Method and system for genetic transformation and polygene editing of diploid wild rice

Abstract

The invention relates to the technical field of biology, and particularly provides a diploid wild rice genetic transformation and polygene editing method and system. According to the method, a wild rice genetic transformation platform is established by optimizing conditions of an induction culture medium, agrobacterium infection and a regeneration culture medium; on the basis, key genes are domesticated; for example, a zinc finger transcription factor coding gene PROG1 for controlling the plant type of wild rice to be converted from creeping growth to upright growth, a gene D2 for determining the size of a tillering angle, a transcription factor coding gene An-1 for controlling the awn length and yield of the wild rice, a key pleiotropic gene DEP1 for controlling the yield of the rice, a coding gene GW2 for controlling the grain width and a gene Bh4 for controlling the glume shell color of the wild rice are used as target genes; a CRISPR / Cas9 mediated diploid wild rice polygene editing system is constructed, a gene editing stable plant is obtained through genetic transformation, an annual and perennial diploid wild rice genome editing technical system is established, and then diploid wild rice stress-resistant novel gene resources and new germplasm with breeding utilization value are rapidly obtained.

Description

Technical Field

[0001] The 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 the main staple food crop in my country and even in the world. Although my country has made great achievements in rice breeding in the past few decades, the accelerated urbanization process has led to a reduction in arable land, frequent extreme weather and pests and diseases, and increased natural consumption, and rice production safety has been greatly threatened. How to ensure the efficient, green and sustainable development of rice production is a serious problem that needs to be solved urgently in my country and even in the world.

[0003] The narrow genetic diversity of existing rice varieties and the serious homogeneity of breeding materials are one of the main limiting factors for efficient, green and sustainable rice production. While the domestication and breeding process has improved the important agronomic traits of crops, it has also caused a large amount of genetic diversity such as stress resistance to be lost. Using gene editing for "de novo domestication" to directly edit the key agronomic trait genes of wild germplasm materials, while improving their important agronomic traits, retaining the stress resistance characteristics lost by existing varieties during the long-term domestication process, will hopefully breed better new crop varieties. At present, de novo domestication has been successfully applied in wild plants such as wild tomatoes and tetraploid wild rice.

[0004] Annual and perennial diploid wild rice has rich genetic resources for stress resistance. It has strong resistance to diseases, insects, salt and other stresses. It is an important germplasm resource for expanding the genetic diversity and germplasm innovation of existing rice varieties. 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 burden of unfavorable genes, which seriously hinders the mining and breeding of excellent genetic resources of diploid wild rice. Through gene editing, de novo domestication and improvement of diploid wild rice will greatly promote the breeding and utilization of its excellent genetic resources and enrich the diversity of germplasm resources of cultivated rice. However, diploid wild rice, especially perennial diploid wild rice, is difficult to genetically transform and gene edit. At present, there are no related reports at home and abroad on the de novo domestication and improvement of diploid wild rice through multi-gene editing.

[0005] Rice is an important food crop in my country. Cultivating high-yield and excellent new rice varieties is particularly important for my country's food production security and social stability. As a simple and efficient editor that can improve crop traits at multiple points, the CRISPR / Cas9 system has been widely used in the genetic improvement and genetic breeding of crops. De novo domestication of diploid wild rice with excellent stress resistance traits, that is, using gene editing technology to directly edit important domestication and improvement-related agronomic trait genes in wild rice, and through the aggregation of domesticated and improved wild rice germplasm with the excellent traits of modern cultivated rice, it is expected to quickly cultivate high-yield, stress-resistant, and green new rice varieties, 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 medium, Agrobacterium infection and regeneration medium; on this basis, to domesticate key genes, such as zinc finger transcription factor encoding genes that control the transformation of wild rice plant type from creeping growth to upright growth PROG1 , determines the tillering angle D2 Genes, 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 that control the color of wild rice husks Bh4 Using genes such as IL-67 as target genes, we constructed a CRISPR / Cas9-mediated diploid wild rice multi-gene editing system, obtained gene-edited stable plants through genetic transformation, established an annual and perennial diploid wild rice genome editing technology system, and thus quickly obtained new diploid wild rice stress-resistant gene resources and new germplasm with breeding value.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: 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 constructed 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; 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; In a specific embodiment, the above gene editing transformation vector is artificially synthesized by interactive tandem of tRNA multiple gRNA expression technology, and the specific steps are as follows: (1) Obtaining the gRNA-tRNA basic fragment by overlapping PCR method: in the first round of PCR, the gRNA basic fragment is obtained by using primers gRNAF1, gRNAR1, and gRNAR2, and the tRNA basic fragment is obtained by 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 for amplification to finally obtain the gRNA-tRNA basic fragment; 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; (2) Using overlapping PCR method to obtain tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragment: The first round of PCR used the gRNA-tRNA basic fragment as a template and used the primer pair Actin-tRNAF1 and PROG1-tRNAR1 to obtain PROG1The tRNA fragment of the gene target sequence was then amplified using the primer pairs PROG1-gRNAF1, An1-tRNAR1 and An1-gRNAF1, PolyA-tRNA-R1 with 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 tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragments and purify and recover them; at the same time, restriction endonucleases are used Pme I digested the vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-PolyA-Nos and recovered the digested product; finally, the recovered product was connected using homologous recombination enzyme 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; the base sequence of the PolyA-R1 is shown in SEQ ID NO.22; (3) Using primer pairs DEP1-gRNAF1, D2-tRNAR1 and D2-gRNAF1, PolyA-tRNA-R1 and DEP1-gRNAF2, PolyA-R1, tRNA-sgRNA3 was obtained by overlapping PCR method ( 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 product is recovered; finally, the recovered product is connected using a homologous recombination enzyme 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; (4) Using primer pairs GW2-gRNAF1, Bh4-tRNAR1 and Bh4-gRNAF1, PolyA-tRNA-R1 and GW2-gRNAF2, PolyA-R1, tRNA-sgRNA5( GW2 )-tRNA-sgRNA6( Bh4 )-tRNA fragment. At the same time, restriction endonucleases were 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-recovered product is connected using homologous recombination enzyme 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 in SEQ ID NO.29; the base sequence of the Bh4-tRNAR1 is shown in SEQ ID NO.30; the base sequence of the Bh4-gRNAF1 is shown in SEQ ID NO.31; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO.32; the base sequence of the GW2-gRNAF2 is shown in SEQ ID NO.33; the base sequence of the PolyA-R1 is shown in SEQ ID NO.34.

[0008] 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.

[0009] Furthermore, the method of genetic transformation and multi-gene editing comprises the following steps: (1) Genetic transformation of diploid wild rice recipient plants to obtain regenerated plants; (2) Detect whether the gene editing of the regenerated plants is successful; (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.

[0010] Furthermore, the step (1) comprises: (1) Selecting a transformation receptor material, wherein the transformation receptor material is a diploid wild rice O. Rufipogon and / or Nivara wild rice O. Nivara ; (2) Induction and subculture of wild rice callus; (3) Agrobacterium-mediated genetic transformation; (4) Regeneration seedling cultivation; (5) After the regenerated plants are acclimated and transplanted, the edited regenerated plants are screened and obtained.

[0011] 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: 1) Induction and subculture of wild rice callus Firstly, the promoting effects of three different reagents, sterile water, 3% H2O2, and 0.1% w / v gibberellin solution, on the germination of wild rice seeds 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.

[0012] 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.

[0013] 2) Agrobacterium-mediated genetic transformation Acetosyringone (AS) is a phenolic compound that can induce the activation and efficient expression of genes in the Vir region on the Ti plasmid or Ri plasmid DNA in Agrobacterium. Different concentrations of AS and the duration of action directly affect the induction effect of AS, and then affect the infection efficiency of Agrobacterium. Based on the above reasons, the technical personnel of the present invention tested and compared the effects of different concentrations of AS (20 µm, 50 µm, 100 µm, 200 µm), infection time (10 min, 20 min, 30 min) and penetrant (0.1% PluronicF68, 0% Pluronic F68) on the infection efficiency of Agrobacterium.

[0014] 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.

[0015] 3) Regeneration seedling cultivation In order to establish an efficient diploid wild rice regeneration system, the technicians of the present invention tested and compared the regeneration effects of four regeneration culture media. The components of the four regeneration culture media are shown in Table 4.

[0016] The composition of the culture medium for the regenerated seedling culture 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.

[0017] 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.

[0018] In addition, the present invention also designs gene editing vector detection specific primer pairs Cas9-6915F, Cas9-7518R and hptF, hptR and Actin-ce, PROG-R to amplify and identify transgenic expression boxes such as Cas9, hptⅡ and sgRNA, respectively. The results show that through genetic separation of offspring, a new diploid wild rice germplasm with no transgene and multiple gene editing is 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; the base sequence of the PROG-R is shown in SEQ ID NO. 40.

[0019] Beneficial effects of the present invention: The present invention establishes a wild rice genetic transformation platform by optimizing the induction medium, Agrobacterium infection and regeneration 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 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 that control 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.

[0020] The present invention obtains multiple gene knockout mutants through CRISPR / Cas9 multi-gene editing technology, and realizes the de novo domestication and improvement of diploid wild rice for the first time, which greatly promotes the breeding and utilization of its excellent gene resources, enriches the diversity of cultivated rice germplasm resources, and overcomes the difficulty of genetic transformation and multi-gene editing of diploid wild rice. It is not only of great significance for the study of the excellent gene function of diploid wild rice, but also has great application value in rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Picture 1 This is a comparison of seed morphology of Zhonghua 11, Nipponbare, and wild rice from different origins.

[0022] Picture 2 This is the map of the pDR5-RUBY-Nos overexpression vector.

[0023] Picture 3 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 induced growth of three perennial common wild rice materials Y409, Y665 and Y702 and three Nivara wild rice materials Y706, Y710 and Y711.

[0024] Picture 4 The growth status of ZH11, Y702, Y706 and other receptor materials on the third round of screening medium after different treatments: The growth status of ZH11 and common wild rice Y702, as well as Nivara wild rice Y706 callus on the third round of screening medium after different treatments. A, B, C, respectively represent the treatment conditions of adding 0.1% penetrant Pluronic F68, AS concentration of 20 µM, infection time of 10, 20 and 30 min, respectively; D, E, F, respectively represent the treatment conditions of not adding penetrant Pluronic F68, AS concentration of 20 µM, infection time of 10, 20 and 30 min, respectively.

[0025] Picture 5 Comparison of transformation efficiency of receptor materials such as ZH11, Y702, and Y706 under 24 groups of Agrobacterium infection experimental conditions: a, comparison of transformation efficiency of ZH11 under 24 different groups of Agrobacterium infection experimental conditions; b, comparison of transformation efficiency of common wild rice Y702 under 24 different groups of Agrobacterium infection experimental conditions; c, comparison of transformation efficiency of Nivara wild rice Y706 under 24 different Agrobacterium infection experimental conditions.

[0026] Picture 6Regeneration of ZH11 and diploid wild rice callus: Growth status of callus tissues such as ZH11, Y409, Y665, Y702, Y706, Y710, and Y711 after being cultured on regeneration medium 1, 2, 3, and 4 for 2 weeks.

[0027] Picture 7 Schematic diagram of editing vector structure: PROG1 + D2 + DEP1 + An-1 + GW2 + Bh4 Schematic diagram of the six-gene editing vector structure.

[0028] 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 the GW2, Bh4 double gene edited common wild rice Y702 and the wild-type Y702; b, comparison of grain length between the harvested grains of the T1 generation strain 848-1-138 of the GW2, Bh4 double gene edited common wild rice Y702 and the wild-type Y702; c, comparison of awn length between the harvested grains of the T1 generation strain 848-1-138 of the GW2, Bh4 double gene edited common wild rice Y702 and the wild-type Y702.

[0029] Picture 9 Grain phenotype of 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 edited line 563-2-59 and the wild type Y711; c, grain width statistics of the edited line of Nivara wild rice Y711; d, GW2 , Bh4, An-1, DEP1, D2 Comparison of awn length between the harvested grains of the five-gene edited T1 generation line 563-2-59 and the wild type Y711; e, the awn length of the 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, Statistical data of awn length of the edited line of Nivara wild rice Y711. DETAILED DESCRIPTION

[0030] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0031] Experimental materials: The diploid wild rice receptor materials used in the following examples mainly include common wild rice O. Rufipogon 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.

[0032] Main reagents and instruments: The restriction enzymes, plant genomic DNA extraction kits, small plasmid kits, large plasmid kits, PCR amplification high-fidelity enzymes, homologous recombination enzymes, and Escherichia coli competent cells used in the following examples were purchased from reagent companies. Reagents used for plant tissue culture were purchased from Beijing Ximeijie Technology Co., Ltd., Beijing Coolbo Technology Co., Ltd., etc. The amplification primers and first-generation sequencing used were completed at Beijing Qingke Biotechnology Co., Ltd. Hainan Branch, Guangzhou Shenggong Biotechnology Co., Ltd., Beijing BGI, Beijing Tianyi Huiyuan Biotechnology Co., Ltd., Sanya BGI First-Generation Sequencing Platform, and Nanshan Biotechnology Co., Ltd.

[0033] pH meter, pipette, clean bench, glass bead sterilizer, high temperature and high pressure steam sterilizer, PCR instrument, constant temperature water bath, gel imaging system.

[0034] Example 1: Establishment and optimization of diploid wild rice genetic transformation system 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 seedling hardening and transplanting.

[0035] In order 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.

[0036] (1) Induction and subculture of wild rice callus First, this study tested and compared the effects of three different reagents on the promotion of wild rice seed germination. During the experiment, except for the different reagents and treatment time, other experimental conditions remained unchanged. After the wild rice seeds were shelled, they were placed in 50 mL centrifuge tubes, and the surface of the seeds was disinfected with 75% ethanol for 5-10 min. Then, they were transferred to the clean bench and disinfected with 20% sodium hypochlorite solution for 20-30 min, and then rinsed with sterile water 5-6 times. Then, the seeds were placed in sterile water, 3% H2O2, and 0.1% gibberellin solution for 12h or 24h, and then rinsed with sterile water 5-6 times. Finally, they were inoculated into NB induction medium, and 3 dishes were planted in each group of seeds, with 12 seeds in each dish. Mark them well, and after culturing in the dark at 30 ℃ for 30 days, the callus induction effect of wild rice was counted and observed. Secondly, in order to explore the 2,4-D concentration suitable for callus induction of common wild rice and Nivara wild rice, this study set up three groups of 2,4-D concentration gradients: 3.5 mg / L, 5 mg / L and 8 mg / L, and tested the induction effect of wild rice callus under different 2,4-D concentrations. Experimental process Other experimental conditions remain unchanged. In the clean bench, the surface-sterilized rice seeds were inoculated into the induction medium containing different concentrations of 2,4-D. Each group of seeds was planted in 3 dishes, and each dish was planted with 12 seeds. After marking, they were placed in the dark at 30 ℃ for 30 days, and the callus induction effect of wild rice was counted and observed. In addition, this study also tested and compared the effects of two different carbon sources, sucrose and maltose, on the induction rate of rice callus. Also keeping other experimental conditions unchanged, the surface-sterilized rice seeds were inoculated into the induction medium containing sucrose or maltose in the clean bench. Each group of seeds was planted in 3 dishes, and each dish was planted with 12 seeds. Mark them well, culture them in the dark at 30 ℃ for 30 days, and then count and observe the callus induction effect of wild rice.

[0037] The induced callus tissue was transferred to the subculture medium NB1 and cultured at 30°C in the dark for 2-3 weeks. The light yellow and dense embryonic callus tissue was selected for subsequent genetic transformation experiments.

[0038] Table 1 Wild rice callus induction scheme

[0039] Experimental results: Treatments with different seed germination agents had significant effects on callus induction of mature wild rice seeds (e.g. Picture 3As shown in the figure, after soaking the seeds in sterile water for 2 h or 12 h, the callus induction effect of mature wild rice seeds was significantly better than that of soaking the seeds in 3% hydrogen peroxide or 0.1% gibberellin for 12 h or 24 h. From the perspective of wild rice seed germination and callus texture, after soaking mature seeds in sterile water for 12 h, the germination rate of mature wild rice seeds was higher, resulting in more callus tissue and a more compact and firm texture ( Picture 3 ). At the same time, increasing the concentration of 2,4-D could not promote the induction of callus tissue in mature seeds of wild rice. Different types of carbon sources such as sucrose or maltose had no significant difference in the induction of callus tissue in mature seeds of wild rice. Finally, the optimal callus induction scheme was determined: after surface disinfection, wild rice seeds were soaked in sterile water for 12 h, 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 ℃ for 30 days. At the same time, if Picture 3 As shown in the figure, three perennial common wild rice materials Y409, Y665 and Y702 and three Nivara wild rice materials Y706, Y710 and Y7113 were found to have high callus induction rates. Among them, among perennial common wild rice, Y665 had the highest induction rate, which could be as high as 65.8%. Among annual Nivara wild rice, Y706 had the highest induction rate, which could be as high as 52.3%.

[0040] (2) Agrobacterium-mediated genetic transformation This study optimized the diploid wild rice Agrobacterium-mediated genetic transformation system from the aspects of whether to add penetrants, optimizing AS concentration and Agrobacterium infection time, and tested and compared the effects of different concentrations of AS (20 µm, 50 µm, 100 µm, 200 µm), infection time (10 min, 20 min, 30 min) and penetrants (0.1% Pluronic F68, 0% Pluronic F68) on the Agrobacterium infection efficiency (Table 2). A total of 24 treatment experiments were set up, as shown in Table 3.

[0041] Table 2 Agrobacterium infection scheme for diploid wild rice

[0042] Table 3 24 experimental treatments of diploid wild rice infected with Agrobacterium tumefaciens

[0043] 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 and cultured overnight at 28°C and 220 rpm until the logarithmic phase (OD 600=0.6-1.0), and transfer an appropriate amount of bacterial culture to 20-30 mL of fresh YEB liquid medium. Cultivate under the same conditions until OD 600 =0.3-0.5. Then centrifuge the activated Agrobacterium at 4500 rpm for 10 min, discard the supernatant and resuspend the bacterial solution with an equal volume of AAM medium containing an appropriate concentration of AS. Immerse the collected callus tissue in the AAM-AS bacterial solution for an appropriate period of time (shake several times during the period to increase the dissolved oxygen content in the solution), discard the bacterial solution and use a gun to suck the bacterial solution dry, then place the tissue block in a plate covered with 4 layers of filter paper and blow dry for about 30 minutes, and finally transfer it to the NB2C co-culture medium covered with a layer of filter paper (to block nutrition), and co-culture at 28 ℃ in the dark for 2-3 days. Then, the callus block was transferred to NBS1 screening medium containing 50 mg / L hygromycin and 400 mg / L timentin for dark culture for 2-3 weeks, and then transferred to NBS2 screening medium containing 50 mg / L hygromycin and 300 mg / L timentin for dark culture for 2-3 weeks. According to the growth of resistant callus, a third round of screening can be selected, and finally bright red positive callus is obtained. The frequency of red callus appearance is counted, and the Agrobacterium transformation efficiency at the resistant callus stage is calculated.

[0044] 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 bombardment calli * 100% 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, the transformation efficiency of each treatment was calculated. The results are shown in Picture 5 shown.

[0045] As can be seen from the figure, the transformation efficiencies of ZH11, perennial common wild rice Y702 and Nivara wild rice Y706 under the same infection conditions are not exactly the same. ZH11, perennial common wild rice Y702 and Nivara wild rice Y706 all achieved higher transformation efficiencies when the penetrant Pluronic F68 was not added, and when the AS concentration was 20 µm, the three wild rice receptor materials could all achieve good transformation efficiencies. Among them, when the penetrant Pluronic F68 was not added, the AS concentration was 20 µm, and the infection time was 10 min, the transformation efficiency of ZH11 could reach a maximum of 81.5%, the transformation efficiency of Nivara wild rice Y706 could reach a maximum of 74.1%, and the transformation efficiency of common wild rice Y702 could reach 69.7%. When the penetrant Pluronic F68 was not added, the AS concentration was 20 µm, and the infection time was extended to 30 min, the transformation efficiency of common wild rice Y702 could reach a maximum of 77.8%.

[0046] (3) Optimization of regeneration system In order to establish an efficient diploid wild rice regeneration system, this study tested and compared the regeneration effects of four regeneration media. The components of the four regeneration media are shown in Table 4. Specifically, the embryonic callus obtained by subculture was inoculated on the regeneration medium, placed under 30 ℃, 60% humidity, 3000 lx light intensity, 16 h light / 8 h dark conditions for 2 weeks, and then transferred to fresh regeneration medium. After another 2 weeks of culture under the same conditions, the regeneration effect of the callus was observed.

[0047] Table 4 Main components of four regeneration culture media

[0048] Among them, the specific composition of regeneration medium 1 is as follows: NB basic 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 basic medium (4.33 g) + MS organic (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 basic medium (4.33 g) + MS organic (10 mL) + maltose (30 g) + NAA (0.1 mg) + ZT (2 mg) per liter. The specific ingredients of regeneration medium 4 are as follows: each liter contains 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); the ingredients 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.

[0049] Experimental results: Picture 6As shown in the figure, callus tissues of ZH11 and common wild rice Y409, Y665, Y702, and Nivara wild rice Y706, Y710, and Y711 all showed green growth points after 2 weeks of culture on the four regeneration media. Among them, cultivated rice ZH11 grew fastest on regeneration medium 4; the growth states of common wild rice Y409, Y665, and Y702 callus tissues on the four regeneration media were not exactly the same. Overall, the regeneration state of Y702 callus tissue on regeneration medium 4 was the best, the regeneration state of Y665 callus tissue was also good, and the regeneration state of Y409 callus tissue was the worst; the growth states of Nivara wild rice Y706, Y710, and Y711 callus tissues on the four regeneration media were relatively similar. In general, callus tissues of ZH11, Y409, Y665, Y702, Y706, Y710, and Y711 had the largest number of calli, the most green growth points, and the fastest growth of regenerated plants on regeneration medium 4. Therefore, regeneration medium 4 was used to cultivate regenerated plants in subsequent experiments of this study.

[0050] Example 2: Construction of editing vector (1) Target selection of target genes 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), and the 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. Then, CRISPR-GE (http: / / skl.scau.edu.cn / targetdesign / ) was used to design the target site. The specific gene structure and target site sequence are shown in Table 5.

[0051] Table 5 Target gene name, chromosome location and target sequence information

[0052] Note: PAM sites are in italics.

[0053] (2) Construction of editing vector S1. Obtaining the gRNA-tRNA basic fragment by overlapping PCR method: in the first round of PCR, the gRNA basic fragment is obtained by using primers gRNAF1, gRNAR1, and gRNAR2, and the tRNA basic fragment is obtained by using primers tRNAF1, tRNAR1, and tRNAR2; in the second round of PCR, the gRNA fragment and the tRNA fragment of the first round of PCR product are mixed in a molar ratio of 1:1 as a template, and the primer pair gRNAF2 / tRNAR3 is used for amplification to finally obtain the gRNA-tRNA basic fragment; 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. Use overlapping PCR to obtain tRNA-sgRNA1 ( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragment: The first round of PCR used the gRNA-tRNA basic fragment as a template and used the primer pair Actin-tRNAF1 and PROG1-tRNAR1 to obtain PROG1 The tRNA fragment of the gene target sequence was then amplified using the primer pairs PROG1-gRNAF1, An1-tRNAR1 and An1-gRNAF1, PolyA-tRNA-R1 with 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 tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragments and purify and recover them; at the same time, restriction endonucleases are used Pme I digested the vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-PolyA-Nos and recovered the digested product; finally, the recovered product was connected using homologous recombination enzyme 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; the base sequence of the PolyA-R1 is shown in SEQ ID NO.22; S3, using primer pairs DEP1-gRNAF1, D2-tRNAR1 and D2-gRNAF1, PolyA-tRNA-R1 and DEP1-gRNAF2, PolyA-R1 to obtain tRNA-sgRNA3 by overlapping PCR method ( 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 product is recovered; finally, the recovered product is connected using a homologous recombination enzyme 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, 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 were 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-recovered product is connected using homologous recombination enzyme 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 in SEQ ID NO.29; the base sequence of the Bh4-tRNAR1 is shown in SEQ ID NO.30; the base sequence of the Bh4-gRNAF1 is shown in SEQ ID NO.31; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO.32; the base sequence of the GW2-gRNAF2 is shown in SEQ IDNO.33; the base sequence of the PolyA-R1 is shown in SEQ ID NO.34.

[0054] Table 6 List of primers used in this application

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] (3) Genotype identification of T0 generation regenerated plants Using the genomic DNA of T0 generation rice plants as template, 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, respectively. The amplified PCR products were sequenced, and the sequencing results were input into the website http: / / dsdecode.scgene.com / to analyze the editing type. For samples with complex results, the PCR products were connected to the cloning vector, and the editing type was finally determined by sequencing the single clone.

[0061] The genotype identification results of the T0 generation regenerated plants are as follows: 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)-tRNA NA-sgRNA6(Bh4)-tRNA-PolyA-Nos-mediated editing of wild rice multiple genes. In this study, 90 wild rice callus tissues 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 obtained regenerated plants were sampled in clusters, 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 connected to the cloning vector, and the editing type was finally determined by sequencing and analyzing the single clones.

[0062] 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-1 There 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.

[0063] Table 7 Genotype analysis of T0 edited plants of common wild rice Y409

[0064] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0065] For the T0 generation regenerated plants of the 37 clusters 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 One strain was gene-edited. 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.

[0066] Table 8 Genotype analysis of T0 edited plants of common wild rice Y665

[0067] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0068] 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, D2 There were two strains with five genes edited, and the specific edited plant genotypes are shown in Table 9.

[0069] Table 9 Genotype analysis of T0 edited plants of common wild rice Y702

[0070] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0071] Of 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 One strain was gene-edited. GW2 , Bh4 There are 3 strains with double gene editing. GW2 , DEP1There 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 genes edited. GW2 , Bh4, An-1, PROG1, DEP1, D2 There was one plant with six genes edited, and the specific edited plant genotype is shown in Table 10.

[0072] Table 10 Genotype analysis of T0 edited plants of Nivara wild rice Y706

[0073] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0074] 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 , Bh4 There 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 genes edited. GW2 , An-1, PROG1, D2 There is one strain with four gene editing. An-1, PROG1, DEP1, D2 Three strains with four gene editing, Bh4, An-1, PROG1, DEP1, D2 There was one plant with five genes edited, and the specific edited plant genotype is shown in Table 11.

[0075] Table 11 Genotype analysis of T0 edited plants of Nivara wild rice Y710

[0076] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0077] For the T0 generation regenerated plants of the 27 clusters 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 One strain was gene-edited. 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 genes edited. GW2 , Bh4, An-1, DEP1 There are 2 strains with four genes edited. 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 genes edited. GW2 , Bh4, An-1, PROG1, DEP1, D2 There were three strains with six genes edited, and the specific edited plant genotypes are shown in Table 12.

[0078] Table 12 Genotype analysis of T0 edited plants of Nivara wild rice Y711

[0079] Bi: biallelic; He: heterozygous; Chi: chimera; WT: wild type.

[0080] (4) Off-target analysis of T0 generation regenerated plants 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 sequencing results of PCR products showed that most of the target sites designed in this experiment had no off-target situation at the predicted off-target sites, and only a few plants had An-1The first off-target site predicted by the target site targeted by 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 investigated later, it is important to eliminate these plants with off-target phenomena to avoid the impact of off-target on the phenotype of the edited plants.

[0081] Table 13 Target and off-target analysis

[0082] (5) Genetic analysis of T1 edited plants The harvested T0 generation seeds were soaked and germinated and then sown in the soil. When the seedlings grew to the 3-leaf stage, they were transplanted to the field for labeling and sampling. The genomic DNA of the T1 generation offspring plants was extracted, and about 200 ng of genomic DNA was used as a template. PCR amplification and sequencing were performed using the corresponding detection primers of each target gene. The segregation of editing events in the T1 generation plants was analyzed based on the sequencing results. 2 The test method was used to test whether the segregation of the editing event 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 strains conformed to Mendel's law of segregation.

[0083] The T0 generation strain 848-1 is a three-gene edited strain of common wild rice Y702. GW2 , Bh4 and An-1 When conducting genetic analysis on the T0 generation strain 848-1, a total of 33 T1 generation plants were tested, including 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 gene detected 9 heterozygous strains, 15 homozygous strains and 9 wild-type strains. According to the statistical data (see Table 14), the genetic segregation ratio of the offspring of the T0 generation strain 848-1 does not conform to Mendel's law of segregation, which also shows that the T0 generation strain 848-1 is a mosaic strain.

[0084] 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, including GW2 All genes were homozygous mutations; Bh4The gene detected 25 strains with double alleles, 12 homozygotes with 5 base deletions and 13 homozygotes with 6 base deletions. According to the statistical data (see Table 14), the genetic segregation ratio of the offspring of the T0 generation strain 825-1 conforms to Mendel's law of segregation.

[0085] Table 14 Genetic analysis of T1 edited plants and segregation of transgenic elements

[0086]

[0087]

[0088]

[0089] Note: The χ2 value indicates whether the T1 generation edited strain tested conforms 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 in good agreement with 1:2:1. For transgenic analysis, '+' indicates that Cas9 / hptII / sgRNA was detected, while '–' indicates that Cas9 / hptII / sgRNA was not detected. Ho: homozygous edited plant; He: heterozygous edited plant; WT: wild type; Bi: biallelic type; Chi: chimera.

[0090] The T0 generation strain 822-1 is a two-gene edited strain of Nivara wild rice Y710. An-1 and PROG1 When conducting genetic analysis on the T0 generation strain 822-1, a total of 23 T1 generation plants were tested, including An-1 The gene detected 13 strains with biallelic genotypes, 4 homozygous with 1 base insertion, and 6 homozygous with 1 base deletion; PROG1 The gene was detected in 2 heterozygous strains, 4 homozygous strains 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 PROG1 The gene was in a mosaic state in the T0 generation strain 822-1, and editing occurred in a small number of cells.

[0091] The T0 generation strain 847-1 is also a second gene-edited 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, includingGW2 The gene detected 25 strains with biallelic genotypes, 13 strains homozygous for 1 base G insertion and 11 strains homozygous for 1 base T insertion; Bh4 All genes are homozygous mutations. According to 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.

[0092] The T0 generation strain 563-2 is a five-gene edited strain of Nivara wild rice Y711. GW2 , Bh4, An-1 , DEP1 and D2 In the genetic analysis of the T0 generation line 563-2, a total of 96 T1 generation plants were tested, including GW2 The gene detected 51 strains with biallelic genotypes, 20 strains homozygous for 3-base deletion and 25 strains homozygous for 1-base deletion; Bh4 The gene detected 48 strains with biallelic genotypes, 22 strains homozygous for 1 base insertion, and 26 strains 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; D2 The gene detected 49 strains with double alleles, 22 strains with 3 base deletions and 25 strains with 1 base deletion. 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 genes in offspring all conform to Mendel's laws.

[0093] The T0 generation strain 564-1 is a six-gene edited strain 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, including GW2 The gene detected 55 strains with biallelic genotypes, 26 strains homozygous for 3-base deletion, and 28 strains homozygous for 1-base deletion; Bh4 The gene detected 56 strains with biallelic genotypes, 29 homozygous for 1 base insertion, and 24 homozygous for 1 base deletion; An-1The 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 heterozygous strains, 24 homozygous strains with 3 base deletions and 25 homozygous strains 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.

[0094] (6) Editing plant traits for improvement To determine whether gene editing technology can be applied to the de novo domestication of diploid wild rice and the improvement of its agronomic traits, this study observed the seed phenotypes of the two-gene-edited T1 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.

[0095] According to reports, GW2 The 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 the color of rice grains. Loss of the 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. The loss of this gene function can significantly shorten the awn length of rice.

[0096] The T1 line 848-1-138 of the common wild rice Y702 GW2 The gene has a homozygous insertion mutation of one base at the target site, making GW2 The gene has a frameshift mutation. GW2 Complete loss of gene function; Bh4 The gene has a site-directed mutation with a one-base deletion 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.

[0097] The T1 line 563-2-59 of Nivara wild rice Y711 GW2 The gene has a homozygous gene mutation with a base deletion at the target site, which makes 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 makes 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, resulting in An-1 The allele produces a frameshift mutation, and the corresponding gene function is lost. Figure 9 As shown in the figure, compared with the wild type Y711, the seed width of the T1 generation line 563-2-59 increased significantly, 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.

[0098] 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, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application, and the changes made 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 gene editing transformation vector is introduced into the cells of the diploid wild rice plant, and then the offspring plants with stable inheritance are obtained by screening; 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 constructed 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; the nucleotide sequence of the six-gene combination knockout vector is composed of SEQ ID NO.1 and SEQ ID NO.2 spliced ​​together; 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 common wild rice O.Rufipogon and / or Nivara wild rice O. Nivara .

2. The method according to claim 1, characterized in that The steps for constructing the gene editing transformation vector are as follows: (1) Obtaining the gRNA-tRNA basic fragment by overlapping PCR method: in the first round of PCR, the gRNA basic fragment is obtained by using primers gRNAF1, gRNAR1, and gRNAR2, and the tRNA basic fragment is obtained by 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 for amplification to finally obtain the gRNA-tRNA basic fragment; 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; (2) Using overlapping PCR method to obtain tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragment: The first round of PCR used the gRNA-tRNA basic fragment as a template and used the primer pair Actin-tRNAF1 and PROG1-tRNAR1 to obtain PROG1 The tRNA fragment of the gene target sequence was then amplified using the primer pairs PROG1-gRNAF1, An1-tRNAR1 and An1-gRNAF1, PolyA-tRNA-R1 with 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 tRNA-sgRNA1( PROG1 )-tRNA-sgRNA2( An-1 )-tRNA fragments and purify and recover them; at the same time, restriction endonucleases are used Pme I digested the vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-PolyA-Nos and recovered the digested product; finally, the recovered product was connected using homologous recombination enzyme 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; the base sequence of the PolyA-R1 is shown in SEQ ID NO.22; (3) Using primer pairs DEP1-gRNAF1, D2-tRNAR1 and D2-gRNAF1, PolyA-tRNA-R1 and DEP1-gRNAF2, PolyA-R1, tRNA-sgRNA3 was obtained by overlapping PCR method ( 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 product is recovered; finally, the recovered product is connected using a homologous recombination enzyme 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; (4) Using primer pairs GW2-gRNAF1, Bh4-tRNAR1 and Bh4-gRNAF1, PolyA-tRNA-R1 and GW2-gRNAF2, PolyA-R1, tRNA-sgRNA5( 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 product is recovered; finally, the recovered product is connected using a homologous recombination enzyme 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 in SEQ ID NO.29; the base sequence of the Bh4-tRNAR1 is shown in SEQ ID NO.30; the base sequence of the Bh4-gRNAF1 is shown in SEQ ID NO.31; the base sequence of the PolyA-tRNA-R1 is shown in SEQ ID NO.32; the base sequence of the GW2-gRNAF2 is shown in SEQ ID NO.33; the base sequence of the PolyA-R1 is shown in SEQ ID NO.

34.

3. The method according to claim 1, characterized in that The following steps are involved: (1) Genetic transformation of diploid wild rice recipient plants to obtain regenerated plants; (2) Detect whether the gene editing of the regenerated plants is successful; (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.

4. The method according to claim 3, characterized in that The step (1) comprises: (1) Selecting a transformation receptor material, wherein the transformation receptor material is a diploid wild rice O.Rufipogon and / or Nivara wild rice O. Nivara ; (2) Induction and subculture of wild rice callus; (3) Agrobacterium-mediated genetic transformation; (4) Regeneration seedling cultivation; (5) After the regenerated plants are acclimated and transplanted, the edited regenerated plants are screened and obtained.

5. The method according to claim 4, characterized in that In step (2), the induction and subculture scheme of wild rice callus 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.

6. The method according to claim 4, characterized in that In step (3), the Agrobacterium-mediated genetic transformation system 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.

7. The method according to claim 4, characterized in that 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, naphthylacetic 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.

8. 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 1-7.

9. Use of the method according to any one of claims 1 to 7 and the method according to claim 8 in the field of diploid wild rice breeding and / or gene function research.

Images