A method and system for genetic transformation and multi-gene editing of diploid wild rice

Abstract

The present invention relates to the field of biotechnology, specifically providing a method and system for genetic transformation and multi-gene editing of diploid wild rice. The present invention establishes a wild rice genetic transformation platform by optimizing the conditions of induction culture medium, Agrobacterium infection, and regeneration culture medium. On this basis, key genes for domestication, such as the gene encoding the zinc finger transcription factor that controls the transition of wild rice plant type from creeping growth to upright growth, are used to transform wild rice. PROG1 , genes that determine tiller angle size D2 , transcription factor encoding genes that control awn length and yield in wild rice An‑1 , key pleiotropic genes controlling rice yield DEP1 , genes encoding grain width GW2 , genes controlling the color of wild rice husks Bh4 For the target gene, a CRISPR / Cas9-mediated diploid wild rice multi-gene editing system was constructed, gene-edited stable plants were obtained through genetic transformation, and a genome editing technology system for annual and perennial diploid wild rice was established, thereby quickly obtaining new stress-resistant gene resources and new germplasm of diploid wild rice with breeding value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method and system for genetic transformation and multi-gene editing of diploid wild rice. Background Art

[0002] Rice is a staple food crop in my country and around the world. While my country has made significant progress in rice breeding over the past few decades, the rapid pace of urbanization has led to a reduction in arable land, frequent extreme weather events and pests and diseases, and increased natural depletion, posing significant threats to rice production safety. Ensuring efficient, green, and sustainable rice production is a pressing challenge for my country and the world.

[0003] The limited genetic diversity of existing rice varieties and the high homogeneity of breeding materials are major limitations to efficient, green, and sustainable rice production. While the domestication and breeding processes have improved key agronomic traits of crops, they have also resulted in a significant loss of genetic diversity, such as stress resistance. Using gene editing for "de novo domestication"—directly editing key agronomic trait genes in wild germplasm—improves these important agronomic traits while retaining the stress resistance lost during long-term domestication. This holds the promise of breeding superior new crop varieties. Currently, de novo domestication has been successfully applied to wild plants such as wild tomatoes and tetraploid wild rice.

[0004] Annual and perennial diploid wild rice possesses a rich genetic resource for stress resistance, with strong resistance to disease, insects, and salt, making it an important germplasm resource for expanding the genetic diversity of existing rice varieties and promoting germplasm innovation. Directly utilizing the excellent genetic resources of diploid wild rice through hybridization and backcrossing requires many years and generations of backcrossing, and it is difficult to get rid of the genetic baggage of unfavorable genes, which seriously hinders the exploration and breeding of excellent genetic resources of diploid wild rice. De novo domestication and improvement of diploid wild rice through gene editing will greatly promote the breeding and utilization of its excellent genetic resources and enrich the diversity of cultivated rice germplasm resources. However, diploid wild rice, especially perennial diploid wild rice, is difficult to genetically transform and gene edit. Currently, there are no reports of de novo domestication and improvement of diploid wild rice through multi-gene editing.

[0005] Rice is an important food crop in my country. Breeding high-yield, high-quality rice varieties is crucial for my country's food security and social stability. The CRISPR / Cas9 system, a simple, efficient, and effective editor for targeted, multi-site crop trait improvement, has been widely applied in crop genetic improvement and breeding. De novo domestication of diploid wild rice with superior stress tolerance involves using gene editing technology to directly edit genes for key domestication-related agronomic traits. By combining the superior traits of domesticated and improved wild rice germplasm with those of modern cultivated rice, it is hoped that new high-yield, stress-resistant, and green rice varieties can be rapidly developed, which is of great significance for ensuring global food security. Summary of the Invention

[0006] The purpose of the present invention is to establish a wild rice genetic transformation platform by optimizing the conditions of induction culture medium, Agrobacterium infection and regeneration culture medium; on this basis, to domesticate key genes, such as the zinc finger transcription factor encoding gene that controls the transformation of wild rice plant type from creeping growth to upright growth PROG1 , determines the size of the tillering angle D2 Genes encoding transcription factors that control awn length and yield in wild rice An-1 , key pleiotropic genes controlling rice yield DEP1 , genes encoding grain width GW2 , genes controlling the color of wild rice husks Bh4 Using genes such as IL-6 as target genes, a CRISPR / Cas9-mediated diploid wild rice multi-gene editing system was constructed, gene-edited stable plants were obtained through genetic transformation, and a genome editing technology system for annual and perennial diploid wild rice was established, thereby quickly obtaining new stress-resistant gene resources and new germplasm of diploid wild rice with breeding value.

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

[0008] The present invention provides a gene editing transformation vector, wherein the gene editing transformation vector is a gene PROG1 Gene, D2 Gene, DEP1 Gene, An-1 Gene, GW2 Gene, Bh4The gene is the target gene, and the six-gene combination knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1(PROG1)-tRNA-sgRNA2(An-1)-tRNA-sgRNA3(DEP1)-tRNA-sgRNA4(D2)-tRNA-sgRNA5(GW2)-tRNA-sgRNA6(Bh4)-tRNA-PolyA-Nos is constructed; the nucleotide sequence of the six-gene combination knockout vector is composed of the two sequences of SEQ ID NO.1 and SEQ ID NO.2, and the splicing order is the last base of SEQ ID NO.1 connected to the first base of SEQ ID NO.2; the PROG1 Gene, D2 Gene, DEP1 Gene, An-1 Gene, GW2 Gene, Bh4 The nucleotide sequence of the gene is shown in SEQ ID NO. 3-8;

[0009] In a specific embodiment, the above-mentioned gene editing transformation vector is artificially synthesized by interactive tandem tRNA multiple gRNA expression technology, and the specific steps are as follows:

[0010] (1) Obtaining the gRNA-tRNA basic fragment using the overlapping PCR method: in the first round of PCR, the gRNA basic fragment is obtained using primers gRNAF1, gRNAR1, and gRNAR2, and the tRNA basic fragment is obtained using primers tRNAF1, tRNAR1, and tRNAR2; in the second round of PCR, the gRNA fragment and tRNA fragment of the first round of PCR products are mixed at a molar ratio of 1:1 as a template, and the primer pair gRNAF2 / tRNAR3 is used to amplify, and finally the gRNA-tRNA basic fragment is obtained; the base sequences of the primers gRNAF1, gRNAR1, and gRNAR2 are shown in SEQ ID NOs. 9-11; the base sequences of the primers tRNAF1, tRNAR1, and tRNAR2 are shown in SEQ ID NOs. 12-14;

[0011] (2) Obtain tRNA-sgRNA1 using overlapping PCR method ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragment: The first round of PCR uses the gRNA-tRNA basic fragment as a template and uses the primer pair Actin-tRNAF1 and PROG1-tRNAR1 to obtain PROG1The tRNA fragment of the gene target sequence was then amplified using the primers PROG1-gRNAF1, An1-tRNAR1 and An1-gRNAF1, PolyA-tRNA-R1 using the gRNA-tRNA basic fragment as a template to obtain sgRNA1 with a linker ( PROG1 )-tRNA and sgRNA2( An-1 )-tRNA fragment; the second round of PCR was performed using the three PCR products obtained in the first round mixed at a molar ratio of 1:1:1 as a template, and primers ActinF1 and PolyA-R1 were used to amplify, and finally tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragments and purify and recover them; at the same time, restriction endonucleases were used Pme I. Enzyme digestion of the vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-PolyA-Nos was performed, and the digestion products were recovered; finally, the recovered products were connected using homologous recombination enzymes to obtain PROG1 + An-1 Double gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-PolyA-Nos; the base sequence of the Actin-tRNAF1 is shown in SEQ ID NO. 15; the base sequence of the PROG1-tRNAR1 is shown in SEQ ID NO. 16; the base sequence of the PROG1-gRNAF1 is shown in SEQ ID NO. 17; the base sequence of the An1-tRNAR1 is shown in SEQ ID NO. 18; the base sequence of the An1-gRNAF1 is shown in SEQ ID NO. 19; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO. 20; the base sequence of the ActinF1 is shown in SEQ ID NO. 21; and the base sequence of the PolyA-R1 is shown in SEQ ID NO. 22;

[0012] (3) tRNA-sgRNA3 was obtained by overlapping PCR using primer pairs DEP1-gRNAF1, D2-tRNAR1 and D2-gRNAF1, PolyA-tRNA-R1 and DEP1-gRNAF2, PolyA-R1. DEP1 )-tRNA-sgRNA4( D2)-tRNA fragment; At the same time, using restriction endonucleases Pme I pair of vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-PolyA-Nos is digested and the digestion products are recovered; finally, the recovered products are connected using homologous recombinase to obtain PROG1 + An-1 + DEP1 + D2 Four-gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-PolyA-Nos; the base sequence of the DEP1-gRNAF1 is shown in SEQ ID NO. 23; the base sequence of the D2-tRNAR1 is shown in SEQ ID NO. 24; the base sequence of the D2-gRNAF1 is shown in SEQ ID NO. 25; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO. 26; the base sequence of the DEP1-gRNAF2 is shown in SEQ ID NO. 27; the base sequence of the PolyA-R1 is shown in SEQ ID NO. 28;

[0013] (4) tRNA-sgRNA5 was obtained by overlapping PCR using primer pairs GW2-gRNAF1, Bh4-tRNAR1 and Bh4-gRNAF1, PolyA-tRNA-R1 and GW2-gRNAF2, PolyA-R1. GW2 )-tRNA-sgRNA6( Bh4 )-tRNA fragment. At the same time, restriction endonucleases are used Pme I pair of vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-PolyA-Nos is digested and the digestion product is recovered. Finally, the above-mentioned recovered products are connected using homologous recombinase to obtain PROG1 + An-1 + DEP1 + D2 + GW2 + Bh4 Six-gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-sgRNA5( GW2 )-tRNA-sgRNA6( Bh4 )-tRNA-PolyA-Nos; the base sequence of the GW2-gRNAF1 is shown as SEQ ID NO.29; the base sequence of the Bh4-tRNAR1 is shown as SEQ ID NO.30; the base sequence of the Bh4-gRNAF1 is shown as SEQ ID NO.31; the base sequence of the PolyA-tRNA-R1 is shown as SEQ ID NO.32; the base sequence of the GW2-gRNAF2 is shown as SEQ ID NO.33; the base sequence of the PolyA-R1 is shown as SEQ ID NO.34.

[0014] The present invention also provides a method for genetic transformation and multi-gene editing of diploid wild rice, that is, introducing a gene editing transformation vector into cells of a diploid wild rice plant, and then screening to obtain heritable, transgenic, and non-transgenic and stably edited isolated offspring plants.

[0015] Furthermore, the method of genetic transformation and multi-gene editing comprises the following steps:

[0016] (1) Genetic transformation of diploid wild rice recipient plants to obtain regenerated plants;

[0017] (2) Detect whether the gene editing of the regenerated plants is successful;

[0018] (3) After the seeds of the successfully edited regenerated plants germinate, they are screened to obtain stably inherited, homozygous transgenic offspring plants, as well as non-transgenic, stably inherited, homozygous edited offspring plants.

[0019] Furthermore, the step (1) includes:

[0020] (1) Selecting a transformation receptor material, wherein the transformation receptor material is a diploid wild rice Oryza sativa O. Rufipogon and / or Nivara wild rice O.Nivara ;

[0021] (2) Induction and subculture of wild rice callus;

[0022] (3) Agrobacterium-mediated genetic transformation;

[0023] (4) Regeneration seedling culture;

[0024] (5) After the regenerated plants are acclimated and transplanted, the edited regenerated plants are screened and obtained.

[0025] In a specific embodiment, the above-mentioned wild rice callus induction and subculture scheme, Agrobacterium-mediated genetic transformation system, and medium optimization process for regenerated seedling culture are as follows:

[0026] 1) Induction and subculture of wild rice callus

[0027] First, the promoting effects of three different reagents, sterile water, 3% H2O2, and 0.1% w / v gibberellin solution, on wild rice seed germination were compared; secondly, the 2,4-D concentration suitable for callus induction of common wild rice and Nivara wild rice was explored, and three groups of 2,4-D concentration gradients were set up: 3.5 mg / L, 5 mg / L and 8 mg / L, and the induction effect of wild rice callus under different 2,4-D concentrations was tested; in addition, the technicians of the present invention also tested and compared the effects of two different carbon sources, sucrose and maltose, on the induction rate of rice callus.

[0028] The final wild rice callus induction and subculture scheme was as follows: after surface disinfection, the wild rice seeds were soaked in sterile water for 12 h, planted on an induction medium containing 3.5 mg / L 2,4-D and sucrose as a carbon source, and cultured in the dark at 30 °C for 30 days.

[0029] 2) Agrobacterium-mediated genetic transformation

[0030] Acetosyringone (AS) is a phenolic compound that can induce the activation and efficient expression of genes in the Vir region of Ti or Ri plasmid DNA within Agrobacterium. Different AS concentrations and exposure times directly affect the induction effect, and thus the infection efficiency of Agrobacterium. Based on these considerations, the present inventors tested and compared the effects of different AS concentrations (20 µM, 50 µM, 100 µM, 200 µM), infection times (10 min, 20 min, 30 min), and penetrants (0.1% Pluronic F68 and 0% Pluronic F68) on the infection efficiency of Agrobacterium.

[0031] The final Agrobacterium-mediated genetic transformation system was as follows: no penetrant Pluronic F68 was added, the AS concentration was 20 μM, and the infection time was 10 min.

[0032] 3) Regeneration seedling cultivation

[0033] In order to establish an efficient diploid wild rice regeneration system, the present inventors tested and compared the regeneration effects of four regeneration culture media. The components of the four regeneration culture media are shown in Table 4.

[0034] The final culture medium composition of the regenerated seedlings was as follows: 100 mL of N6 element, 10 mL of subculture B stock solution, 10 mL of iron salt, 10 mL of vitamins, 10 g of sucrose, 20 g of maltose, 0.2 mg of naphthaleneacetic acid, 2 mg of kinetin, 2 mg of 6-benzyladenine, and 0.2 mg of auxin per liter.

[0035] The components of the successive generation B stock solution include: 10 g / L manganese sulfate tetrahydrate, 2 g / L zinc sulfate heptahydrate, 3 g / L boric acid, 0.75 g / L potassium iodide, 0.25 g / L sodium molybdate dihydrate, 0.025 g / L cobalt chloride hexahydrate, and 0.025 g / L copper sulfate pentahydrate.

[0036] In addition, the present invention also designed gene editing vector detection specific primer pairs Cas9-6915F, Cas9-7518R and hptF, hptR and Actin-ce, and PROG-R to amplify and identify transgenic expression cassettes such as Cas9, hptⅡ, and sgRNA, respectively. The results showed that through genetic segregation of offspring, a new diploid wild rice germplasm with no transgenes and multiple gene editing was successfully obtained. The base sequence of the Cas9-6915F is shown in SEQ ID NO. 35; the base sequence of the Cas9-7518R is shown in SEQ ID NO. 36; the base sequence of the hptF is shown in SEQ ID NO. 37; the base sequence of the hptR is shown in SEQ ID NO. 38; the base sequence of the Actin-ce is shown in SEQ ID NO. 39; and the base sequence of the PROG-R is shown in SEQ ID NO. 40.

[0037] Beneficial effects of the present invention:

[0038] The present invention establishes a wild rice genetic transformation platform by optimizing the induction culture medium, Agrobacterium infection and regeneration culture medium conditions; on this basis, the key genes for domestication, such as the zinc finger transcription factor encoding gene that controls the transformation of wild rice plant type from creeping growth to upright growth, are used to transform wild rice into wild rice. PROG1 , genes that determine tiller angle size D2 , transcription factor encoding genes that control awn length and yield in wild rice An-1 , key pleiotropic genes controlling rice yield DEP1, genes encoding grain width GW2 , genes controlling the color of wild rice husks Bh4 For the target gene, a CRISPR / Cas9-mediated diploid wild rice multi-gene editing system was constructed, gene-edited stable plants were obtained through genetic transformation, and a genome editing technology system for annual and perennial diploid wild rice was established.

[0039] This study, using CRISPR / Cas9 multi-gene editing technology to generate multiple gene knockout mutants, represents the first implementation of de novo domestication and improvement of diploid wild rice using multi-gene editing. This significantly promotes the breeding and utilization of its superior genetic resources, enriches the diversity of cultivated rice germplasm resources, and overcomes the difficulties of genetic transformation and multi-gene editing in diploid wild rice. This approach is not only of great significance for functional research on superior genes in diploid wild rice but also has significant application value in rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Picture 1 This is a comparison of seed morphology among Zhonghua 11, Nipponbare, and wild rice from different origins.

[0041] Picture 2 This is the map of the pDR5-RUBY-Nos overexpression vector.

[0042] Picture 3 Figure 2. Wild rice seeds and callus induction status: a. Comparison of seed appearance morphology of three common wild rice materials Y409, Y665 and Y702 and three Nivara wild rice materials Y706, Y710 and Y711 with ZH11; b. Callus induction growth of three perennial common wild rice materials Y409, Y665 and Y702 and three Nivara wild rice materials Y706, Y710 and Y711.

[0043] Picture 4 Figures 2 and 3 show the growth of ZH11, Y702, and Y706 calli on the third-round screening medium after different treatments. Figures 2 and 3 show the growth of ZH11, Y702, and Y706 calli on the third-round screening medium after different treatments. A, B, and C represent the treatments with 0.1% penetrant Pluronic F68, an AS concentration of 20 µM, and infection times of 10, 20, and 30 minutes, respectively. D, E, and F represent the treatments without Pluronic F68, an AS concentration of 20 µM, and infection times of 10, 20, and 30 minutes, respectively.

[0044] Picture 5Comparison of the transformation efficiency of receptor materials such as ZH11, Y702, and Y706 under 24 groups of Agrobacterium infection experimental conditions: a, comparison of the transformation efficiency of ZH11 under 24 different groups of Agrobacterium infection experimental conditions; b, comparison of the transformation efficiency of common wild rice Y702 under 24 different groups of Agrobacterium infection experimental conditions; c, comparison of the transformation efficiency of Nivara wild rice Y706 under 24 different groups of Agrobacterium infection experimental conditions.

[0045] Picture 6 Regeneration of ZH11 and diploid wild rice callus: Growth status of callus tissues such as ZH11, Y409, Y665, Y702, Y706, Y710, and Y711 after culturing on regeneration medium 1, 2, 3, and 4 for 2 weeks.

[0046] Picture 7 Schematic diagram of editing vector structure: PROG1 + D2 + DEP1 + An-1 + GW2 + Bh4 Schematic diagram of the six-gene editing vector structure.

[0047] Picture 8 The grain phenotypes of the T1 generation edited strain 848-1-138 of common wild rice Y702: a, comparison of grain width between the harvested grains of the T1 generation strain 848-1-138 of common wild rice Y702 with the GW2 and Bh4 double genes edited and the wild-type Y702; b, comparison of grain length between the harvested grains of the T1 generation strain 848-1-138 of common wild rice Y702 with the GW2 and Bh4 double genes edited and the wild-type Y702; c, comparison of awn length between the harvested grains of the T1 generation strain 848-1-138 of common wild rice Y702 with the wild-type Y702.

[0048] Picture 9 The grain phenotype of the T1 edited line 563-2-59 of Nivara wild rice Y711: a. GW2 、 Bh4, An-1, DEP1, D2 Comparison of grain width between the harvested grains of the five-gene edited T1 generation line 563-2-59 and the wild type Y711; b, Nivala wild rice Y711 GW2 、 Bh4, An-1, DEP1, D2 Comparison of grain length between the five-gene edited T1 generation line 563-2-59 and the wild type Y711; c, grain width statistics of the edited lines of Nivara wild rice Y711; d, grain length of the edited lines of Nivara wild rice Y711 GW2 、 Bh4, An-1, DEP1, D2Comparison of awn length between the harvested grains of the five-gene edited T1 generation line 563-2-59 and the wild type Y711; e, Nivara wild rice Y711 GW2 、 Bh4, An-1, DEP1, D2 Comparison of the appearance morphology of the harvested grains of the five-gene edited T0 generation line 563-2 and the wild type Y711; f, awn length statistical data of the edited line Y711 of Nivara wild rice. DETAILED DESCRIPTION

[0049] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0050] Experimental materials: The diploid wild rice receptor materials used in the following examples mainly include common wild rice O. Rufipogon and Nivara wild rice O.Nivara ,in O. Rufipogon Eight materials were tested, namely Y409, Y416, Y665, Y675, Y698, Y702, Y704 and Y713 ( Picture 1 ), O.Nivara Seven materials were tested, namely Y645, Y676, Y683, Y706, Y707, Y710 and Y711 ( Picture 1 All wild rice materials used were provided by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.

[0051] Key Reagents and Instruments: Restriction enzymes, plant genomic DNA extraction kits, mini- and maxi-plasmid kits, high-fidelity PCR amplification enzymes, homologous recombination enzymes, and competent E. coli cells used in the following examples were purchased from reagent companies. Plant tissue culture reagents were purchased from Beijing Cimejie Technology Co., Ltd. and Beijing Coolbo Technology Co., Ltd. Amplification primers and first-generation sequencing were performed at Beijing Qingke Biotechnology Co., Ltd. (Hainan Branch), Guangzhou Sangon Biotechnology Co., Ltd., Beijing BGI, Beijing Tianyi Huiyuan Biotechnology Co., Ltd., Sanya BGI First-Generation Sequencing Platform, and Nanshan Biotechnology Co., Ltd.

[0052] pH meter, pipette, clean bench, glass bead sterilizer, high temperature and high pressure steam sterilizer, PCR instrument, constant temperature water bath, gel imaging system.

[0053] Example 1: Establishment and optimization of diploid wild rice genetic transformation system

[0054] The basic process of the diploid wild rice genetic transformation system includes: callus induction and subculture, Agrobacterium activation and infection, co-cultivation, resistant callus screening, callus regeneration into seedlings, seedling rooting, and finally hardening and transplanting.

[0055] To facilitate the screening of subsequent genetic transformation conditions, this study used the phenomenon that RUBY gene expression can make positive callus tissue appear red, and selected the pDR5-RUBY-Nos overexpression vector for the evaluation of transformation efficiency at the callus stage ( Picture 2 The Agrobacterium tumefaciens strain used was EHA105.

[0056] (1) Induction and subculture of wild rice callus

[0057] First, this study tested and compared the effects of three different reagents on promoting wild rice seed germination. All experimental conditions remained unchanged, except for the reagents and treatment time. After dehulling, the wild rice seeds were placed in 50 mL centrifuge tubes. The seeds were surface-disinfected with 75% ethanol for 5-10 minutes. The seeds were then transferred to a clean bench and disinfected with 20% sodium hypochlorite solution for 20-30 minutes. The seeds were then rinsed 5-6 times with sterile water. The seeds were then soaked in sterile water, 3% H₂O₂, and 0.1% gibberellin solution for 12 or 24 hours, followed by 5-6 rinses with sterile water. Finally, the seeds were inoculated into NB induction medium, with three plates per plate containing 12 seeds per plate. The tubes were labeled and incubated in the dark at 30°C for 30 days. The results of wild rice callus induction were then counted and observed. Secondly, to explore the optimal 2,4-D concentration for callus induction in common wild rice and Nivara wild rice, this study tested three 2,4-D concentration gradients: 3.5 mg / L, 5 mg / L, and 8 mg / L. The wild rice callus induction effects of these concentrations were evaluated. All other experimental conditions remained unchanged. Surface-sterilized rice seeds were inoculated in an induction medium containing varying concentrations of 2,4-D in a clean bench. Three plates of 12 seeds were plated per plate for each group. The plates were labeled and incubated in the dark at 30°C for 30 days. The callus induction effects of wild rice were then counted and observed. Furthermore, this study compared the effects of two different carbon sources, sucrose and maltose, on the callus induction rate of rice. Again, maintaining all other experimental conditions, surface-sterilized rice seeds were inoculated in an induction medium containing either sucrose or maltose in a clean bench. Three plates of 12 seeds were plated per plate for each group. Mark the cells, culture them in the dark at 30°C for 30 days, and then count and observe the callus induction effect of wild rice.

[0058] The induced callus tissue was transferred to the subculture medium NB1 and cultured in the dark at 30°C for 2-3 weeks. The light yellow and dense embryonic callus tissue was selected for subsequent genetic transformation experiments.

[0059] Table 1 Wild rice callus induction scheme

[0060]

[0061] Experimental results:

[0062] Treatments with different seed germination agents had significant effects on callus induction in mature wild rice seeds (e.g. Picture 3 As shown in the figure, the callus induction effect of mature wild rice seeds after soaking in sterile water for 2 or 12 hours was significantly better than that after soaking in 3% hydrogen peroxide or 0.1% gibberellin for 12 or 24 hours. In terms of wild rice seed germination and callus texture, the germination rate of mature wild rice seeds was higher after soaking in sterile water for 12 hours, resulting in more callus tissue and a more compact and firm texture ( Picture 3 ). At the same time, increasing the 2,4-D concentration did not promote the induction of callus tissue in mature wild rice seeds. Different types of carbon sources, such as sucrose or maltose, had no significant difference in the induction of callus tissue in mature wild rice seeds. The optimal callus induction scheme was finally determined: after surface disinfection, the wild rice seeds were soaked in sterile water for 12 hours, and the seeds were planted on an induction medium containing 3.5 mg / L 2,4-D and sucrose as carbon sources, and cultured in the dark at 30°C for 30 days. At the same time, if Picture 3 As shown, three perennial common wild rice accessions, Y409, Y665, and Y702, and three Nivara wild rice accessions, Y706, Y710, and Y7113, showed high callus induction rates. Among the perennial common wild rice, Y665 had the highest induction rate, reaching 65.8%. Among the annual Nivara wild rice, Y706 had the highest induction rate, reaching 52.3%.

[0063] (2) Agrobacterium-mediated genetic transformation

[0064] This study optimized the Agrobacterium-mediated genetic transformation system for diploid wild rice by considering the addition of penetrants, optimizing AS concentration, and Agrobacterium infection time. The effects of different AS concentrations (20 µM, 50 µM, 100 µM, and 200 µM), infection times (10 min, 20 min, and 30 min), and penetrants (0.1% Pluronic F68 and 0% Pluronic F68) on Agrobacterium infection efficiency were tested and compared (Table 2). A total of 24 treatments were set up, as shown in Table 3.

[0065] Table 2 Agrobacterium infection scheme for diploid wild rice

[0066]

[0067] Table 3 24 experimental treatments of diploid wild rice infected with Agrobacterium

[0068]

[0069] Specifically, in this study, Agrobacterium EHA105 carrying the pDR5-RUBY-Nos overexpression plasmid was streaked on a plate for 2-3 days, and then a single colony was picked and placed in an appropriate amount of YEB liquid medium. The culture was shaken overnight at 28°C and 220 rpm until the logarithmic phase (OD 600 = 0.6-1.0), aspirate an appropriate amount of bacterial solution and transfer it to 20-30 mL fresh YEB liquid medium, and culture under the same conditions until OD 600 =0.3-0.5. The activated Agrobacterium was then centrifuged at 4500 rpm for 10 minutes. The supernatant was discarded and the culture suspension was resuspended in an equal volume of AAM medium containing an appropriate concentration of AS. The collected callus tissue was immersed in the AAM-AS culture suspension for an appropriate time (shaking may be performed several times during this time to increase the dissolved oxygen content in the solution). The culture suspension was discarded and then aspirated dry with a pipette. The tissue pieces were then placed on a plate covered with four layers of filter paper and air-dried for approximately 30 minutes. Finally, the tissue pieces were transferred to NB2C co-culture medium covered with a layer of filter paper (to block nutrients) and incubated in the dark at 28°C for 2-3 days. Next, transfer the callus pieces to NBS1 selection medium containing 50 mg / L hygromycin and 400 mg / L timentin and culture them in the dark for 2-3 weeks. Then, transfer them to NBS2 selection medium containing 50 mg / L hygromycin and 300 mg / L timentin and culture them in the dark for another 2-3 weeks. Depending on the growth of resistant calli, a third round of screening may be performed to obtain bright red positive calli. The frequency of red calli appearance was counted to calculate the Agrobacterium transformation efficiency at the resistant callus stage.

[0070] The calculation formula is as follows: Transformation efficiency at the resistant callus stage = number of red calli / total number of infected calli or total number of gene gun bombarded calli * 100%

[0071] Experimental results: After RUBY overexpression, the resistant callus showed red color, indicating the transformation effect of different treatments, such as Picture 4 By counting the frequency of red callus appearance, the transformation efficiency of each treatment was calculated. The results are shown in Picture 5 shown.

[0072] As shown in the figure, the transformation efficiencies of ZH11, perennial common wild rice Y702, and Nivara wild rice Y706 under the same infection conditions varied. ZH11, perennial common wild rice Y702, and Nivara wild rice Y706 all achieved higher transformation efficiencies when the penetrant Pluronic F68 was not added. At an AS concentration of 20 µM, all three wild rice receptors achieved good transformation efficiencies. When the penetrant Pluronic F68 was not added, the AS concentration was 20 µM, and the infection time was 10 minutes, the transformation efficiency of ZH11 reached a maximum of 81.5%, that of Nivara wild rice Y706 reached a maximum of 74.1%, and that of common wild rice Y702 reached 69.7%. When the penetrant Pluronic F68 was not added, the AS concentration was 20 µM, and the infection time was extended to 30 minutes, the transformation efficiency of common wild rice Y702 reached a maximum of 77.8%.

[0073] (3) Optimization of regeneration system

[0074] To establish an efficient diploid wild rice regeneration system, this study tested and compared the regeneration effects of four regeneration media. The compositions of the four regeneration media are shown in Table 4. Specifically, embryogenic calli obtained by subculture were inoculated onto regeneration media and cultured for two weeks at 30°C, 60% humidity, 3000 lx light intensity, and 16 h light / 8 h dark. The culture media were then transferred to fresh regeneration media and cultured under the same conditions for another two weeks. The regeneration effect of the calli was then observed.

[0075] Table 4 Main components of four regeneration culture media

[0076]

[0077] The specific composition of regeneration medium 1 is as follows: NB minimal medium (4.1 g) + sucrose (15 g) + maltose (15 g) + NAA (0.5 mg) + KT (0.5 mg) + 6-BA (2 mg) per liter. The specific composition of regeneration medium 2 is as follows: MS minimal medium (4.33 g) + MS organic medium (10 mL) + maltose (30 g) + NAA (0.2 mg) + KT (4 mg) + 6-BA (3 mg) per liter. The specific composition of regeneration medium 3 is as follows: MS minimal medium (4.33 g) + MS organic medium (10 mL) + maltose (30 g) + NAA (0.1 mg) + ZT (2 mg) per liter. The specific components of regeneration medium 4 are as follows: N6 macroelements (100 mL) + succession B stock solution (10 mL) + iron salt (10 mL) + vitamins (10 mL) + sucrose (10 g) + maltose (20 g) + NAA (0.2 mg) + KT (2 mg) + 6-BA (2 mg) + IAA (0.2 mg) per liter; the components of the succession B stock solution include: manganese sulfate tetrahydrate 10 g / L, zinc sulfate heptahydrate 2 g / L, boric acid 3 g / L, potassium iodide 0.75 g / L, sodium molybdate dihydrate 0.25 g / L, cobalt chloride hexahydrate 0.025 g / L, and copper sulfate pentahydrate 0.025 g / L.

[0078] Experimental results: Picture 6 As shown, calli of ZH11, common wild rice Y409, Y665, and Y702, and Nivara wild rice Y706, Y710, and Y711 all showed green growth points after two weeks of culture on the four regeneration media. Among them, cultivated rice ZH11 grew fastest on regeneration medium 4. Common wild rice Y409, Y665, and Y702 calli showed varying growth patterns on the four regeneration media. Overall, Y702 calli showed the best regeneration pattern on regeneration medium 4, while Y665 calli also showed good regeneration patterns. Y409 calli showed the worst regeneration pattern. Nivara wild rice Y706, Y710, and Y711 calli showed similar growth patterns on the four regeneration media. In general, calli such as ZH11, Y409, Y665, Y702, Y706, Y710, and Y711 showed the greatest callus proliferation, the most green growth points, and the fastest regenerated plant growth on regeneration medium 4. Therefore, regeneration medium 4 was used in all subsequent experiments of this study to cultivate regenerated plants.

[0079] Example 2: Construction of editing vector

[0080] (1) Target selection of target genes

[0081] The nucleotide sequences of wild rice homologous genes PROG1, D2, DEP1, An-1, GW2, and Bh4 were retrieved from the EnsemblPlants database (http: / / plants.ensembl.org / Triticum_aestivum / Info / Index). Target gene sequences were determined in the recipient common wild rice materials Y409, Y665, and Y702, and the Nivara wild rice materials Y706, Y710, and Y711. Target sites were then designed using CRISPR-GE (http: / / skl.scau.edu.cn / targetdesign / ). The specific gene structures and target sequences are shown in Table 5.

[0082] Table 5 Target gene name, chromosome location and target sequence information

[0083]

[0084] Note: PAM sites are in italics.

[0085] (2) Construction of editing vector

[0086] S1. Obtaining the gRNA-tRNA basic fragment using the overlapping PCR method: In the first round of PCR, the gRNA basic fragment is obtained using primers gRNAF1, gRNAR1, and gRNAR2, and the tRNA basic fragment is obtained using primers tRNAF1, tRNAR1, and tRNAR2; in the second round of PCR, the gRNA fragment and tRNA fragment of the first round of PCR products are mixed at a molar ratio of 1:1 as a template, and the primer pair gRNAF2 / tRNAR3 is used to amplify, and finally the gRNA-tRNA basic fragment is obtained; the base sequences of the primers gRNAF1, gRNAR1, and gRNAR2 are shown in SEQ ID NOs. 9-11; the base sequences of the primers tRNAF1, tRNAR1, and tRNAR2 are shown in SEQ ID NOs. 12-14;

[0087] S2, using overlapping PCR method to obtain tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragment: The first round of PCR uses the gRNA-tRNA basic fragment as a template and uses the primer pair Actin-tRNAF1 and PROG1-tRNAR1 to obtain PROG1The tRNA fragment of the gene target sequence was then amplified using the primers PROG1-gRNAF1, An1-tRNAR1 and An1-gRNAF1, PolyA-tRNA-R1 using the gRNA-tRNA basic fragment as a template to obtain sgRNA1 with a linker ( PROG1 )-tRNA and sgRNA2( An-1 )-tRNA fragment; the second round of PCR was performed using the three PCR products obtained in the first round mixed at a molar ratio of 1:1:1 as a template, and primers ActinF1 and PolyA-R1 were used to amplify, and finally tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragments and purify and recover them; at the same time, restriction endonucleases were used Pme I. Enzyme digestion of the vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-PolyA-Nos was performed, and the digestion products were recovered; finally, the recovered products were connected using homologous recombination enzymes to obtain PROG1 + An-1 Double gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-PolyA-Nos; the base sequence of the Actin-tRNAF1 is shown in SEQ ID NO. 15; the base sequence of the PROG1-tRNAR1 is shown in SEQ ID NO. 16; the base sequence of the PROG1-gRNAF1 is shown in SEQ ID NO. 17; the base sequence of the An1-tRNAR1 is shown in SEQ ID NO. 18; the base sequence of the An1-gRNAF1 is shown in SEQ ID NO. 19; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO. 20; the base sequence of the ActinF1 is shown in SEQ ID NO. 21; and the base sequence of the PolyA-R1 is shown in SEQ ID NO. 22;

[0088] S3, tRNA-sgRNA3 was obtained by overlapping PCR using primer pairs DEP1-gRNAF1, D2-tRNAR1 and D2-gRNAF1, PolyA-tRNA-R1 and DEP1-gRNAF2, PolyA-R1 ( DEP1 )-tRNA-sgRNA4( D2)-tRNA fragment; At the same time, using restriction endonucleases Pme I pair of vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-PolyA-Nos is digested and the digestion products are recovered; finally, the recovered products are connected using homologous recombinase to obtain PROG1 + An-1 + DEP1 + D2 Four-gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-PolyA-Nos; the base sequence of the DEP1-gRNAF1 is shown in SEQ ID NO. 23; the base sequence of the D2-tRNAR1 is shown in SEQ ID NO. 24; the base sequence of the D2-gRNAF1 is shown in SEQ ID NO. 25; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO. 26; the base sequence of the DEP1-gRNAF2 is shown in SEQ ID NO. 27; the base sequence of the PolyA-R1 is shown in SEQ ID NO. 28;

[0089] S4, tRNA-sgRNA5 was obtained by overlapping PCR using primer pairs GW2-gRNAF1, Bh4-tRNAR1 and Bh4-gRNAF1, PolyA-tRNA-R1 and GW2-gRNAF2, PolyA-R1. GW2 )-tRNA-sgRNA6( Bh4 )-tRNA fragment. At the same time, restriction endonucleases are used Pme I pair of vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-PolyA-Nos is digested and the digestion product is recovered. Finally, the above-mentioned recovered products are connected using homologous recombinase to obtain PROG1 + An-1 + DEP1 + D2 + GW2 + Bh4 Six-gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-sgRNA5( GW2 )-tRNA-sgRNA6( Bh4 )-tRNA-PolyA-Nos, the nucleotide sequence is composed of SEQ ID NO.1 and SEQ ID NO.2 ( Picture 7 ); the base sequence of the GW2-gRNAF1 is shown as SEQ ID NO.29; the base sequence of the Bh4-tRNAR1 is shown as SEQ ID NO.30; the base sequence of the Bh4-gRNAF1 is shown as SEQ ID NO.31; the base sequence of the PolyA-tRNA-R1 is shown as SEQ ID NO.32; the base sequence of the GW2-gRNAF2 is shown as SEQ ID NO.33; the base sequence of the PolyA-R1 is shown as SEQ ID NO.34.

[0090] Table 6 List of primers used in this application

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] (3) Genotype identification of T0 generation regenerated plants

[0098] Using genomic DNA from T0 generation rice plants as templates, amplification was performed using primer pairs such as GW2-testF1 / GW2-testR1, DEP1-testF1 / DEP1-testR1, D2-testF1 / D2-testR1, An-testF1 / An-testR1, PROG-testF1 / PROG-testR1, and Bh4-testF1 / Bh4-testR1. The amplified PCR products were sequenced, and the sequencing results were entered into the website http: / / dsdecode.scgene.com / for analysis of the editing type. For samples with complex results, the PCR products were ligated to the cloning vector, and the editing type was finally determined by sequencing the single clones.

[0099] The genotype identification results of the T0 generation regenerated plants are as follows:

[0100] For the PROG1+An-1+DEP1+D2+GW2+Bh4 six-gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1(PROG1)-tRNA-sgRNA2(An-1)-tRNA-sgRNA3(DEP1)-tRNA-sgRNA4(D2)-tRNA-sgRNA5(GW2)-tR NA-sgRNA6(Bh4)-tRNA-PolyA-Nos-mediated multi-gene editing of wild rice. In this study, 90 wild rice calli were transformed, and 26 clusters of Y409 T0 regenerated plants, 37 clusters of Y665 T0 regenerated plants, 36 clusters of Y702 T0 regenerated plants, 34 clusters of Y706 T0 regenerated plants, 55 clusters of Y710 T0 regenerated plants and 27 clusters of Y711 T0 regenerated plants were obtained. First, the regenerated plants were clustered and sampled, and the target genes were preliminarily detected using Hi-tom high-throughput sequencing. The regenerated plants showing editing events were sampled individually, and then amplified separately. PROG1 、 D2 、 DEP1 、 An-1 、 GW2 、 Bh4 The target regions of the six target genes were analyzed by PCR products, and the sequencing results were analyzed using DSDcode online software. For samples with complex sequencing results, the PCR products were ligated to the cloning vector, and the editing type was finally determined by sequencing and analyzing the single clones.

[0101] Of the 26 T0 regenerated plants of Y409 obtained, 13 regenerated plants with editing events were identified, totaling 17 plants, of which only GW2 There are 2 strains that have been gene edited, only An-1There are 2 strains that have been gene edited, only D2 There are 4 strains that have been gene-edited. GW2 、 Bh4 There is one strain with double gene editing. GW2 、 D2 There are 7 strains with double gene editing. GW2 、 D2, DEP1 There was one plant with three genes edited, and the specific edited plant genotype is shown in Table 7.

[0102] Table 7 Genotype analysis of T0 edited plants of common wild rice Y409

[0103]

[0104] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0105] Among the 37 T0 regenerated plants of Y665 obtained, a total of 50 regenerated plants with editing events were identified, of which only GW2 There are 18 strains that have been gene-edited, only DEP1 There is only one gene-edited strain. D2 There is one gene-edited strain. GW2 、 Bh4 There are 23 strains with double gene editing. GW2 、 DEP1 There are 6 strains with double gene editing. GW2 、 D2, DEP1 There was one plant with three genes edited, and the specific edited plant genotype is shown in Table 8.

[0106] Table 8 Genotype analysis of T0 edited plants of common wild rice Y665

[0107]

[0108] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0109] Among the 36 T0 regenerated plants of Y702 obtained, a total of 14 regenerated plants with editing events were identified, of which only GW2 There are 2 strains that have been gene edited, only D2 There are 2 strains that have been gene-edited. GW2 、 DEP1 There are 3 strains with double gene editing. GW2 、 Bh4, An-1 There are 3 strains with triple gene editing. GW2 、 Bh4, DEP1, D2 There is one strain with four gene editing, GW2 、 Bh4, An-1, DEP1 There is one strain with four gene editing, GW2 、 Bh4, An-1, DEP1, D2There were two plants with five genes edited, and the specific edited plant genotypes are shown in Table 9.

[0110] Table 9 Genotype analysis of T0 edited plants of common wild rice Y702

[0111]

[0112] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0113] Among the 34 T0 regenerated plants of Y706 obtained, a total of 35 regenerated plants with editing events were identified, of which only GW2 There are 10 strains that have been gene-edited, only DEP1 There is one gene-edited strain. GW2 、 Bh4 There are 3 strains with double gene editing. GW2 、 DEP1 There are 2 strains with double gene editing. GW2 、 PROG1 There is one strain with double gene editing. GW2 、 An-1 There are 8 strains with double gene editing. GW2 、 D2 There are 3 strains with double gene editing. GW2 、 An-1, PROG1 There is one strain with three gene editing. Bh4 、 DEP1, D2 There is one strain with three gene editing. GW2 、 Bh4, PROG1, DEP1 There is one strain with four gene editing, GW2 、 Bh4, DEP1, D2 There is one strain with four gene editing, GW2 、 Bh4, An-1, PROG1, DEP1 There are 2 strains with five gene editing. GW2 、 Bh4, An-1, PROG1, DEP1, D2 There was one plant with six genes edited, and the specific edited plant genotypes are shown in Table 10.

[0114] Table 10 Genotype analysis of T0 edited plants of Nivara wild rice Y706

[0115]

[0116] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0117] Among the 55 T0 regenerated plants of Y710 obtained, a total of 49 regenerated plants with editing events were identified, of which only Bh4 There are 23 strains that have been gene-edited, only PROG1 There are 7 strains that have been gene-edited. GW2 、 Bh4There are 4 strains with double gene editing. An- 1 、 PROG1 There are 3 strains with double gene editing. GW2 、 DEP1, D2 There is one strain with three gene editing. GW2 、 An-1, PROG1 There is one strain with three gene editing. GW2 、 An-1, DEP1 There is one strain with three gene editing. GW2 、 An-1, D2 There is one strain with three gene editing. GW2 、 Bh4, An-1, PROG1 There is one strain with four gene editing, GW2 、 Bh4, An-1, D2 There are 2 strains with four gene editing. GW2 、 An-1, PROG1, D2 There is one strain with four gene editing, An-1, PROG1, DEP1, D2 3 strains with four gene editing, Bh4, An-1, PROG1, DEP1, D2 There was one plant with five genes edited, and the specific edited plant genotypes are shown in Table 11.

[0118] Table 11 Genotype analysis of T0 edited plants of Nivara wild rice Y710

[0119]

[0120] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0121] Among the 27 T0 regenerated plants of Y711 obtained, a total of 36 regenerated plants with editing events were identified, of which only Bh4 There are 9 strains that have been gene-edited, only PROG1 There are 8 strains that have been gene-edited, only DEP1 There is one gene-edited strain. PROG1, DEP1 There is one strain with double gene editing. GW2 、 Bh4, D2 There is one strain with three gene editing. GW2 、 Bh4, An-1, PROG1 There are 4 strains with four gene editing. GW2 、 Bh4, An-1, DEP1 There are 2 strains with four gene editing. GW2 、 Bh4, An-1, D2 There are 5 strains with four gene editing. GW2 、 Bh4, An-1, DEP1, D2 There are 2 strains with five gene editing. GW2 、 Bh4, An-1, PROG1, DEP1, D2 There were three plants with six genes edited, and the specific edited plant genotypes are shown in Table 12.

[0122] Table 12 Genotype analysis of T0 edited plants of Nivara wild rice Y711

[0123]

[0124] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0125] (4) Off-target analysis of T0 generation regenerated plants

[0126] Through the search on the CRISPR-GE (http: / / skl.scau.edu.cn / offtarget / ) website, PROG1 、 D2 、 DEP1 、 An-1 、 GW2 and Bh4 Two potential off-target sites were selected for each target for off-target detection. The PCR product sequencing results showed that most of the target targets designed in this experiment had no off-target situation at the predicted off-target sites, and only a few plants had An-1 The first off-target site predicted by the target site of the gene has off-target phenomenon, and the results are shown in Table 13. In view of this situation, when the agronomic traits of the edited plants are evaluated later, these plants with off-target phenomena should be eliminated to avoid the impact of off-target on the phenotype of the edited plants.

[0127] Table 13 Target and off-target analysis

[0128]

[0129] (5) Genetic analysis of T1 edited plants

[0130] The harvested T0 generation seeds were soaked and germinated before being sown in the soil. When the seedlings reached the 3-leaf stage, they were transplanted to the field for labeling and sampling. Genomic DNA from T1 generation offspring plants was extracted. Approximately 200 ng of genomic DNA was used as a template, and PCR amplification and sequencing were performed using the corresponding detection primers for each target gene. The segregation of editing events in T1 generation plants was analyzed based on the sequencing results. 2 The test method was used to test whether the segregation of the editing events conformed to Mendel's law of inheritance. The results are shown in Table 14. Statistical analysis showed that the genetic segregation ratios of the offspring of most edited lines conformed to Mendel's law of segregation.

[0131] The T0 generation strain 848-1 is a three-gene edited strain of common wild rice Y702. GW2 、 Bh4 and An-1When conducting genetic analysis on the T0 generation strain 848-1, a total of 33 T1 generation plants were tested, of which GW2 Three heterozygous, eight homozygous (i1-T or i1-G) and 22 wild-type strains were detected; Bh4 Three heterozygous, seven homozygous, and 23 wild-type strains were detected; An-1 The genetic analysis revealed 9 heterozygous, 15 homozygous, and 9 wild-type strains. Statistical data (Table 14) indicated that the genetic segregation ratio of the offspring of the T0 generation strain 848-1 did not conform to Mendel's law of segregation, indicating that the T0 generation strain 848-1 was a mosaic strain.

[0132] The T0 generation strain 825-1 is a two-gene edited strain of Nivara wild rice Y706. GW2 and Bh4 When conducting genetic analysis on the T0 generation strain 825-1, a total of 51 T1 generation plants were tested, of which GW2 All genes were homozygous mutations; Bh4 Twenty-five strains were found to have biallelic genotypes, 12 were homozygous for a 5-base deletion, and 13 were homozygous for a 6-base deletion. Statistical data (Table 14) showed that the genetic segregation ratio of the offspring of the T0 generation strain 825-1 conformed to Mendel's law of segregation.

[0133] Table 14 Genetic analysis and transgenic element segregation of T1 edited plants

[0134]

[0135]

[0136]

[0137]

[0138] Note: The χ2 value indicates whether the T1 generation edited lines tested conform to Mendel's law of genetic segregation. ** P >0.5, which is very consistent with 1:2:1. *0.1 < P < 0.5, which is consistent with a 1:2:1 ratio. For transgenic analysis, '+' indicates detection of Cas9 / hptII / sgRNA, while '–' indicates no detection of Cas9 / hptII / sgRNA. Ho: homozygous edited plant; He: heterozygous edited plant; WT: wild type; Bi: biallelic; Chi: chimeric.

[0139] The T0 generation strain 822-1 is a two-gene edited strain of Nivara wild rice Y710. An-1 andPROG1 When conducting genetic analysis on the T0 generation strain 822-1, a total of 23 T1 generation plants were tested, of which An-1 The gene detected 13 strains with biallelic genotypes, 4 homozygous strains with 1 base insertion, and 6 homozygous strains with 1 base deletion; PROG1 The gene was detected in 2 heterozygous, 4 homozygous and 17 wild-type strains. According to the statistical data (see Table 14), the T0 generation strain 822-1 An-1 The genetic segregation ratio of offspring conforms to Mendel's law. PROG1 The segregation ratio of genes does not conform to Mendel's law, which may be due to PROG1 The gene was in a mosaic state in the T0 generation strain 822-1, and editing occurred in a small number of cells.

[0140] The T0 generation strain 847-1 is also a second gene editing strain of Nivara wild rice Y710. GW2 and Bh4 When conducting genetic analysis on the T0 generation strain 847-1, a total of 49 T1 generation plants were tested, of which GW2 Twenty-five strains were detected with biallelic genotypes, 13 of which were homozygous for a single G insertion and 11 were homozygous for a single T insertion; Bh4 According to the statistical data (see Table 14), the T0 generation strain 847-1 GW2 and Bh4 The genetic segregation ratio of offspring conforms to Mendel's law of segregation.

[0141] The T0 generation line 563-2 is a five-gene edited line of Nivara wild rice Y711. GW2 、 Bh4, An-1 、 DEP1 and D2 When conducting genetic analysis on the T0 generation line 563-2, a total of 96 T1 generation plants were tested, of which GW2 The gene detected 51 strains with biallelic genotypes, 20 strains homozygous for 3 base deletions, and 25 strains homozygous for 1 base deletion; Bh4 The gene detected 48 strains with biallelic genotypes, 22 homozygous for 1 base insertion, and 26 homozygous for 1 base deletion; An-1 The gene detected 50 strains with biallelic genotypes, 22 strains homozygous for 1 base insertion, and 24 strains homozygous for 33 base insertions; DEP1 The gene detected 45 heterozygous strains, 24 homozygous strains with 1 base insertion, and 27 wild-type strains; D2The gene detected 49 strains with double alleles, 22 strains with 3 base deletion homozygotes and 25 strains with 1 base deletion homozygotes. According to the statistical data (see Table 14), the T0 generation strain 847-1 GW2 、 Bh4, An-1 、 DEP1 and D2 The genetic segregation ratios of the offspring of the genes all conform to Mendel's law.

[0142] The T0 generation line 564-1 is a six-gene edited line of Nivara wild rice Y711. GW2 、 Bh4, An-1 、PROG1、 DEP1 and D2 When conducting genetic analysis on the T0 generation line 564-1, a total of 109 T1 generation plants were tested, of which GW2 The gene detected 55 strains with biallelic genotypes, 26 strains with homozygous deletion of 3 bases, and 28 strains with homozygous deletion of 1 base; Bh4 The gene detected 56 strains with biallelic genotypes, 29 homozygous for 1 base insertion, and 24 homozygous for 1 base deletion; An-1 The gene detected 57 strains with biallelic genotypes, 27 strains homozygous for 1 base insertion, and 25 strains homozygous for 33 base insertions; PROG1 The gene detected 52 heterozygous strains, 25 homozygous strains with 1 base insertion, and 32 wild-type strains; DEP1 The gene detected 57 heterozygous strains, 26 homozygous strains with 1 base insertion, and 26 wild-type strains; D2 The gene detected 59 heterozygotes, 24 homozygotes with 3 base deletions and 25 homozygotes with 1 base deletion. According to the statistical data (see Table 14), the T0 generation strain 564-1 GW2 、 Bh4, An-1 、PROG1、 DEP1 and D2 The genetic segregation ratio of offspring conforms to Mendel's law of segregation.

[0143] (6) Editing plant traits for improvement

[0144] To determine whether gene editing technology can be applied to the de novo domestication of diploid wild rice and improve its agronomic traits, this study observed the phenotypes of harvested seeds from the two-gene-edited T1 generation line 848-1-138 of the common wild rice Y702 and the five-gene-edited line 563-2-59 of the Nivara wild rice Y711. Figure 8 and 9 shown.

[0145] According to reports, GW2The gene is one of the major genes controlling rice grain width, and loss of its function can significantly increase rice grain width. Bh4 The gene is one of the key genes controlling rice grain color. Loss of this gene function can cause the color of rice grains to change from black to yellow or white. An-1 The gene is one of the main genes controlling the awn length of rice grains. Loss of this gene function can significantly shorten the awn length of rice.

[0146] The T1 generation of common wild rice Y702 line 848-1-138 GW2 The gene has a homozygous insertion mutation of one base at the target site, which GW2 The gene has a frameshift mutation. GW2 Complete loss of gene function; Bh4 The gene has a site-directed mutation with a deletion of one base at the target site. Bh4 The allele produces a frameshift mutation, and the corresponding gene function is completely lost. Figure 8 As shown, compared with the wild type Y702, the seed width of T0 line 137 was significantly increased and the seed color also changed significantly.

[0147] The T1 generation of Nivara wild rice Y711 line 563-2-59 GW2 The gene has a homozygous gene mutation with a base deletion at the target site, which GW2 The gene has a frameshift mutation. GW2 loss of gene function; Bh4 The gene has a homozygous gene mutation with a base deletion at the target site, which Bh4 The allele produces a frameshift mutation, and the corresponding gene function is completely lost; An-1 The gene has a biallelic mutation with a deletion of 1 base or an insertion of 33 bases at the target site, which An-1 The allele produces a frameshift mutation, and the corresponding gene function is lost. Figure 9 As shown, compared with wild-type Y711, the seed width of the T1 generation line 563-2-59 was significantly increased, the seed color changed from dark yellow to light yellow, and the seed awn length was significantly shortened. In summary, gene editing technology can be applied to the de novo domestication of diploid wild rice and directly improve wild rice.

[0148] For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, can be equivalent parameters, concentration and conditions, implement the present invention in a wide range. Although the present invention provides special embodiments, it should be understood that the present invention can be further improved. In a word, by the principle of the present invention, the application is intended to include any variation, purposes or improvements to the present invention, including departing from the disclosed range in the application, and the changes performed with conventional techniques known in the art.

Claims

1. A method for genetic transformation and multi-gene editing of diploid wild rice, characterized in that: The following steps are involved: (1) Selecting a transformation receptor material, wherein the transformation receptor material is wild rice O.Nivara ; (2) Induction and subculture of wild rice callus; (3) Agrobacterium-mediated genetic transformation of gene editing transformation vectors; (4) Regeneration seedling culture; (5) After the regenerated plants are acclimated and transplanted, the edited regenerated plants are screened; The gene editing transformation vector is a gene PROG1 Gene, D2 Gene, DEP1 Gene, An-1 Gene, GW2 Gene, Bh4 The gene is the target gene, and the six-gene combination knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1(PROG1)-tRNA-sgRNA2(An-1)-tRNA-sgRNA3(DEP1)-tRNA-sgRNA4(D2)-tRNA-sgRNA5(GW2)-tRNA-sgRNA6(Bh4)-tRNA-PolyA-Nos is constructed; the nucleotide sequence of the six-gene combination knockout vector is composed of SEQ ID NO.1 and SEQ ID NO.2 spliced ​​together; the PROG1 Gene, D2 Gene, DEP1 Gene, An-1 Gene, GW2 Gene, Bh4 The nucleotide sequence of the gene is shown in SEQ ID NO. 3-8; The diploid wild rice plant is Oryza nivara O.Nivara ; The steps for constructing the gene editing transformation vector are as follows: S1 uses overlapping PCR to obtain the gRNA-tRNA basic fragment: in the first round of PCR, the gRNA basic fragment is obtained using primers gRNAF1, gRNAR1, and gRNAR2, and the tRNA basic fragment is obtained using primers tRNAF1, tRNAR1, and tRNAR2; in the second round of PCR, the gRNA fragment and tRNA fragment of the first round of PCR products are mixed at a molar ratio of 1:1 as a template, and the primer pair gRNAF2 / tRNAR3 is used to amplify, and finally the gRNA-tRNA basic fragment is obtained; the base sequences of the primers gRNAF1, gRNAR1, and gRNAR2 are shown in SEQ ID NOs. 9-11; the base sequences of the primers tRNAF1, tRNAR1, and tRNAR2 are shown in SEQ ID NOs. 12-14; S2 used overlapping PCR to obtain tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragment: The first round of PCR uses the gRNA-tRNA basic fragment as a template and uses the primer pair Actin-tRNAF1 and PROG1-tRNAR1 to obtain PROG1 The tRNA fragment of the gene target sequence was then amplified using the primers PROG1-gRNAF1, An1-tRNAR1 and An1-gRNAF1, PolyA-tRNA-R1 using the gRNA-tRNA basic fragment as a template to obtain sgRNA1 with a linker ( PROG1 )-tRNA and sgRNA2( An-1 )-tRNA fragment; in the second round of PCR, the three PCR products obtained in the first round were mixed at a molar ratio of 1:1:1 as templates, and primers ActinF1 and PolyA-R1 were used to amplify, and finally tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragments and purify and recover them; at the same time, restriction endonucleases were used Pme I. Enzyme digestion of the vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-PolyA-Nos was performed, and the digestion products were recovered; finally, the recovered products were connected using homologous recombination enzymes to obtain PROG1 + An-1 Double gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-PolyA-Nos; the base sequence of the Actin-tRNAF1 is shown in SEQ ID NO. 15; the base sequence of the PROG1-tRNAR1 is shown in SEQ ID NO. 16; the base sequence of the PROG1-gRNAF1 is shown in SEQ ID NO. 17; the base sequence of the An1-tRNAR1 is shown in SEQ ID NO. 18; the base sequence of the An1-gRNAF1 is shown in SEQ ID NO. 19; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO. 20; the base sequence of the ActinF1 is shown in SEQ ID NO. 21; and the base sequence of the PolyA-R1 is shown in SEQ ID NO. 22; S3 obtained tRNA-sgRNA3 by overlapping PCR using primer pairs DEP1-gRNAF1, D2-tRNAR1 and D2-gRNAF1, PolyA-tRNA-R1 and DEP1-gRNAF2, PolyA-R1 ( DEP1 )-tRNA-sgRNA4( D2 )-tRNA fragment; At the same time, using restriction endonucleases Pme I pair of vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-PolyA-Nos is digested and the digestion products are recovered; finally, the recovered products are connected using homologous recombinase to obtain PROG1 + An-1 + DEP1 + D2 Four-gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-PolyA-Nos; the base sequence of the DEP1-gRNAF1 is shown in SEQ ID NO.23; the base sequence of the D2-tRNAR1 is shown in SEQ ID NO.24; the base sequence of the D2-gRNAF1 is shown in SEQ ID NO.25; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO.26; The base sequence of the DEP1-gRNAF2 is shown in SEQ ID NO. 27; the base sequence of the PolyA-R1 is shown in SEQ ID NO. 28; S4 obtained tRNA-sgRNA5 by overlapping PCR using primer pairs GW2-gRNAF1, Bh4-tRNAR1 and Bh4-gRNAF1, PolyA-tRNA-R1 and GW2-gRNAF2, PolyA-R1 ( GW2 )-tRNA-sgRNA6( Bh4 )-tRNA fragment; At the same time, using restriction endonucleases Pme I pair of vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-PolyA-Nos is digested and the digestion products are recovered; finally, the recovered products are connected using homologous recombinase to obtain PROG1 + An-1 + DEP1 + D2 + GW2 + Bh4 Six-gene knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA-sgRNA3( DEP1 )-tRNA-sgRNA4( D2 )-tRNA-sgRNA5( GW2 )-tRNA-sgRNA6( Bh4 )-tRNA-PolyA-Nos; the base sequence of the GW2-gRNAF1 is shown as SEQ ID NO.29; the base sequence of the Bh4-tRNAR1 is shown as SEQ ID NO.30; the base sequence of the Bh4-gRNAF1 is shown as SEQ ID NO.31; the base sequence of the PolyA-tRNA-R1 is shown as SEQ ID NO.32; the base sequence of the GW2-gRNAF2 is shown as SEQ ID NO.33; the base sequence of the PolyA-R1 is shown as SEQ ID NO.

34.

2. The method according to claim 1, characterized in that The following steps are also included: (6) Detect whether the gene editing of the regenerated plants is successful; (7) After the seeds of the successfully edited regenerated plants germinate, they are screened to obtain stably inherited, homozygous transgenic offspring plants, as well as non-transgenic, stably inherited, homozygous edited offspring plants.

3. The method according to claim 1, characterized in that In step (2), the wild rice callus induction and subculture scheme is as follows: after surface disinfection, the wild rice seeds are soaked in sterile water for 12 h, the seeds are sown on an induction medium containing 3.5 mg / L 2,4-D and sucrose as a carbon source, and cultured in the dark at 30°C for 30 days.

4. The method according to claim 1, wherein In step (3), the genetic transformation system of the Agrobacterium-mediated gene editing transformation vector is as follows: no penetrant Pluronic F68 is added, the concentration of the acetosyringone solution is 20 μM, and the infection time is 10 min.

5. The method according to claim 1, wherein In step (4), the components of the culture medium for culturing the regenerated seedlings include: N6 element, subculture B stock solution, iron salt, vitamins, sucrose, maltose, naphthaleneacetic acid, kinetin, 6-benzyladenine, and auxin; the components of the subculture B stock solution include: 10 g / L manganese sulfate tetrahydrate, 2 g / L zinc sulfate heptahydrate, 3 g / L boric acid, 0.75 g / L potassium iodide, 0.25 g / L sodium molybdate dihydrate, 0.025 g / L cobalt chloride hexahydrate, and 0.025 g / L copper sulfate pentahydrate.

6. A method for obtaining new diploid wild rice germplasm with multiple gene editing, characterized in that: The progeny plant T1 obtained by the method according to any one of claims 1 to 5.

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