CN118291533A - Method for changing flowering period of rice by gene editing and application - Google Patents
Method for changing flowering period of rice by gene editing and application Download PDFInfo
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- CN118291533A CN118291533A CN202410521718.9A CN202410521718A CN118291533A CN 118291533 A CN118291533 A CN 118291533A CN 202410521718 A CN202410521718 A CN 202410521718A CN 118291533 A CN118291533 A CN 118291533A
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Abstract
The invention belongs to the field of crops, and particularly relates to a method for changing the flowering period of rice by gene editing and application thereof, and more particularly, the invention provides an application of simultaneously editing rice DTH7 and DTH8 genes by a gene editing tool in changing the flowering period of rice.
Description
Technical Field
The invention belongs to the technical field of crop gene editing breeding, and particularly relates to a method for changing the growth period of rice varieties by gene editing to influence the flowering of rice and application thereof.
Background
The crops such as rice and the like take a plurality of months from seed sowing to grain harvesting, after emergence of seedlings, nutrition growth and development are firstly carried out, environmental factors, especially the change of temperature and sunlight length are sensed, and then the crops are transferred into flowering, fruiting and maturation in a reproductive growth stage, and the length of the whole growth stage or the maturation stage is an extremely important agricultural production property. Some plants need long sunlight stimulation of a certain number of days to be changed from vegetative growth to reproductive growth, and flowers and fruits are long sunlight plants; while some plants need short-day stimulation for a certain number of days to bloom, which is a short-day plant. The rice is used as a short-day plant, is very sensitive to the sunlight length, the time for the complete maturation of different varieties is from about 120 days of early maturation to about 150 days of late maturation, the varieties with proper maturation periods must be selected in different ecological areas, the local sunlight heat accumulation cannot be fully utilized by the early maturation varieties to reduce the yield, the excessive late maturation varieties cannot be timely matured before frosting, and the absolute yield can be caused when serious. The northeast area of China, especially Heilongjiang province, longitudinally spans ten geographic latitudes, the ecological environment is extremely complex and various, and the temperature is divided into six temperature accumulating zones from higher than 2700 ℃,2500-2700 ℃,2300-2500 ℃,2100-2300 ℃ and 2100-1900 ℃ to lower than 1900 ℃ according to the number of annual temperature accumulating zones, wherein the first to fourth temperature accumulating zones are suitable for rice planting, but the special adaptation variety of each temperature accumulating zone must be planted.
The growth period is taken as one of the most important agronomic traits of rice, not only is important attention and application in breeding practice, but also genetic and molecular mechanisms of control of the rice are intensively studied. Flowering marks the transition of plants from nutrition to reproductive growth, directly determines the length of the growth period, is a complex quantitative trait controlled by a plurality of gene loci, and is regulated by illumination and temperature in the environment. About 70 or more flowering regulatory genes have been identified by studies on natural variation, mutant, transgene, etc. of rice, wherein Hd3a is the main flowering gene, and the expression of two developmental genes MADS14 and MADS15 is directly controlled to induce flowering. Under short-day conditions, hd3a is positively regulated by Hd1 and Ehd1, while Hd1 experiences spectra and photoperiod through Phys and GI, and Ehd1 is positively or negatively regulated by multiple genes. Under long-day conditions, hd3a and another flowering gene RFT1 are positively regulated by Ehd1 and negatively regulated by Hd1, hd1 also senses photoperiod through GI, ehd1 is directly or indirectly positively or negatively regulated by a plurality of genes, and in addition, a few other genes can also directly influence the expression of Hd3 a. Wherein Ghd7 and DTH8 inhibit flowering by negatively regulating Ehd1, and DTH7 (PRR 37) inhibits flowering by directly negatively regulating Hd3a （Brambilla and Fornara, 2013, J Integrative Plant Biol 55: 410-418;Hori et al., 2016,TheorAppl Genet 29: 2241-2252）.
The summer suitable for plant growth in high latitude areas is short, but the sunshine time per day is long, the temperature is quickly reduced after the change into short sunshine, and the requirements of late-maturing varieties on short sunshine days and accumulated temperature are difficult to meet. However, through long-term ecological adaptive breeding, early maturing rice varieties adapting to different heat accumulating zones are bred, and genomic sequencing analysis discovers that the varieties often contain haplotypes of the main flowering regulation gene mutation, and the mutation is disclosed as a genetic gene basis for leading the rice to be early matured, wherein the haplotypes of different genetic mutations have a superposition effect, namely, the earlier maturing varieties have more mutations. Theoretically, if the expression level of the main flowering regulatory genes is regulated by accurate mutation, it is possible to improve the good late maturing varieties without the early maturing haplotype into early maturing varieties, keep other original good characters to the maximum extent, and rapidly select and breed the early maturing varieties suitable for a wider heat accumulating zone (Zhang et al, 2015,New Phytologist 208:1056-1066; ye et al, 2018,Front Plant Sci 9:35).
The gene editing technology utilizes Cas9 endonuclease of the acquired immune system CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) of streptococcus pyogenes Streptococcus pyogenes to form a ribozyme complex with guide gRNA, precisely recognizes a target site through base pairing between an RNA sequence with the length of 20 bp in the gRNA and an edited DNA sequence, selectively cuts off specific genes in cells, and realizes gene editing through two inherent DNA repair processes of the cells. The Non-homologous end-linked Non-homologous end joining (NHEJ) has high repair efficiency, does not need a template, and can introduce insertion or deletion mutation in the cutting point linking process, so that the gene is inactivated, the final product after the editing offspring are subjected to genetic separation does not contain any exogenous DNA, and the Non-transgenic product can avoid the supervision and approval process and cost of complex and expensive transgenic products and is suitable for rapid production and application. The homology dependent repair Homologydependent repair (HDR) pathway requires the provision of a pre-designed DNA fragment as a template that can be site-directed inserted into a preselected site to modify the target gene, although the repair is precise, but is inefficient (Jinek et al, 2012, science 337:816-821).
The gene editing technology has been rapidly developed in recent years, and the multiple gRNA synchronous expression technology can simultaneously edit multiple loci of multiple genes in a high-efficiency NHEJ pathway (Hsieh-FENG AND YANG 2020, aBIOTECH 1: 123-134); single base editing techniques include cytosine editors (Cytosine base editor, CBE) and adeno glance sideways at-in editors (Adenine base editor, ABE) that can implement specific editing (Komor et al., 2016, Nature 533: 420–424; Nishida et al., 2016, Science 353: aaf8729;Gaudelli et al., 2017, Nature 551: 464–471); mutation SpCas9 of a single base through irreversible C-T or a-G conversion, respectively, to recognize NGH (h=a, C, T) outside NGG, pan-site editing (Hu et al., 2018, Nature 556: 57–63; Kleinstiveret al., 2015, Nature 523: 481–485; Nishimasu et al., 2018, Science 361: 1259–1262; Walton etal., 2020, Science 368：290–296); novel gene editing tool enzymes of PAM sequences such as NGN, etc., such as Cas12a (Cpf 1), etc., having more diverse recognition sites and editing functional characteristics (Zetsche et al., 2015, cell 163:1-13; murovec et al., 2017, plant Biotechnol J15:917-926); the guided editing technique (PRIME EDITING) has made breakthrough progress in precise gene editing, and can realize various types of precise gene editing including base conversion, substitution, deletion of small fragments, insertion, substitution, and the like (Anzalone et al., 2019, nature 576:149-157). Various gene editing techniques are widely applied in the field of plant research, and realize knockout editing of various plants and numerous genes and few precise design editing (Li et al., 2015Plant Physiol 169: 960–970; Ma et al., 2016, Mol Plant 9: 961–974; Chen etal., 2019, Annu Rev Plant Biol 70: 667–97; Mao et al., 2019, Natl Sci Rev 6: 421-437).
The more the well-known Rice variety in japan starts to be planted in 1953, becomes a new main-pushed variety in the county of new lager and is formally named as the more light (Koshihikari), by virtue of its excellent quality, the most widely planted Rice variety in japan is still planted in 2016 even after being planted for more than 50 years, and the planting area in 2016 occupies 36.2% of the planting area in japan (Kobayashi et al, 2018, rice 11:15), which is worth being introduced as a breeding resource material of excellent quality for improving the properties such as the quality of northeast Rice in China. However, the growth period of the sunlight is longer, and even in the first heat accumulating zone with the highest heat accumulating temperature in the Heilongjiang, the sunlight can not normally bloom and fruit, so that the breeding application of the sunlight is severely restricted. The invention aims at the light-crossing base material, aims at 4 rice growth period control genes DTH7 (LOC_Os07 g49460, also called Hd 2), DTH8 (LOC_Os08 g07740, also called Hd 5), ghd7 (LOC_Os07 g15770, also called Hd 4) and Hd1 (LOC_Os06 g 16370) which inhibit flowering under long-day conditions, utilizes the gene editing technology to carry out various combination editing on different sites of different genes so as to obtain an improved strain with shortened growth period, obtains unexpected effects, and invents a method for effectively shortening the rice growth period on the premise of not affecting other favorable traits.
Disclosure of Invention
According to the invention, a CRISPR/Cas9 plant gene editing system is established, gene editing genetic transformation vectors aiming at rice flowering control genes DTH7 (LOC_Os 07g 49460) and DTH8 (LOC_Os 08g 07740) are designed and constructed, the rice variety is transformed by using an agrobacterium-mediated method to be brighter, multiple types of editing of nucleotide sequences of a regeneration plant DTH7 gene target region and a DTH8 gene target region are realized, the gene editing efficiency is up to 100%, and finally, a homozygous editing rice strain which shortens the growth period by more than 45 days, is non-transgenic and is inherited stably is obtained, and compared with other flowering control gene combinations, the flowering combined gene plant edited by the invention grows normally, and no obvious phenotypic defect is observed.
The invention covers the complete process from the design and construction of an editing vector to the editing and confirmation of a transformed plant offspring gene, and the main steps of the adopted technical scheme include the control of the selection of a target trait gene, the design and construction of a gene editing transformation vector, the genetic transformation of rice cultivars, the molecular biological analysis of transformation events and the gene editing and confirmation, and the screening of non-transgenic homozygous editing offspring of regenerated plants, thereby realizing the efficient editing of the target trait gene by a gene editing method.
Specifically, the technical scheme provided by the invention is as follows:
The main implementation scheme provided by the invention is that the DTH7 gene and the DTH8 gene of the rice are edited simultaneously by a gene editing tool and applied to change the flowering period of the rice, so that the growth period of the rice is shortened.
As some embodiments, the gene editing tools include the CRISPR/Cas9 system, and its derivative tools cytosine editor, adeno glance sideways at-n editor, or guide editor, or gene editing systems based on different Cas12 enzymes, or other gene editing tools. .
Furthermore, the gene editing tool is a CRISPR/Cas9 system, the gene editing transformation vector designed based on the CRISPR/Cas9 system contains three gene expression units ZmU pro, gRNA is AtU-26 term,ZmUBI1 pro:SpCas9:PsE9 term, bsaI restriction sites are reserved between 35S pro:HYG:35S term,ZmU6 pro and gRNA, one or more gRNA sequences are inserted by a DNA ligase or Gibson cloning method after linearizing the vector, and the gene editing vector aiming at any target site of DTH7 and DTH8 genes is formed.
Furthermore, gRNAs are designed for the recognizable sites of different regions of the DTH7 and DTH8 genes, and a plurality of gRNAs are inserted between ZmU pro and gRNA frameworks of the gene editing transformation vector by a cloning method after being connected mutually by tRNA technology.
Preferably, the designed gene editing transformation vector inserts a tRNA-OsDTH 7-G1-gRNA-tRNA-OsDTH-G1 fragment artificially synthesized in an interactive arrangement of tRNA and gRNA backbone between ZmU pro and gRNA, the sequence of the fragment is shown as SEQ ID NO: shown at 7.
Furthermore, the genetic transformation method for changing the flowering period of the rice is to introduce the designed gene editing transformation vector into rice cells through agrobacterium mediation, and then screen to obtain a non-transgenic and stable genetic homozygous editing rice line.
Specifically, the genetic transformation method comprises the following steps:
s1: genetic transformation is carried out on the receptor rice cells to obtain regenerated plants;
s2: detecting whether gene editing of the regenerated plant is successful or not;
S3: and screening to obtain a progeny plant which can be inherited, is non-transgenic and is stably edited.
Further, the step S1 includes: s1-1, inducing rice callus; s1-2, subculturing rice callus; s1-3 infection of callus; s1-4, screening and identifying resistant callus; s1-5, differentiating the callus into seedlings; s1-6, rooting culture; s1-7, domesticating and transplanting to obtain regenerated plants.
Further, the step S2 includes:
s2-1, collecting a resistant callus or a tender leaf tissue of a regenerated plant, extracting genomic DNA and carrying out PCR amplification;
S2-2, designing PCR primers covering a certain length of the upper and lower streams of a cutting site according to sequences of target genes DTH7 and DTH8 and expected cutting point positions of Cas9, and amplifying target gene fragments;
S2-3, judging whether the gene editing is successful or not according to the difference between the amplified target gene fragment and the target gene fragment which is not amplified by the genetically transformed plant.
Furthermore, the obtained offspring plants can be used in rice breeding, more specifically, offspring plants based on gene editing and screening have stable characters, normal growth and development, early flowering period and shortened growth period, and other objective characters can be obtained by further adopting means such as gene editing breeding, molecular breeding or hybridization breeding.
Further, the DTH7 genes in the offspring plants R81-1-1-12, R81-2-1-9, R81-5-3-3, R89-2-3-1 and R89-2-4-3 obtained by screening are respectively shown as SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO:45 and SEQ ID NO:46, the DTH8 gene is shown as SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO:50 and SEQ ID NO: 51.
Drawings
FIG. 1 is a schematic diagram of four flowering control gene structures and gene editing sites of rice (in the figure, (a) the coding structure of four flowering control genes in sequence: DTH7 (LOC_Os07g49460.1) consists of 10 exons and 9 introns, one site of exon 4 is selected from designed OsDTH7-G1 gRNA, and primers OsDh7-F2 and OsDh7-R2 are respectively designed at the upper and lower streams for PCR specific amplification and sequencing analysis of the editing region 1329 bp DNA fragment; DTH8 (LOC_Os08g07740.1) has simple structure, no intron, one site is selected and designed for OsDTH8-G1 gRNA at the upstream and downstream, primers OsDh-F1 and OsDh-R1 are designed for PCR specific amplification and sequencing analysis editing region 1329 bp DNA respectively, ghd7 (LOC_Os07G 15770) is composed of 2 exons and 1 intron, one site is selected and designed for OsGhd7-G3 and OsGhd7-G3R gRNA, primers OsG-F4 and OsG-R4 are designed for PCR specific amplification and sequencing analysis editing region 826 bp DNA respectively at the upstream and downstream, hd1 (LOC_Os06G 16370) is composed of 2 exons and 1 intron, one site is selected and designed for LOC_Os07-G1 gRNA, one site is designed for PCR specific amplification and sequencing analysis editing region OsHd-G3R RNA, one site is designed for PCR specific amplification and sequencing analysis region 826-d 1-G3R 4 is designed for PCR specific amplification and sequencing analysis editing region 826-bp DNA respectively at the downstream, and 3d 1 (LOC_Os06G 16370) is designed for PCR specific amplification and sequencing analysis region 678-G3 b 1 gRNA, and the other sites are designed for PCR region-3 b-d 3 and 3d 1 gRNA is designed for PCR domain-3 and 3-d 3 b, and 3d is designed for the DNA domain is sequentially and 3-3 and 3-d 3 and 3d is designed and a region, the relative positions of the gRNAs are shown).
FIG. 2 is a schematic diagram of a gene editing transformation vector KF99 (in the figure, T-DNA RB is the right border of T-DNA; zmU pro is the maize U6 snRNA gene promoter; atU term is the Arabidopsis U6 snRNA gene terminator; zmUBI pro is the maize ubiquitin gene promoter; cas9 is the endonuclease of the S.pyogenes Streptococcus pyogenes acquired immune system CRISPR; psE9 term is the pea ribulose-1, 5-bisphosphate carboxylase/oxygenase small subunit E9 protein gene terminator; caMV35S pro is the cauliflower mosaic virus 35S promoter; hygR is hygromycin phosphotransferase; caMV35S term is the cauliflower mosaic virus 35S terminator; T-DNA LB is the left border of T-DNA).
FIG. 3 is a diagram of PCR identification of T1 progeny plants of the gene editing event (in the diagram, genomic DNA of leaves of representative T1 plants of each event is used as a template, the expected 540 bp fragment is amplified by PCR by using Cas9 gene specific primers Cas9-F1 and Cas9-R1 respectively, or the expected 402 bp fragment is amplified by PCR by using HygR gene specific primers Hyg-F1 and Hyg-R1, and non-transgenic plants are not successfully amplified; the expected approximately 1329 bp fragment was amplified by PCR using OsDTH gene editing region specific primers OsDh-F2 and OsDh7-R2, the expected approximately 1329 bp fragment by PCR using OsDTH8 gene editing region specific primers OsDh8-F1 and OsDh8-R1, the expected approximately 826 bp fragment by PCR using OsGhd gene editing region specific primers OsG-F4 and OsG7-R4, the expected approximately 801 fragment by PCR using OsHd1 gene editing region specific primers OsHd-F3 and OsHd1-R3, and each DNA sequence editing situation was analyzed by sequencing, the labeled DNA fragment lengths were 2000, 1000, 750, 500, and 250 bp, the wt and wt+KF88 or KF114 or KF99, respectively, were positive controls of wild type negative for the more negative and small amounts of DNA added, (a) R55 gene editing family, (b) R66 gene editing family, (c) R81 and R89 gene editing family.
FIG. 4 is a partial DNA sequence sequencing diagram of different gene editing families in a gene target region (in the diagram, (a) DNA sequences near the editing sites of R55 gene editing family OsGhd-G3 and OsHd-G1, and (b) DNA sequences near the editing sites of R66 gene editing family OsGhd 7-G3R).
FIG. 5 is a DNA sequence diagram of R81 and R89 gene editing families OsDTH-G1 and OsDTH-G1 near the editing site.
FIG. 6 is a graph showing the growth state of wild type Yuanguang and gene editing family R81-1-1-12 plants on the rice experimental base in Haerbin Alcity region at 8 months of 2022.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to the drawings and examples, but the present invention is not limited to the scope of the examples. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. The reagents and materials used in the present invention are commercially available.
Example 1
1. Construction of Gene editing transformation vector
According to the data analysis of the rice genome sequencing database, oryza sativa v7.0, annotated according to the U.S. department of energy (https:// phytozome. Jgi. Doe. Gov/pz/portal. Html), the rice flowering control gene DTH7 (LOC_Os 07g49460, hd 2) is of complex gene structure, consisting of 10 exons and 9 introns (FIG. 1 a), comprising the total length 12519 bp of 5' UTR and 3' UTR (LOC_Os 07g49460|Chr7:29616704..29629223 forward), the expected coding sequence starts from the third exon and is of length 2229 bp, encoding a long protein containing 742 amino acid residues, according to the search result analysis of the protein sequence of the national center of biotechnology information (https:// blast. Ncbi. Lm. Nih. Gov/blast. Cgi), the amino terminal of which contains the amino acid domain responsible for signal reception Response regulator receiver (UTR: amino acid sequence of the U.S. 3' UTR) and the amino acid sequence of the gene sequence of interest (CCT 1-682).
The rice flowering control gene DTH8 (LOC_Os 08g07740, hd 5) has simple gene structure and no intron (figure 1 a), the total length of 5'UTR and 3' UTR is only 1718 bp (LOC_Os 08g07740|Chr8:4333716..4335434 reverse), the coding sequence length is 894 bp, the coding sequence length is 297 amino acid residue-containing long protein, histone-like transcription factor CBF/NF-Y domain (amino acids 61-126) is a pseudohistone transcription factor.
The gene Ghd7 (LOC_Os 07g15770, hd 4) of the rice flowering control gene consists of 3 exons and 2 introns (figure 1 a), the total length excluding the 5'UTR and the 3' UTR is 2784 bp (LOC_Os 07g15770|Chr7:9152401..9155185 reverse), the coding sequence is 774 bp, the long protein containing 257 amino acid residues is coded, and the carboxyl end is provided with a CCT motif (amino acids 190-232, figure 1 b) containing a cell nucleus positioning signal, and the gene is a transcription regulatory factor.
Rice flowering control gene Hd1 (LOC_Os 06g16370, hd 1) consists of 2 exons and 1 intron (FIG. 1 a), does not include the total length of 5'UTR and 3' UTR is 2285 bp (LOC_Os 06g16370|Chr6:9336358..9338643 forward), the coding sequence length 1188 bp codes for a protein containing 395 amino acid residues in length, the amino terminus of which contains B-Box-type zinc finger (B-Box type zinc finger domain, amino acids 33-77) responsible for regulatory signal reception, and the carboxy terminus of which has a CCT motif (amino acids 326-369, FIG. 1B) containing a nuclear localization signal, is a transcriptional regulator.
"Os" is abbreviated herein to Oryza sativa rice, namely, rice DTH7, rice DTH8, rice Ghd7 and rice Hd1 gene sequences, abbreviated to OsDTH, osDTH8, osGhd7 and OsHd gene sequences.
According to the above OsDTH, osDTH8, osGhd and OsHd1 gene sequences, the coding sequences of the respective important functional domains and the nearby regions are selected, the N (20) NGG sequence is screened as the gRNA target site of the potential SpCas9 by using DNA analysis software Geneious Prime (GraphPad Software LLC), the gRNA with the best expected activity is selected (Doench et al., 2016, nat Biotechnol 34:184-191), and the gRNA recognition sequences OsDTH7-G1, osDTH-G1, osGhd-G3 and the complementary sequences OsGhd7-G3r and OsHd-G1 (SEQ ID NO:1-5, FIG. 1 a) are respectively designed in combination with consideration of the positions, the base sequences, the expected editing change effects and the like. Although simply designing grnas in the regions upstream of OsDTH, osGhd7, and OsHd1 genes also enables gene editing (fig. 1) and obtains a phenotype effect (Cui et al.,2019, Theor Appl Genet 132: 1887-1896; Zhou et al., 2024, Plant Biotechnol J 22: 751-758), with a shortened growth period, the gene editing efficiency and the obtained phenotype effect are different, and a plurality of target genes need to be edited simultaneously to obtain a superposition effect with a significantly shortened growth period, and multi-gene editing often affects other important agronomic traits of rice, resulting in impaired growth and development or reduced yield, which cannot be applied in breeding practice.
The invention discloses a gene editing agrobacterium transformation DNA vector special for rice, which is independently developed in North Dahan reclamation and rich species industry, and comprises three gene expression units ZmU pro, wherein gRNA is AtU-26 term,ZmUBI1pro:SpCas9:PsE9 term and 35S pro:HYG:35S term, bsaI cleavage sites are reserved between ZmU pro and gRNA, one or more gRNA sequences can be inserted by using methods such as DNA ligase or Gibson cloning after linearization of the vector to form a basic gene editing vector aiming at any target site (Ma Rui and the like, patent publication No. CN 113604501B), specific sequence information is detailed in KF80 transformation vectors in a gene editing method of a indica rice modified strain aroma control gene and application thereof of patent CN 113604501B.
The designed gene editing vector KF88 contains a tRNA-OsGhd-G3-gRNA-tRNA-OsHd-G1-gRNA-tRNA-OsDTH-G1 fragment (SEQ ID NO: 6) artificially synthesized in an interactive arrangement mode of tRNA and gRNA frameworks, and 20 bp additional sequences are respectively arranged at two ends of the fragment and are homologous with the 3 '-end of the ZmU6 promoter of the basic gene editing vector and the 5' -end sequence of the gRNA framework; the designed gene editing vector KF99 (figure 2) contains a tRNA-OsDTH 7-G1-gRNA-tRNA-OsDTH-G1 (SEQ ID NO: 7) fragment artificially synthesized in an interactive arrangement mode of tRNA and gRNA frameworks, and the two ends of the fragment are respectively provided with the same 20 bp additional sequence homologous to the 3 '-end of the ZmU promoter of the basic gene editing vector and the 5' -end sequence of the gRNA frameworks; the designed gene editing vector KF114 only contains OsGhd-G3 r of a gRNA, and only needs to synthesize a short nucleotide with the same 20 bp additional sequence at two ends (SEQ ID NO: 8); three synthetic DNA fragments or short nucleotides were inserted between the ZmU promoter and the gRNA backbone of the basic gene editing vector by NEBuilder cloning method (NEW ENGLAND Biolabs), and constructed as gene editing vectors KF88, KF99, and KF114, respectively. Sequencing each gene editing vector by using a ZmU promoter specific primer ZmU-F2 (SEQ ID NO: 9), confirming that the vector is error-free, transferring into an agrobacterium strain EHA105 for rice transformation, and preserving in a refrigerator at the temperature of minus 80 ℃ for later use.
The later experimental result shows that the editing efficiency of each gRNA locus designed by the invention is up to 100%, the unexpected phenotypic effect is obtained, the flowering period of 1 or 2 genes which are about to be more light in Haerbin Alcheng area is about 45 days in advance, normal maturing is realized, and other characters are not adversely affected by some editing combinations, so that the method for quickly and effectively shortening the rice growth period is invented on the premise of not affecting other characters.
2. Genetic transformation of japonica rice varieties
The gene editing can be realized by transforming the gene editing vector into cells for expression, and the healthy mature seeds of the glabrous rice are used as explants by using an agrobacterium-mediated transformation method, so that the vector KF88 for editing OsGhd, osHd1 and OsDTH genes, the vector KF114 for editing OsGhd7 genes and the vector KF99 for editing OsDTH and OsDTH8 genes are respectively transformed into cells, and the DNA sequences near the identification sites of the target genes are accurately modified, and various basic culture medium formulas used in genetic transformation experiments are shown in table 1.
TABLE 1 composition of basic culture medium for genetic transformation experiment of Agrobacterium tumefaciens and preparation method thereof
| Medium name | Formula composition and preparation method |
| Agrobacterium culture medium YEP | 5 G/l NaCl, 10 g/l yeast extract, 10 g/l tryptone, pH 7.0. The solid YEP culture medium is added with 1.2% of agar powder, and the culture medium is sterilized at 121 ℃ for 20 minutes. |
| Induction medium | 10 XN 6 minimal medium 3.98 g/L, 200 XN 6 vitamin 5 ml/L, sucrose 30 g/L, acid hydrolyzed casein 1 g/L, proline 4 g/L, auxin 2, 4-D2.5 mg/L, plant gel (Wako) 4 g/L, pH 5.8, sterilization at 121℃for 20 minutes. |
| Infection suspension culture medium | 10 X AAI macroelement 100 ml, 1000 x AAI microelement 1 ml, 100 x ferric salt 10ml, 200 x AAI vitamin 5ml, 20 x AAI amino acid 50ml, sucrose 68.5 g/L, glucose 36 g/L, acid hydrolysis casein 0.5g/L, pH 5.2, 121 ℃ sterilization 20 minutes, cooling and adding 1000 x acetyl syringone 1 ml. |
| Co-culture medium | 10 XN 6 basic culture medium 3.98 g/L, 200 XN 6 vitamin 5 ml/L, sucrose 30 g/L, glucose 10 g/L, auxin 2, 4-D2.5 mg/L, plant gel 4 g/L, pH 5.2, 121 ℃ sterilization 20 minutes, cooling and adding 1000 Xacetyl syringone 1 ml. |
| Screening media | 10 XN 6 minimal medium 3.98 g/L, 200 XN 6 vitamin 5 ml/L, sucrose 30 g/L, acid hydrolyzed casein 1 g/L, proline 4 g/L, auxin 2, 4-D2.5 mg/L, vegetable gel 4 g/L, pH 5.8, 121 ℃ sterilization 20 minutes, cooling after adding carbenicillin 400 mg/L, hygromycin 50 mg/L. |
| Differentiation medium | 10 XMS minimal medium 4.74 g/L, sucrose 30 g/L, acid hydrolyzed casein 1 g/L, sorbitol 20 g/L, acid hydrolyzed casein 1 g/L, auxin NAA 1.0 mg/L, IAA 1.0 mg/L, cytokinin 6-BA 0.2 mg/L, zeatin 2.3 mg/L, plant gel 4 g/L, pH5.8, sterilization at 121℃for 20 minutes. |
| Rooting culture medium | 10 XMS minimal medium 2.37 g/L, sucrose 20 g/L, plant gel 3 g/L, pH 5.8, 121 ℃ sterilization 20 minutes. |
| 200 XN 6 vitamin | Inositol 20 g/L, thiamine hydrochloride 0.2 g/L, nicotinic acid 0.1 g/L, pyridoxine hydrochloride 0.1 g/L, glycine 0.4 g/L, split charging and preserving at-20deg.C for use. |
| 1000 X acetosyringone | Dissolving 0.392 g acetosyringone into 10 ml DMSO (dimethyl sulfoxide), filtering with 0.2 μm filter membrane, sterilizing, and preserving at-20deg.C. |
| 10 X AAI macroelements | Magnesium sulfate MgSO 4.7H2 O5 g/L, calcium chloride CaCI 2.2H2 O1.5 or CaCI 2 1.14.14 g/L, sodium dihydrogen phosphate NaH 2PO4.H2 O1.5 or NaH 2PO4.2H2 O1.7 g/L, potassium chloride KCl 29.5 g/L, split charging and preserving at-20deg.C for later use. |
| 1000 X AAI trace elements | Manganese sulfate MnSO 4.4H2 O or MnSO 4. H2 O10 or 7.58 g/L, zinc sulfate ZnSO 4.7H2 O2 g/L, boric acid H 3BO3 g/L, potassium iodide KI 0.75 g/L, sodium molybdate Na 2MoO4.2H2 O0.25 g/L, cobalt chloride CoCl 2.6H2 O0.025 g/L, copper sulfate CuSO 4.5H2 O0.025 g/L, and packaging and storing at-20 ℃ for later use. |
| 200 X AAI vitamins | Inositol 20 g/L, thiamine hydrochloride 2g/L, nicotinic acid 0.2 g/L, pyridoxine hydrochloride 0.2 g/L, split charging and preserving at-20 ℃ for standby. |
| 200 X AAI amino acids | 17.5 G/L glutamine, 5.3 g/L aspartic acid, 3.48 g/L arginine and 0.15 g/L glycine, and is packaged and stored at-20 ℃ for later use. |
| 100×Ferric salt 600 ml | Ferrous sulfate FeSO 4.7H2 O2.78 g is dissolved in 300 ml of water, na 2EDTA.2H2 O3.73 g is heated to about 70 ℃ and dissolved in 300 ml of water, and the two solutions are mixed in equal amounts and stored at 4 ℃ for later use. |
| 2,4-D1 mg/ml | 100 Mg of 2,4-D was dissolved with a small amount of 1N sodium hydroxide, the volume was fixed to 100 ml, and the mixture was stored at 4 ℃. |
| 6-BA1 mg/ml | 100 Mg of 6-BA was dissolved with a small amount of 1N sodium hydroxide, the volume was set to 100 ml and stored at 4 ℃. |
| NAA1 mg/ml | 100 Mg NAA was dissolved with a small amount of 1N sodium hydroxide, and the volume was set to 100 ml and stored at 4 ℃. |
| IAA1 mg/ml | 100 Mg IAA was dissolved with a small amount of 1N sodium hydroxide, and the volume was set to 100 ml and stored at 4 ℃. |
| Zeatin 1 mg/ml | 100 Mg of zeatin is dissolved in 95% ethanol, the volume is fixed to 100ml, and the mixture is stored at 4 ℃. |
| Hygromycin 50 mg/ml | Dissolving 5g hygromycin in 100ml water, filtering and sterilizing with 0.22 μm filter membrane, subpackaging with 2 ml centrifuge tube, and preserving at-20deg.C. |
| Kanamycin 50 mg/ml | 5 G kanamycin was dissolved in 100 ml water, filtered and sterilized with 0.22 μm filter, and sub-packed in 2 ml centrifuge tubes and stored at-20 ℃. |
| Carbenicillin 250 mg/ml | After dissolution 5g of carboxin were sterilized by filtration through a 0.22 μm filter in 20 ml water, and sub-packed in 2 ml centrifuge tubes and stored at-20 ℃. |
| 1N sodium hydroxide | Dissolve 5.6 g sodium hydroxide in 100 ml water and store at room temperature. |
The specific operation flow is as follows:
1. Induction of rice callus: about 200 mature, full and healthy rice seeds are picked for each transformation experiment, glumes are stripped, the rice seeds are placed in a 100ml sterile glass bottle and washed 3 times by 75% ethanol, equal volumes of sterile water and 10% sodium hypochlorite NaClO are added, two drops of Tween 20 are added, shaking sterilization treatment is carried out for 20 minutes, and the rice seeds are washed 3 to 5 times by the sterile water until foams are washed off. The surface moisture of the seeds is sucked by sterile filter paper, horizontally placed on an induction culture medium, and dark-cultured in a constant temperature incubator at 33 ℃ for 3-4 weeks until the calli grow out from the embryos.
2. Subculture of rice callus: healthy callus with vigorous stripping growth is selected, placed on a new induction culture medium for subculture for 1-2 weeks, and propagated and the vigor of the callus is maintained.
3. Infection of callus: the agrobacterium glycerol storage tube carrying the target gene vector is taken out from a low-temperature refrigerator at the temperature of minus 80 ℃, a small amount of agrobacterium is streaked and inoculated on a solid YEP culture medium flat plate containing 50 mg/L kanamycin, and the agrobacterium is cultivated in the dark at the temperature of 28 ℃ for 2 to 3 days. And (3) picking single colony, inoculating 5ml of YEP liquid culture medium, culturing overnight at 28 ℃ in a dark way, and taking a small amount of bacterial liquid to extract plasmid DNA for carrier-specific PCR detection to confirm that the strain carries a target gene carrier. Meanwhile, part of bacterial liquid is coated with YEP culture medium for plate culture observation (the residual culture medium can be temporarily stored at-80 ℃ C. For later direct use). The agrobacterium used for infection must be free of contamination by bacteria, and the whole plate is required to be smooth and free of production of particles or other colors of fungi or mold and other bacteria to ensure the purity of the obtained agrobacterium and to ensure no contamination in subsequent work by strict aseptic manipulation.
After the agrobacteria flat plate is determined to be pollution-free, directly scraping the thalli into a 100ml triangular flask containing 25 ml of suspension infection culture medium, culturing for 2-3 hours on a 100-120 rpm rotary shaking table with the constant temperature of 25 ℃, sampling and measuring an OD 600 value, and using the suspension infection culture medium to adjust the OD 600 value of the agrobacteria to be 0.1-0.2 for callus infection. Selecting small granular healthy callus, placing the small granular healthy callus in a 250 ml sterile glass bottle, adding agrobacterium for hand-shake infection for 3-5 minutes, then sucking the bacterial liquid on the surface of the callus by using sterile filter paper, placing the bacterial liquid on the filter paper on the surface of a co-culture medium, placing a piece of sterile filter paper on the co-culture medium in advance to prevent the agrobacterium from growing, and performing co-culture in the dark at 28 ℃ for 3 days.
4. Screening and identification of resistant calli: after 3 days of co-cultivation, the rice calli were transferred to sterile 250 ml glass flasks and washed several times with sterile water until the water was clear and transparent, and finally washed with a final concentration of about 250 mg/l carbenicillin on a 100 rpm shaker for 0.5 hours. Transferring the callus to a culture dish, sucking the surface moisture of the callus with sterile filter paper, placing the callus in a sterile operation super clean workbench for air drying for 2.5 hours, sub-packaging the callus on a screening culture medium, and culturing in a constant temperature incubator at 33 ℃ for 3-4 weeks in a dark way.
The screening medium contains 400 mg/L of carbenicillin to inhibit the growth of agrobacterium, 50 mg/L of hygromycin is used for screening transformed cells, non-transformed cells stop growing and gradually die on the screening medium, and after 3-4 weeks of culture, the successfully transformed cells can grow resistant callus. After the growth of the resistant callus, sampling the callus in proper amount, extracting genome DNA, and carrying out PCR identification by using a primer specific to a transformation vector, wherein each PCR positive callus is an independent transformation event. If necessary, the edited DNA fragment can be amplified by PCR with primers specific to the target gene, and then sequenced directly or after cloning to analyze the effect of gene editing.
5. Callus differentiation into shoots: the positive and growing resistant calli identified by PCR are transferred into a culture flask containing a differentiation medium, kept under 16 hours of illumination every day, and after 2-3 weeks of culture at 28 ℃, green spots are visible to differentiate, and after about 2 weeks, part of green spots can differentiate into seedlings and grow and elongate.
6. Rooting culture: transplanting the differentiated seedlings with the height of about 2-5 cm and normal morphology into a culture flask containing rooting culture medium, illuminating for 16 hours per day, culturing for 1-2 weeks at 28 ℃ to strengthen the seedlings, and differentiating healthy root systems.
7. Domestication and transplanting of the resistant regenerated plants: when the root system of the seedling grows to be about 3-4 cm and is stronger, opening a sealing film of a culture bottle, adding a small amount of distilled water, hardening the seedling for 24 hours, carefully clamping the plant out by using forceps to avoid damaging stems and roots, flushing a culture medium attached to the root by using clear water, transplanting the plant into a seedling tray containing vermiculite nutrient soil, covering the seedling tray to keep humidity, culturing in a 28 ℃/24 ℃ artificial climate chamber or a greenhouse under 16/8 hours of illumination until new leaves grow out, transplanting the seedling into a larger flowerpot for soaking water culture, and applying a proper amount of fertilizer until flowers and fruits are obtained. During this period, leaves of these T0 plants were sampled, and DNA was extracted for transgene identification and gene editing analysis.
3. Gene edit identification of transformation events
The resistant regenerated plants must be detected by molecular biology to confirm whether genetic transformation is successful, and a common simple and reliable detection method is PCR amplification. Sampling a small amount of callus or tender leaf tissue of regenerated plant with resistance transformation event, extracting genome DNA for PCR amplification, designing carrier-specific DNA primer according to the transformation carrier DNA sequence, obtaining the plant with specific amplified fragment as transformation positive, and further detecting the editing effect of target gene. According to the target gene DNA sequence and the expected Cas9 cleavage point position, PCR primers with the length of about 300 bp parts are designed to cover the upper and downstream of the cleavage site, target gene fragments with the length of about 400-800bp are amplified, and the same upstream or downstream primers are directly used for sequencing after purification. If the DNA fragment sequence of the regenerated plant is not different from the wild type sequence, no gene editing is carried out; if the detected DNA sequence shows a sleeve peak near the expected Cas9 cleavage site, the sequenced PCR fragment is a mixed template, and the regenerated plant is heterozygous edited; if the regenerated plant DNA fragment has a definite sequence difference with the wild type, the homozygous gene is edited. The PCR fragment of the target gene of the heterozygous editing plant can be cloned by using a PCR fragment cloning kit, at least 3 cloning vectors are selected for specific primer sequencing, and the specific editing change of the target gene sequence is confirmed.
The analysis and comparison of DNA sequences involved in the molecular biology experiments in the invention, the cloning construction design of DNA vectors, the design of PCR primers and CRISPR GRNA, and the like are all completed by using DNA analysis software Geneious Prime (Biomatters Ltd.).
Sampling rice resistant callus, or regenerated T0 plant leaves, extracting genome DNA by a SDS (Sodium dodecyl sulphate) -based DNA rapid extraction method, performing Cas9 gene-specific PCR detection, and identifying transformation event. The PCR reaction system adopts Quick Taq HS DyeMix (DTM-101) kit (TOYOBOLife Science), the 20 microliter PCR reaction system comprises 10 microliter 2x Quick Taq HS DyeMix, 1.0 microliter 10 pmol/microliter primer Cas9-F1 (SEQ ID NO: 10) and 1.0 microliter 10 pmol/microliter primer Cas9-R1 (SEQ ID NO: 11), 6.0 microliter sterile water, and finally 2.0 microliter 50 ng/microliter of genomic DNA of the sample is added. The PCR reaction conditions were 95℃for 5 minutes, then 95℃for 30 seconds, 60℃for 1 minute, 72℃for 1 minute for 30 cycles, and finally 72℃for 7 minutes and maintained at 4 ℃. The PCR amplification product was separated by 1% agarose gel electrophoresis, and samples successfully amplified the 540 bp long fragment specific for the Cas9 gene (SEQ ID NO: 12) were judged to be transgene positive. Similar PCR analysis can also be performed by using primers Hyg-F1 and Hyg-R1 (SEQ ID NO:13, 14) specific to the vector hygromycin gene, and samples successfully amplified with the HygR gene-specific 402 bp long fragment (SEQ ID NO: 15) are judged to be transgenic positive.
A target fragment (SEQ ID NO: 18) of 1329 bp length near the editing site of the OsDTH7-G1 gene can be amplified from a Cas9 positive sample by a similar PCR amplification method using OsDTH7 (LOC-Os 07G 49460) specific primers OsDh7-F2 and OsDh-R2 (SEQ ID NO:16, 17). Using OsDTH gene (LOC-Os 08G 07740) specific primers OsDh8-F1 and OsDh-R1 (SEQ ID NO:19, 20), a target fragment (SEQ ID NO: 21) of 1329 bp length near the OsDTH-G1 gene editing site can be amplified. Using OsGhd gene (LOC_Os 07G 15770) specific primers OsG7-F4 and OsG7-R4 (SEQ ID NO:22, 23), a target fragment (SEQ ID NO: 24) of OsGhd7-G3 or its complementary strand OsGhd7-G3R, which is 826 bp long near the gene editing site, can be amplified. Using OsHd gene (LOC_Os 06G 16370) specific primers OsHd1-F3 and OsHd1-R3 (SEQ ID NO:25, 26), a target fragment 801. 801 bp long near the OsHd-G1 gene editing site (SEQ ID NO: 27) can be amplified. These PCR fragments were directly sequenced in one direction using the above PCR primers, respectively, and if a set of peaks exists near the expected gene editing site, even the downstream DNA could not be sequenced accurately, i.e., it was judged that DNA sequence editing occurred at that site. The PCR fragment TOPO of the edited sample is cloned into pCR2.1 vector (Thermo FISHER SCIENTIFIC), and 3 clones are sent to sequence each sample, so that specific DNA editing sequence changes can be accurately analyzed.
The transformation efficiency of each gene editing vector on the beyond-light rice is not high, the identification is the actual transformation event by carrying out the PCR analysis on the callus samples of the resistance event obtained by screening, the gene editing efficiency reaches 100% by confirming that the events are all subjected to gene editing by the sequencing analysis of the target gene fragments, and the analysis results are summarized in Table 2.
TABLE 2 genetic transformation and editing efficiency of the regulatory genes for the growth period of the genetic editing light
| Experiment number | Gene editing vector | Number of infected calli | Resistance event | Conversion efficiency% | PCR Positive event | Gene editing event | Gene editing efficiency% | Regeneration event |
| R55 | KF88 | 400 | 1 | 0.25 | 1 | 1 | 100 | 4 |
| R66 | KF114 | 500 | 9 | 1.8 | 9 | 9 | 100 | 6 |
| R81 | KF99 | 1000 | 5 | 0.50 | 5 | 5 | 100 | 3 |
| R89 | KF99 | 1800 | 2 | 0.11 | 2 | 2 | 100 | 2 |
4. Screening of offspring plants from Gene editing events
Transplanting the T0 plant for realizing gene editing into a greenhouse for culture, naturally selfing, setting maturing, harvesting the T1 generation seeds, and sowing in the greenhouse at proper time. When seedlings were about three leaves in size, a small amount of leaf samples were taken to extract genomic DNA, and detailed PCR detection and target gene sequencing analysis were performed (FIG. 3). Because rice selfing follows Mendelian genetic segregation, the genome-inserted gene editing vector fragment and the edited target gene are not generally on the same chromosome, and are separated from each other independently, part of T1 plants no longer contain any editing vector DNA and are non-transgenic offspring, wherein the target genes of part of the plants keep an editing state, and about 16T 1 plants possibly have one plant which does not carry the gene editing vector fragment and is subjected to homozygous editing. If stable non-transgenic homozygous editing plants can be obtained in the T1 generation, the analysis and identification experiment can be expanded after the T2 generation seeds are harvested. If the non-transgenic homozygous edited plant is not obtained, the T2 generation seeds of the heterozygous edited plant are planted in a greenhouse, natural genetic separation is carried out, and the non-transgenic homozygous edited T2 generation plant is selected through similar analysis.
The isolated T1 progeny plants were first PCR amplified using the same Cas9 and HygR gene specific primers described above, and checked for whether the plants still carried the gene editing vector fragment, and non-transgenic progeny plants that could not be amplified were selected (fig. 3). Then, a target fragment of the gene edit region 1329 bp length was amplified using the same OsDTH7 gene-specific primers OsDh7-F2 and OsDh7-R2 as described above (FIG. 3), and the result was directly subjected to one-way sequencing analysis using the OsDh7-F2 primer, and compared with the wild-type sequence of the OsDTH7 gene (SEQ ID NO: 18), thereby confirming the OsDTH7 gene edit condition. A target fragment 1329 bp long in the gene editing region was amplified using the same OsDTH gene-specific primers OsDh8-F1 and OsDh-R1 as described above (FIG. 3), and the result was directly subjected to one-way sequencing analysis using the OsDh8-F1 primer, and compared with the wild type sequence of the OsDTH gene (SEQ ID NO: 21), thereby confirming the condition of OsDTH gene editing. A target fragment having a length of 826 bp in the gene editing region was amplified using the same OsGhd gene-specific primers OsG7-F4 and OsG7-R4 as described above (FIG. 3), and the result was directly subjected to one-way sequencing analysis using the OsG-F4 primer, and the result was aligned with the wild type sequence of OsGhd gene (SEQ ID NO: 24), whereby the condition of OsDTH8 gene editing was confirmed. A target fragment of gene editing region 801 bp was amplified using the same OsHd1 gene-specific primers OsHd1 to F3 and OsHd1 to R3 as described above (FIG. 3), and was analyzed by direct unidirectional sequencing using OsHd1 to F3 primers, and the result was aligned with the wild type sequence of OsHd1 gene (SEQ ID NO: 27), thereby confirming the condition of gene editing of OsHd.
Combining the PCR amplification and target gene editing region DNA fragment sequencing analysis results, co-screening to obtain 4 independent families R55-1-2-9, R55-1-4-10, R55-1-5-5, R55-1-6-17 of non-transgenic homozygous editing R55 plants, wherein the genes of OsGhd7 and OsHd are edited (SEQ ID NO:28-31 and SEQ ID NO: 32-35), and the difference sequence only occurs at the expected editing sites (FIG. 4 a); 6 independent families of non-transgenic, homozygous edited R66 plants were obtained, osGhd genes were edited (SEQ ID NOS: 36-41), the differential sequence only occurred at the expected editing sites (FIG. 4 b), 5 independent families of non-transgenic, homozygous edited R81 and R89 plants were obtained, osDTH and OsDTH genes were all edited (SEQ ID NOS: 42-46 and SEQ ID NOS: 47-51), and the differential sequence only occurred at the expected editing sites (FIG. 5). The target site editing profiles of the individual gene editing families are summarized in Table 3, where the R66 gene editing family OsGhd-G3R sites show the complementary strand sequences. Because the DNA sequence editing only occurs in the protein coding region where the expected editing site is located, namely, CRISPR-Cas9 mediated specific gene editing is performed, the frame shift mutation of each target gene is caused, the normal expression of each protein functional domain sequence is particularly blocked, and the functions of each gene in the edited rice family are effectively knocked out.
TABLE 3 target gene sequence variation of fertility gene editing families
5. Phenotypic analysis of offspring plants from gene editing events
The Heilongjiang province vertically spans ten geographic latitudes (about 43.5-53.5 degrees in North latitude), the ecological environment is extremely complex and various, and the annual heat accumulation number is divided into six heat accumulation zones or ecological areas from higher than 2700 ℃,2500-2700 ℃,2300-2500 ℃,2100-2300 ℃ and 2100-1900 ℃ to lower than 1900 ℃, each area is reduced by about 200 ℃ compared with the last heat accumulation zone, and the maturation period is correspondingly reduced by about 7 days. The rice experimental base of the reclamation and seed-enlarging industry is arranged in the Archimedes area of Harbin, is positioned at the north of 45 degrees north latitude and belongs to the first heat accumulation zone. The non-transgenic homozygous editing material is planted and observed in a special land block in an isolated mode, each material is planted in a single row or in a partition mode, the plant spacing is 10 cm, each row is about 45 plants, meanwhile, wild type parents are planted in the same area as a control, and comprehensive field performance and growth period change are observed. The rice variety generally suitable for being planted in the Archimedes is in heading and flowering in the last ten days of 7 months to 8 months, the date that about 10% of the rice head ends of the plants in the whole area or row of each material are exposed out of leaf sheaths is the heading period of the family, the date that about 50% of the rice head ends in the whole area or row are exposed out of leaf sheaths is the heading period, and the date that about 80% of the rice head ends in the whole area or row are exposed out of leaf sheaths is the heading period. And calculating and recording the days required by heading and flowering according to the planting date and the heading period of each material, and comparing with a control to judge whether the growth period is effectively shortened.
And planting R55 non-transgene, homozygous edited T2 separation offspring in a special isolated plot of a rice experimental base in an Alcity area of Harbin in 2021, and planting R66, R81 and R89 non-transgene, homozygous edited T2 separation offspring in 2022. Each family is transplanted with 5 meters long single row, the plant spacing is 10 cm, 45 plants are all planted, the wild type is used as a control, comprehensive field performance and growth period change are observed, each gene editing family grows and develops normally, the successive heading is started in the next ten days of 7 months, the date that the leaf sheath is exposed at the top end of the rice head of about 10% of the plants in the whole row is recorded as the heading period of the family, the day of heading and flowering of each material is calculated according to the planting date and heading period of each material, and each editing material needs about 100 days from planting to heading (see table 4). The main cultivation area of the light is located in the middle and south of Japan of about 33-38 degrees of North latitude, the growth period is long, the wild type planted on 18 days of 4 months is light as a contrast, the heading is not started until 10 days of 9 months, 145 days are required from the planting to heading, and enough time is not needed to finish the fruiting before the frost is formed on the beginning of 10 months.
Since each editing family can heading and bloom in the next ten days of 7 months, the required time is shortened by up to about 45 days compared with the comparative wild type, the editing effect is extremely remarkable, and all four growth period control genes of DTH7, DTH8, ghd7 and Hd1 do not need to be knocked out simultaneously, and only one Ghd7 gene or two genes of DTH7 and DTH8 need to be knocked out to achieve similar effect (see Table 4), unexpected, and adverse side effects caused by excessive gene knockouts can be avoided as much as possible.
Each editing family effectively shortens the growth period, and can be fully planted in the first or even the second heat accumulating zone of Heilongjiang and matured and firm. But the phenotype difference of each editing combination is obvious while the growth period is obviously shortened, and some materials such as R55 family plants editing Ghd7 and Hd1 genes are uneven in heading, small in inflorescence, firm and sparse and extremely easy to lodge; r66 family plants only editing Ghd7 genes have small inflorescences, are firm and sparse, and seriously influence the yield; whereas R81 and R89 family plants editing the DTH7 and DTH8 genes grew normally, no significant phenotypic defect was observed for the current best editing combination, and could be harvested at 10 months of pre-frost maturity (FIG. 6).
TABLE 4 flowering phase Change in fertility Gene editing families
| Gene editing family | Sowing time | Initiation of spike period | Days required | Harvest time |
| R55-1-2-9 | 2021.4.16 | 2021.7.24 | 99 | 2021.10.8 |
| R55-1-4-10 | 2021.4.16 | 2021.7.25 | 100 | 2021.10.8 |
| R55-1-5-5 | 2021.4.16 | 2021.7.23 | 98 | 2021.10.8 |
| R55-1-6-17 | 2021.4.16 | 2021.7.25 | 100 | 2021.10.8 |
| Average of | 99.25 | |||
| R66-1-1-4 | 2022.4.18 | 2022.7.21 | 94 | 2022.10.8 |
| R66-1-1-11 | 2022.4.18 | 2022.7.25 | 98 | 2022.10.8 |
| R66-2-1-23 | 2022.4.18 | 2022.7.23 | 96 | 2022.10.8 |
| R66-3-2-19 | 2022.4.18 | 2022.7.27 | 100 | 2022.10.8 |
| R66-9-1-9 | 2022.4.18 | 2022.7.27 | 100 | 2022.10.8 |
| R66-2-1-27 | 2022.4.18 | 2022.7.25 | 98 | 2022.10.8 |
| Average of | 97.67 | |||
| R81-1-1-12 | 2022.4.18 | 2022.7.26 | 99 | 2022.10.8 |
| R81-2-1-9 | 2022.4.18 | 2022.7.27 | 100 | 2022.10.8 |
| R81-5-3-3 | 2022.4.18 | 2022.7.28 | 101 | 2022.10.8 |
| R89-2-3-1 | 2022.4.18 | 2022.7.28 | 101 | 2022.10.8 |
| R89-2-4-3 | 2022.4.18 | 2022.7.28 | 101 | 2022.10.8 |
| Average of | 100.4 | |||
| Surging light | 2022.4.18 | 2022.9.10 | 145 | Immature plant |
The gene editing families are novel germplasm resource materials created by a gene editing method, can be directly used as a fertility period improved strain for comprehensive property evaluation, and can also be used as a special material for fertility period improvement for conventional breeding improvement of other rice varieties. Specific primers can be designed according to the unique flowering control gene fragment editing sequence of each family, and specific PCR molecular markers can be developed for identifying and tracking offspring plants of each editing family.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Description of DNA sequence in the present invention:
SEQ ID NO 1: OsDTH7-G1 gRNA 20 bp
SEQ ID NO 2: OsDTH8-G1 gRNA 20 bp
SEQ ID NO 3: OsGhd7-G3 gRNA 20 bp
SEQ ID NO 4: OsGhd7-G3r gRNA 20 bp
SEQ ID NO 5: OsHd1-G1 gRNA 20 bp
SEQ ID NO 6: Synthesized tRNA-OsGhd7-G3-gRNA-tRNA-OsHd1-G1-gRNA-tRNA-OsDTH8-G1 fragment 483 bp
SEQ ID NO 7: synthesized tRNA-OsDTH7-G1-gRNA-tRNA-OsDTH8-G1 fragment 311 bp
SEQ ID NO 8: synthesized OsGhd7-G3r gRNA oligo 60 bp
SEQ ID NO 9: ZmU6-F2 oligo 22 bp
SEQ ID NO 10: Cas9-F1 oligo 24 bp
SEQ ID NO 11: Cas9-R1 oligo 20 bp
SEQ ID NO 12: Cas9 PCR fragment 540 bp
SEQ ID NO 13: Hyg-F1 oligo 22 bp
SEQ ID NO 14: Hyg-R1 oligo 22 bp
SEQ ID NO 15: HygR PCR fragment 402 bp
SEQ ID NO 16: OsDh7-F2 oligo 21 bp
SEQ ID NO 17: OsDh7-R2 oligo 22 bp
SEQ ID NO 18: Oryza sativa Japonica DTH7 gene LOC_Os07g49460 PCR fragment 1329 bp
SEQ ID NO 19: OsDh8-F1 oligo 22 bp
SEQ ID NO 20: OsDh8-R1 oligo 22 bp
SEQ ID NO 21: Oryza sativa Japonica DTH8 gene LOC_Os08g07740 PCR fragment 1329 bp
SEQ ID NO 22: OsG7-F4 oligo 24 bp
SEQ ID NO 23: OsG7-R4 oligo 22 bp
SEQ ID NO 24: Oryza sativa Japonica Ghd7 gene LOC_Os07g15770 PCR fragment 826 bp
SEQ ID NO 25: OsHd1-F3 oligo 22 bp
SEQ ID NO 26: OsHd1-R3 oligo 22 bp
SEQ ID NO 27: Oryza sativa Japonica Hd1 gene LOC_Os06g16370 PCR fragment 801 bp
SEQ ID NO 28: R55-1-2-9 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 821 bp
SEQ ID NO 29: R55-1-4-10 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 824 bp
SEQ ID NO 30: R55-1-5-5 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 824 bp
SEQ ID NO 31: R55-6-1-17 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 821 bp
SEQ ID NO 32: R55-1-2-9 edited event Hd1 gene LOC_Os06g16370 PCR fragment 799 bp
SEQ ID NO 33: R55-1-4-10 edited event Hd1 gene LOC_Os06g16370 PCR fragment 799 bp
SEQ ID NO 34: R55-1-5-5 edited event Hd1 gene LOC_Os06g16370 PCR fragment 798 bp
SEQ ID NO 35: R55-6-1-17 edited event Hd1 gene LOC_Os06g16370 PCR fragment 798 bp
SEQ ID NO 36: R66-1-1-4 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 827 bp
SEQ ID NO 37: R66-1-1-11 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 824 bp
SEQ ID NO 38: R66-2-1-23 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 828 bp
SEQ ID NO 39: R66-2-1-27 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 825 bp
SEQ ID NO 40: R66-3-2-19 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 813 bp
SEQ ID NO 41: R66-9-1-9 edited event Ghd7 gene LOC_Os07g15770 PCR fragment 827 bp
SEQ ID NO 42: R81-1-1-12 edited event DTH7 gene LOC_Os07g49460 PCR fragment 1276 bp
SEQ ID NO 43: R81-2-1-9 edited event DTH7 gene LOC_Os07g49460 PCR fragment 1330 bp
SEQ ID NO 44: R81-5-3-3 edited event DTH7 gene LOC_Os07g49460 PCR fragment 1325 bp
SEQ ID NO 45: R89-2-3-1 edited event DTH7 gene LOC_Os07g49460 PCR fragment 1330 bp
SEQ ID NO 46: R89-2-4-3 edited event DTH7 gene LOC_Os07g49460 PCR fragment 1330 bp
SEQ ID NO 47: R81-1-1-12 edited event DTH8 gene LOC_Os08g07740 PCR fragment 1328 bp
SEQ ID NO 48: R81-2-1-9 edited event DTH8 gene LOC_Os08g07740 PCR fragment 1031 bp
SEQ ID NO 49: R81-5-3-3 edited event DTH8 gene LOC_Os08g07740 PCR fragment 1327 bp
SEQ ID NO 50: R89-2-3-1 edited event DTH8 gene LOC_Os08g07740 PCR fragment 1323 bp
SEQ ID NO 51: R89-2-4-3 edited event DTH8 gene LOC_Os08g07740 PCR fragment 1330 bp 。
Claims (11)
1. The application of the DTH7 and DTH8 genes of the rice in changing the flowering period of the rice is edited simultaneously by a gene editing tool.
2. The use of claim 1, wherein the gene editing tool comprises a CRISPR/Cas9 system, and its derivative tools cytosine editor, adeno glance sideways at-or guide-editor, or a gene editing system based on a different Cas12 enzyme, or other gene editing tools.
3. The use according to claim 2, wherein the gene editing tool is a CRISPR/Cas9 system, the gene editing transformation vector designed based on the CRISPR/Cas9 system contains three gene expression units ZmU pro gRNA: atU-26 term,ZmUBI1 pro:SpCas9:PsE9 term and 35S pro:HYG:35S term,ZmU6 pro leaving BsaI cleavage sites between the gRNA, linearizing the vector and inserting one or more gRNA sequences by DNA ligase or Gibson cloning to form a gene editing vector for any target site of the DTH7 and DTH8 genes.
4. The use according to claim 3, characterized in that a plurality of grnas are designed for the recognition sites of different regions of the DTH7 and DTH8 genes, which are inserted between ZmU pro and the gRNA backbone of the gene-editing transformation vector by cloning after being interconnected by tRNA technology.
5. The use according to claim 4, wherein the designed gene editing transformation vector inserts between ZmU pro and gRNA a fragment of tRNA-OsDTH 7-G1-gRNA-tRNA-OsDTH-G1, which fragment has the sequence set forth in SEQ ID NO: shown at 7.
6. The use according to any one of claims 3 to 5, wherein the genetic transformation method for altering the flowering phase of rice comprises introducing the designed gene editing transformation vector into rice cells via agrobacterium mediation, and then selecting to obtain a non-transgenic, stable genetic homozygous editing rice line.
7. The use according to claim 6, wherein the genetic transformation method comprises the steps of:
s1: genetic transformation is carried out on the receptor rice cells to obtain regenerated plants;
s2: detecting whether gene editing of the regenerated plant is successful or not;
S3: and screening to obtain a progeny plant which can be inherited, is non-transgenic and is stably edited.
8. The use according to claim 7, wherein said step S1 comprises:
S1-1, inducing rice callus;
s1-2, subculturing rice callus;
s1-3 infection of callus;
s1-4, screening and identifying resistant callus;
S1-5, differentiating the callus into seedlings;
S1-6, rooting culture;
s1-7, domesticating and transplanting to obtain regenerated plants.
9. The use according to claim 8, wherein said step S2 comprises,
S2-1, collecting a resistant callus or a tender leaf tissue of a regenerated plant, extracting genomic DNA and carrying out PCR amplification;
S2-2, designing PCR primers covering a certain length of the upper and lower streams of a cutting site according to sequences of target genes DTH7 and DTH8 and expected cutting point positions of Cas9, and amplifying target gene fragments;
S2-3, judging whether the gene editing is successful or not according to the difference between the amplified target gene fragment and the target gene fragment which is not amplified by the genetically transformed plant.
10. Use of a progeny plant obtained by use according to any one of claims 7-9 in rice breeding.
11. The use according to claim 10, wherein the DTH7 genes in the progeny plants R81-1-1-12, R81-2-1-9, R81-5-3-3, R89-2-3-1, R89-2-4-3 obtained by screening are as shown in SEQ ID NOs: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO:45 and SEQ ID NO:46, the DTH8 gene is shown as SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO:50 and SEQ ID NO: 51.
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