gene editing method for converting the color of the rice pericarp based on CRISPR/Cas9 in converting the color of the rice pericarp is provided.

Further, the color of the rice pericarp is changed into that of white pericarp or that of red pericarp.

The invention has the beneficial effects that:

by the method, the free conversion of the red and white of the peel color of the rice variety can be realized. The method has the advantages of simplicity, convenience, rapidness, accuracy and the like. The red peel rice variety is usually strong in adaptability but poor in quality; white rice varieties are good in quality but not strong in adaptability. By freely switching each other, better varieties can be cultured. In addition, the red peel contains procyanidine, so that the procyanidine can effectively inhibit various cancers and other health-care functions clinically, and is gradually favored by more and more people. The technology has good commercial application prospect.

Drawings

FIG. 1 monoclonal PCR of CRISPR/Cas9 expression vector

Wherein "M" is a D2000 label; "+" indicates that the plasmid was a positive control; "-" denotes ddH₂O is negative control;

1-6 pRGEB32-Rc1 monoclonal bacteria

FIG. 2 alignment of pRGEB32-Rc1 vector with the original plasmid pRGEB32 sequence

Wherein "gRNA spacer-Rc 1" is the target position 1

FIG. 3 Agrobacterium genetic transformation of mature embryo callus of oryza sativa (Nipponbare) and weedy rice (WRL-162)

Wherein FIG. 3(a) induces callus; (b) subculturing the callus; (c) agrobacterium and callus co-infected by Agrobacterium

Culturing; (d) screening the callus on a hygromycin culture medium; (e) differentiation of callus on hygromycin medium; (f) supporting culture; (g)

domesticating in a barrel; (h) and (5) plant growing.

FIG. 4T of Rice and weedy Rice₀Molecular identification of plant generation

Hygromycin (Hpt), Cas9, and UBI are all sequences on the pREGB32 vector;

FIG. 4(a) shows rice, 22 seedlings were regenerated in total, hygromycin (Hpt), Cas9 and UBI were tested, 9 of them ( samples 1,2,3, 4, 16, 17, 18, 20 and 21 in the figure) were positive plants, and further Rc gene was sequenced, and 5 of them in which Rc mutation occurred were found to be numbers 16, 17, 18, 20 and 21, respectively, and named: rc 1-557-16, Rc 1-557-17, Rc 1-557-18, Rc1-557-20, Rc 1-557-21;

fig. 4(b) shows oryza sativa, 20 seedlings were regenerated, hygromycin (Hpt), Cas9 and UBI were tested, 9 plants were positive plants ( samples 1,2,3, 8, 9, 15, 16, 17 and 18 in the figure), and the Rc gene was further sequenced, and 2 plants in which the Rc mutation occurred were found to be nos. 1 and 18, respectively, and were named: rc1-162-1 and Rc 1-162-18.

FIG. 5T₀Generation mutant seeds and caryopsis.

Wherein, FIG. 5(a) shows seeds and caryopsis of Nipponbare; and seeds and caryopsis of the Rc1 vector rice mutant;

FIG. 5(b) is a representation of seeds and caryopsis of weedy rice; and seeds and caryopses of the Rc1 vector weedy rice mutant.

Detailed Description