CN118291533A - Method and application of gene editing to change rice flowering period - Google Patents

Abstract

The present invention belongs to the field of crops, and in particular, relates to a method and application of changing the flowering period of rice by gene editing. More specifically, the present invention provides an application of simultaneously editing rice DTH7 and DTH8 genes by gene editing tools in changing the flowering period of rice. The present invention establishes a CRISPR/Cas9-based plant gene editing system, designs and constructs a gene editing genetic transformation vector for rice flowering control genes DTH7 and DTH8, transforms rice by an Agrobacterium-mediated method, and realizes simultaneous multiple editing of nucleotide sequences of DTH7 gene target regions and DTH8 gene target regions of regenerated plants. The gene editing efficiency is as high as 100%, and finally a homozygous edited rice strain with a growth period shortened by more than 45 days, non-transgenic, and stable inheritance is obtained. Compared with editing other flowering control gene combinations, the flowering combination gene plants edited by the present invention grow and develop normally, and no significant phenotypic defects are observed.

Description

Method and application of gene editing to change rice flowering period

Technical Field

The present invention belongs to the technical field of crop gene editing breeding, and in particular relates to a method for changing the growth period of rice varieties by gene editing genes that affect rice flowering, and its application.

Background technique

It takes several months for crops such as rice to grow from seed sowing to grain harvesting. After germination, the first step is vegetative growth and development. After sensing environmental factors, especially changes in temperature and daylight length, the plants enter the reproductive growth stage to bloom, bear fruit, and mature. The length of the entire growth period or maturity period is an extremely important agricultural production trait. Some plants need a certain number of days of long-day stimulation to switch from vegetative growth to reproductive growth, bloom and bear fruit, and are long-day plants; while some plants need a certain number of days of short-day stimulation to bloom, and are short-day plants. As a short-day plant, rice is very sensitive to the length of daylight. Different varieties require a full maturity time ranging from about 120 days for early-maturing to about 150 days for late-maturing. Different ecological zones must select varieties with appropriate maturity periods. Varieties that mature too early cannot fully utilize the local sunshine and accumulated temperature, resulting in reduced production, while varieties that mature too late cannot mature in time before frost, which can cause a total loss of production in severe cases. my country's Northeast region, especially Heilongjiang Province, spans ten geographical latitudes with an extremely complex and diverse ecological environment. It is divided into six accumulated temperature zones according to the annual accumulated temperature, ranging from above 2700℃, 2500-2700℃, 2300-2500℃, 2100-2300℃, 2100-1900℃, to below 1900 degrees. Among them, the first to fourth accumulated temperature zones are suitable for rice cultivation, but special adapted varieties must be planted for each accumulated temperature zone.

As one of the most important agronomic traits of rice, the growth period has not only received special attention and application in breeding practice, but also the genetic and molecular mechanisms controlling it have been studied in depth. Flowering marks the transition of plants from vegetative to reproductive growth, directly determines the length of the growth period, and is a complex quantitative trait controlled by multiple gene loci and regulated by light and temperature in the environment. Through the study of natural variation, mutants, and transgenic rice, more than 70 flowering regulatory genes have been identified, among which Hd3a is the main flowering gene, which directly controls the expression of two developmental genes MADS14 and MADS15 to induce flowering. Under short-day conditions, Hd3a is positively regulated by Hd1 and Ehd1, while Hd1 senses the spectrum 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 the photoperiod through GI, while Ehd1 is directly or indirectly positively or negatively regulated by many genes. In addition, a few other genes can also directly affect the expression of Hd3a. Among them, Ghd7 and DTH8 inhibit flowering by negatively regulating Ehd1, and DTH7 (PRR37) inhibits flowering by directly negatively regulating Hd3a (Brambilla and Fornara, 2013, J Integrative Plant Biol 55: 410-418; Hori et al., 2016, Theor Appl Genet 29: 2241-2252).

The summer in high-latitude areas is short and suitable for plant growth, but the daily sunshine is long. After the transition to short-day sunshine, the temperature drops rapidly, making it difficult to meet the needs of late-maturing varieties for short-day sunshine and accumulated temperature. However, through long-term ecological adaptation breeding, early-maturing rice varieties adapted to different accumulated temperature zones have been selected. Genomic sequencing analysis found that these varieties often contain haplotypes of the above-mentioned major flowering regulatory gene mutations, revealing that these mutations are the genetic basis for early rice maturation. Haplotypes of different gene mutations have an additive effect, that is, the earlier maturing varieties have more mutations. Theoretically, if precise mutations are used to regulate the expression levels of these main flowering regulatory genes, it is possible to improve the excellent late-maturing varieties that do not contain early-maturing haplotypes to early-maturing varieties, retain other original excellent traits to the maximum extent, and quickly select early-maturing varieties that can adapt to a wider temperature zone (Zhang et al., 2015, New Phytologist 208: 1056–1066; Ye et al., 2018, Front Plant Sci 9: 35).

Gene editing technology uses the Cas9 endonuclease of CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats), the acquired immune system of Streptococcus pyogenes, to form a nucleoproteinase complex with the guide gRNA. The 20 bp-long RNA sequence in the gRNA and the edited DNA sequence can accurately identify the target site through base pairing, selectively cut off specific genes in the cell, and achieve gene editing through two inherent DNA repair processes in the cell. Non-homologous end joining (NHEJ) has high repair efficiency and does not require a template. The process of linking the cut point will introduce insertion or deletion mutations, thereby inactivating the gene. After genetic separation of the edited offspring, the final product does not contain any exogenous DNA and is a non-transgenic product. It can avoid the complex and expensive regulatory approval process and costs of transgenic products and is suitable for rapid production and application. The homology-dependent repair (HDR) pathway requires a pre-designed DNA fragment as a template, which can be inserted into a pre-selected site to modify the target gene. Although the repair is precise, the efficiency is very low (Jinek et al., 2012, Science 337: 816–821).

Gene editing technology has developed rapidly in recent years. Multiple gRNA synchronous expression technology can simultaneously perform efficient NHEJ editing of multiple genes and multiple sites (Hsieh-Feng and Yang 2020, aBIOTECH 1: 123–134); single-base editing technology includes cytosine base editor (CBE) and adenine base editor (ABE), which can achieve specific editing of single bases through irreversible C-T or A-G conversion, respectively (Komor et al., 2016, Nature 533: 420–424; Nishida et al., 2016, Science353: aaf8729; Gaudelli et al., 2017, Nature 551: 464–471); mutating SpCas9 to recognize NGH (H=A,C,T) other than NGG, pan-site editing of PAM sequences such as NGN (Hu et al., 2018, Nature 556: 57–63; Kleinstiveret al., 2015, Nature 523: 481–485; Nishimasu et al., 2018, Science 361: 1259–1262; Walton et al., 2020, Science 368：290–296); New gene editing enzymes such as Cas12a (Cpf1) have more diverse recognition sites and editing functions (Zetsche et al., 2015, Cell 163: 1–13; Murovec et al., 2017, Plant Biotechnol J 15：917–926); Prime editing technology has made breakthrough progress in precise gene editing, which can achieve various types of precise gene editing including base conversion, substitution, small fragment deletion, insertion, replacement, etc. (Anzalone et al., 2015, Cell 163: 1–13; Murovec et al., 2017, Plant Biotechnol J 15：917–926). al.,2019, Nature 576: 149–157). Various gene editing technologies have been widely used in the field of plant research, achieving knockout editing of many plants and many genes as well as a few precise design editing (Li et al., 2015 Plant Physiol 169: 960–970; Ma et al., 2016, Mol Plant 9: 961–974; Chen et al., 2019, AnnuRev Plant Biol 70: 667–97; Mao et al., 2019, Natl Sci Rev 6: 421-437).

Koshihikari, a famous Japanese rice variety, has been planted since 1953. In 1956, it became the main variety promoted in Xinxiang County and was officially named Koshihikari. With its excellent quality, even after more than 50 years of large-scale planting, it is still the largest rice variety planted and promoted in Japan. In 2016, the planting area accounted for 36.2% of the national rice planting area in Japan (Kobayashi et al., 2018, Rice 11: 15). It is worthy of introduction as a high-quality breeding resource material for improving the quality and other traits of Northeast my country's rice. However, Koshihikari has a long growth period, and it cannot bloom and bear fruit normally even in the first accumulated temperature zone with the highest accumulated temperature in Heilongjiang, which seriously restricts its breeding application. The present invention uses Koshihikari as the basic material, and targets four rice growth period control genes DTH7 (LOC_Os07g49460, also known as Hd2), DTH8 (LOC_Os08g07740, also known as Hd5), Ghd7 (LOC_Os07g15770, also known as Hd4), and Hd1 (LOC_Os06g16370) that inhibit flowering under long-day conditions. The gene editing technology is used to perform multiple combination editing on different sites of different genes in order to obtain improved varieties with shortened growth periods, and unexpected results are achieved. A method for effectively shortening the growth period of rice without affecting other favorable traits is invented.

Summary of the invention

The present invention establishes a CRISPR/Cas9-based plant gene editing system, designs and constructs gene editing genetic transformation vectors for rice flowering control genes DTH7 (LOC_Os07g49460) and DTH8 (LOC_Os08g07740), and transforms the rice variety Koshihikari using an Agrobacterium-mediated method, thereby achieving simultaneous multiple editing of nucleotide sequences in the target regions of the DTH7 gene and the DTH8 gene of the regenerated plants, with a gene editing efficiency of up to 100%, and ultimately obtaining a homozygous edited rice strain with a growth period shortened by more than 45 days, non-transgenic, and stable inheritance. Compared with editing other flowering control gene combinations, the flowering combination gene plants edited by the present invention grow and develop normally, and no significant phenotypic defects are observed.

The present invention covers the complete process from editing vector design and construction to gene editing confirmation of transformed plant offspring. The main steps of the technical scheme used include controlling the selection of target trait genes, designing and constructing gene editing transformation vectors, genetic transformation of rice cultivars, molecular biological analysis of transformation events and gene editing confirmation, and screening of non-transgenic homozygous edited offspring of regenerated plants, thereby realizing efficient editing of target trait genes using gene editing methods.

Specifically, the technical solution provided by the present invention is as follows:

The main implementation scheme provided by the present invention is to simultaneously edit rice DTH7 and DTH8 genes through gene editing tools and apply them to change the flowering period of rice and shorten the growth period of rice.

As some embodiments, the gene editing tool includes the CRISPR/Cas9 system, and its derivative tools cytosine editor, adenine editor or guide editor, or a gene editing system 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: ZmU6 pro:gRNA:AtU6-26 term, ZmUBI1 pro:SpCas9:PsE9 term and 35S pro:HYG:35S term. A BsaI restriction site is left between ZmU6 pro and gRNA. After the vector is linearized, one or more gRNA sequences are inserted using DNA ligase or Gibson cloning method to form a gene editing vector for any target site of the DTH7 and DTH8 genes.

Furthermore, gRNAs were designed for recognizable sites in different regions of the DTH7 and DTH8 genes, and multiple gRNAs were cross-linked through tRNA technology and then inserted between the ZmU6 pro and gRNA backbone of the gene editing transformation vector using a cloning method.

Preferably, the designed gene editing transformation vector inserts an artificially synthesized tRNA-OsDTH7-G1-gRNA-tRNA-OsDTH8-G1 fragment between ZmU6 pro and gRNA in an interactive arrangement of tRNA and gRNA backbones, and the sequence of the fragment is shown in SEQ ID NO: 7.

Furthermore, the genetic transformation method for changing the flowering period of rice is to introduce the designed gene editing transformation vector into rice cells through Agrobacterium-mediated, and then screen to obtain non-transgenic, stably inherited homozygous edited rice varieties.

Specifically, the genetic transformation method comprises the following steps:

S1: Genetic transformation of recipient rice cells to obtain regenerated plants;

S2: Detect whether the gene editing of the regenerated plants is successful;

S3: Screening for heritable, non-transgenic and stably edited offspring plants.

Furthermore, the step S1 includes: S1-1 induction of rice callus; S1-2 subculture of rice callus; S1-3 infection of callus; S1-4 screening and identification of resistant callus; S1-5 differentiation of callus into seedlings; S1-6 rooting culture; and S1-7 acclimatization and transplantation to obtain regenerated plants.

Furthermore, the step S2 includes:

S2-1 Collect resistant callus or young leaf tissue of regenerated plants, extract genomic DNA for PCR amplification;

S2-2 Based on the sequences of the target genes DTH7 and DTH8 and the expected Cas9 cut-off point, PCR primers covering a certain length upstream and downstream of the cut-off site were designed to amplify the target gene fragments;

S2-3 determines whether the gene editing is successful based on the difference between the amplified target gene fragment and the target gene fragment amplified in the plant that has not been genetically transformed.

Furthermore, the offspring plants obtained by the present invention can be used in rice breeding. More specifically, the offspring plants with stable traits, normal growth and development, early flowering period, and shortened growth period screened after gene editing can be further used for gene editing breeding, molecular breeding or hybrid breeding to enable the offspring plants to obtain other target traits.

Furthermore, 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 shown as SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively, and the DTH8 genes are shown as SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the structure and gene editing sites of four flowering control genes in rice (in the figure: (a) shows the coding structure of the four flowering control genes in turn. DTH7 (LOC_Os07g49460.1) consists of 10 exons and 9 introns. A site in exon 4 was selected to design OsDTH7-G1 gRNA. The upstream and downstream primers OsDh7-F2 and OsDh7-R2 were designed for PCR specific amplification and sequencing analysis of the 1329 bp DNA fragment in the editing region; DTH8 (LOC_Os08g07740.1) has a simple structure and no introns. A site upstream of its coding sequence was selected to design OsDTH8-G1 gRNA. The upstream and downstream primers OsDh8-F1 and OsDh8-R1 were designed for PCR specific amplification and sequencing analysis of the 1329 bp editing region. DNA fragments; Ghd7 (LOC_Os07g15770) consists of 2 exons and 1 intron, and a site in exon 3 was selected to design OsGhd7-G3 and OsGhd7-G3r gRNAs. The upstream and downstream primers OsG7-F4 and OsG7-R4 were designed for PCR specific amplification and sequencing analysis of the 826 bp DNA fragment in the editing region; Hd1 (LOC_Os06g16370) consists of 2 exons and 1 intron, and a site in exon 2 was selected to design OsHd1-G1 gRNAs. The upstream and downstream primers OsHd1-F3 and OsHd1-R3 were designed for PCR specific amplification and sequencing analysis of the 801 bp DNA fragment in the editing region; dth8-a, dth8-b, ghd7-1,ghd7-b, hd1-a, hd1-b is another publicly known gRNA recognition site, all located outside the functional domain; (b) The protein functional domain structures of the four flowering control genes, showing the relative positions of each gRNA).

Figure 2 is a schematic diagram of the gene editing transformation vector KF99 (in the figure: T-DNA RB is the right border of T-DNA; ZmU6pro is the promoter of the maize U6 snRNA gene; AtU6 term is the terminator of the Arabidopsis U6 snRNA gene; ZmUBI1 pro is the promoter of the maize ubiquitin gene; Cas9 is the nuclease of the acquired immune system CRISPR of Streptococcus pyogenes; PsE9 term is the terminator of the pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit E9 protein gene; CaMV35S pro is the promoter of the cauliflower mosaic virus 35S; HygR is the hygromycin phosphotransferase; CaMV35S term is the terminator of the cauliflower mosaic virus 35S; T-DNA LB is the left border of T-DNA).

Figure 3 is a PCR identification diagram of T1 offspring plants of gene editing events (in the figure: using the genomic DNA of leaves of representative T1 plants of each event as a template, the expected 540 bp fragment was amplified by PCR using the Cas9 gene specific primers Cas9-F1 and Cas9-R1, or the expected 402 bp fragment was amplified by PCR using the HygR gene specific primers Hyg-F1 and Hyg-R1, and the plants that failed to amplify were non-transgenic plants; then, the expected fragment of about 1329 bp was amplified by PCR using the OsDTH7 gene editing region specific primers OsDh7-F2 and OsDh7-R2, the expected fragment of about 1329 bp was amplified by PCR using the OsDTH8 gene editing region specific primers OsDh8-F1 and OsDh8-R1, and the expected fragment of about 826 bp was amplified by PCR using the OsGhd7 gene editing region specific primers OsG7-F4 and OsG7-R4. bp fragment, the expected fragment of about 801 bp was amplified by PCR using the OsHd1 gene editing region specific primers OsHd1-F3 and OsHd1-R3, and the editing status of each DNA sequence was analyzed by sequencing; the lengths of the marker DNA fragments were 2000, 1000, 750, 500, and 250 bp, respectively; wt and wt+KF88 or KF114 or KF99 were the wild-type Koshihikari negative and positive controls with a small amount of plasmid DNA added, respectively; (a) R55 gene-edited family; (b) R66 gene-edited family; (c) R81 and R89 gene-edited families).

Figure 4 shows the sequencing diagram of some DNA sequences in the gene target region of different gene-edited lines (in the figure: (a) DNA sequence near the OsGhd7-G3 and OsHd1-G1 editing sites of the R55 gene-edited lineage; (b) DNA sequence near the OsGhd7-G3r editing site of the R66 gene-edited lineage).

Figure 5 shows the DNA sequence diagram near the editing sites of OsDTH7-G1 and OsDTH8-G1 in the R81 and R89 gene-edited lines.

Figure 6 shows the growth status of wild-type Koshihikari and gene-edited family R81-1-1-12 plants at the Rice Experimental Base in Acheng District, Harbin on August 11, 2022.

Detailed ways

The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the embodiments. The experimental methods in the following embodiments that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The reagents and raw materials used in the present invention are all commercially available.

Example 1

1. Construction of gene editing transformation vector

According to the analysis of the Oryza sativa v7.0 rice genome sequencing database collected and annotated by the U.S. Department of Energy (https://phytozome.jgi.doe.gov/pz/portal.html), the rice flowering control gene DTH7 (LOC_Os07g49460, Hd2) has a complex gene structure, consisting of 10 exons and 9 introns (Figure 1a), with a total length of 12519 bp including 5'UTR and 3'UTR (LOC_Os07g49460|Chr7:29616704..29629223forward). The expected coding sequence starts from the third exon, is 2229 bp long, and encodes a protein containing 742 amino acid residues. According to the retrieval results of the protein sequence of the National Center for Biotechnology Information of the United States (https://blast.ncbi.nlm.nih.gov/Blast.cgi), its amino terminus contains the Response regulator responsible for regulating signal reception. The receiver domain (amino acids 64-174) has a CCT motif containing a nuclear localization signal at the carboxyl terminus (amino acids 682-724, Figure 1b), which is a transcriptional regulatory factor.

The rice flowering control gene DTH8 (LOC_Os08g07740, Hd5) has a simple gene structure and no introns (Figure 1a). The total length including the 5’UTR and 3’UTR is only 1718 bp (LOC_Os08g07740|Chr8:4333716..4335434reverse). The coding sequence is 894 bp long, encoding a 297 amino acid residue long protein containing a histone-like transcription factor CBF/NF-Y domain (amino acids 61-126), which is a pseudo-histone transcription factor.

The rice flowering control gene Ghd7 (LOC_Os07g15770, Hd4) consists of 3 exons and 2 introns (Figure 1a). Excluding the 5’UTR and 3’UTR, the total length is 2784 bp (LOC_Os07g15770|Chr7:9152401..9155185 reverse). The coding sequence is 774 bp long, encoding a protein containing 257 amino acid residues. The carboxyl terminus has a CCT motif containing a nuclear localization signal (amino acids 190-232, Figure 1b), which is a transcriptional regulatory factor.

The rice flowering control gene Hd1 (LOC_Os06g16370, Hd1) consists of 2 exons and 1 intron (Figure 1a). Excluding the 5’UTR and 3’UTR, the total length is 2285 bp (LOC_Os06g16370|Chr6:9336358..9338643forward). The coding sequence is 1188 bp long, encoding a protein with 395 amino acid residues. Its amino terminus contains a B-Box-type zinc finger (amino acids 33-77) responsible for regulatory signal reception, and its carboxyl terminus has a CCT motif containing a nuclear localization signal (amino acids 326-369, Figure 1b), which is a transcriptional regulatory factor.

In this article, "Os" is the abbreviation of Oryza sativa rice, that is, rice DTH7, rice DTH8, rice Ghd7 and rice Hd1 gene sequences, abbreviated as OsDTH7, OsDTH8, OsGhd7 and OsHd1 gene sequences.

Based on the above OsDTH7, OsDTH8, OsGhd7 and OsHd1 gene sequences, the coding sequences of their respective important functional domains and nearby regions were selected, and the DNA analysis software Geneious Prime (GraphPad Software LLC) was used to screen the N(20)NGG sequence as a potential SpCas9 gRNA target site, and the gRNA with the best expected activity was selected (Doench et al., 2016, Nat Biotechnol 34: 184–191). Taking into account their location, base sequence, expected editing change effect, etc., the gRNA recognition sequences OsDTH7-G1, OsDTH8-G1, OsGhd7-G3 and their complementary sequences OsGhd7-G3r, and OsHd1-G1 (SEQ ID NO: 1-5, Figure 1a) were designed respectively. Although simply designing gRNA in the upstream region of the OsDTH8, OsGhd7, and OsHd1 genes can also achieve gene editing (Figure 1) and obtain a phenotypic effect of shortening the growing period (Cui et al., 2019, Theor Appl Genet 132: 1887-1896; Zhou et al., 2024, Plant Biotechnol J22: 751-758), the gene editing efficiency and the phenotypic effects obtained vary. It is necessary to edit multiple target genes at the same time to obtain the cumulative effect of significantly shortening the growing period. Multi-gene editing often affects other important agronomic traits of rice, leading to growth and development disorders or reduced yield, and cannot be applied in breeding practice.

Beidahuang Kenfeng Seed Industry independently developed a rice-specific gene-editing Agrobacterium transformation DNA vector, which contains three gene expression units: ZmU6 pro:gRNA:AtU6-26 term, ZmUBI1pro:SpCas9:PsE9 term, and 35S pro:HYG:35S term. A BsaI restriction site is left between ZmU6 pro and gRNA. The vector can be linearized and one or more gRNA sequences can be inserted using DNA ligase or Gibson cloning to form a basic gene editing vector for any target site (Ma Rui et al., patent publication number CN113604501B). For specific sequence information, please refer to the KF80 transformation vector in patent CN113604501B "Gene editing method and application of aroma control gene in improved indica rice strains". Each gene editing transformation vector described in the present invention is constructed based on the disclosed basic gene editing vector.

The designed gene editing vector KF88 contains the tRNA-OsGhd7-G3-gRNA-tRNA-OsHd1-G1-gRNA-tRNA-OsDTH8-G1 fragment (SEQ ID NO: 6) artificially synthesized with the tRNA and gRNA backbones arranged alternately, and the two ends of the fragment have 20 bp additional sequences homologous to the 3' end of the ZmU6 promoter of the basic gene editing vector and the 5' end of the gRNA backbone; the designed gene editing vector KF99 (Figure 2) contains the tRNA-OsDTH7-G1-gRNA-tRNA-OsDTH8-G1 (SEQ ID NO: 7) fragment artificially synthesized with the tRNA and gRNA backbones arranged alternately, and the two ends of the fragment have the same 20 bp additional sequences homologous to the ZmU6 promoter of the basic gene editing vector. The 3' end and the 5' end sequence of the gRNA backbone are homologous; the designed gene editing vector KF114 contains only one gRNA, OsGhd7-G3r, and only needs to synthesize a short nucleotide with the same 20 bp additional sequence at both ends (SEQ ID NO: 8); the three synthesized DNA fragments or short nucleotides were inserted between the ZmU6 promoter and the gRNA backbone of the basic gene editing vector through the NEBuilder cloning method (New England Biolabs), and the gene editing vectors KF88, KF99 and KF114 were constructed respectively. Each gene editing vector was sequenced with the ZmU6 promoter-specific primer ZmU6-F2 (SEQIDNO: 9), and after confirmation, it was transferred to the rice transformation Agrobacterium strain EHA105 and stored in a -80℃ refrigerator for future use.

The later experimental results of the present invention show that the editing efficiency of each designed gRNA site is as high as 100%, and an unexpected phenotypic effect is achieved. Only by knocking out 1 or 2 genes, the flowering period of Koshihikari in Acheng area of Harbin is advanced by about 45 days, and normal fruiting and maturity are achieved. In addition, some editing combinations are not found to have adverse effects on other traits, thus inventing a method for quickly and effectively shortening the growth period of rice without affecting other traits.

2. Genetic transformation of japonica rice varieties

Gene editing can only be achieved by transforming the gene editing vector into cells for expression. Using the Agrobacterium-mediated transformation method, healthy mature seeds of Koshihikari rice were used as explants. The vectors KF88 for editing OsGhd7, OsHd1 and OsDTH8 genes, KF114 for editing OsGhd7 genes, and KF99 for editing OsDTH7 and OsDTH8 genes were transformed into cells respectively to accurately modify the DNA sequences near the recognition sites of the above target genes. The various basic culture medium formulas used in the genetic transformation experiments are listed in Table 1.

Table 1 Composition and preparation method of basic culture medium used in Agrobacterium genetic transformation experiment of japonica rice

Culture medium name Formula composition and preparation method Agrobacterium culture medium YEP NaCl 5 g/L, yeast extract 10 g/L, tryptone 10 g/L, pH 7.0. Add 1.2% agar powder to make solid YEP medium, sterilize at 121 ℃ for 20 minutes. Induction medium 10× N6 basic medium 3.98 g/L, 200× N6 vitamins 5 ml/L, sucrose 30 g/L, acid hydrolyzed casein 1 g/L, proline 4 g/L, auxin 2,4-D 2.5 mg/L, plant gel (Wako) 4 g/L, pH 5.8, sterilization at 121℃ for 20 min. Infection suspension culture medium 100 ml of 10× AAI macroelements, 1 ml of 1000× AAI trace elements, 10 ml of 100× iron salts, 5 ml of 200× AAI vitamins, 50 ml of 20× AAI amino acids, 68.5 g/L of sucrose, 36 g/L of glucose, 0.5 g/L of acid hydrolyzed casein, pH 5.2, sterilize at 121 ℃ for 20 minutes, add 1 ml of 1000× acetosyringone after cooling. Co-culture medium 10× N6 basic medium 3.98 g/L, 200× N6 vitamin 5 ml/L, sucrose 30 g/L, glucose 10 g/L, auxin 2,4-D 2.5 mg/L, plant gel 4 g/L, pH 5.2, sterilize at 121℃ for 20 minutes, add 1000× acetosyringone 1 ml after cooling. Screening medium 10× N6 basic medium 3.98 g/L, 200× N6 vitamins 5 ml/L, sucrose 30 g/L, acid hydrolyzed casein 1 g/L, proline 4 g/L, auxin 2,4-D 2.5 mg/L, plant gel 4 g/L, pH 5.8, sterilize at 121℃ for 20 minutes, add carbenicillin 400 mg/L and hygromycin 50 mg/L after cooling. Differentiation medium 10× MS basic 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, pH 5.8, sterilization at 121 ℃ for 20 minutes. Rooting medium 10× MS basic medium 2.37 g/L, sucrose 20 g/L, plant gel 3 g/L, pH 5.8, sterilization at 121 ℃ for 20 minutes. 200× N6 Vitamins 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, divided into portions and stored at -20°C for later use. 1000×Acetosyringone Dissolve 0.392 g of acetosyringone in 10 ml of DMSO (dimethyl sulfoxide), filter through a 0.2 μm filter membrane for sterilization, and store at -20°C for later use. 10×AAI macroelements Magnesium sulfate MgSO ₄ .7H ₂ O 5 g/L, calcium chloride CaCI ₂ .2H ₂ O 1.5 or CaCI ₂ 1.14 g/L, sodium dihydrogen phosphate NaH ₂ PO ₄ .H ₂ O 1.5 or NaH ₂ PO ₄ .2H ₂ O 1.7 g/L, potassium chloride KCl 29.5 g/L, packaged and stored at -20°C for future use. 1000×AAI trace elements Manganese sulfate MnSO ₄ .4H ₂ O or MnSO ₄ . H ₂ O 10 or 7.58 g/L, zinc sulfate ZnSO ₄ .7H ₂ O 2 g/L, boric acid H ₃ BO ₃ 3 g/L, potassium iodide KI 0.75 g/L, sodium molybdate Na ₂ MoO ₄ .2H ₂ O 0.25 g/L, cobalt chloride CoCl ₂ .6H ₂ O 0.025 g/L, copper sulfate CuSO ₄ .5H ₂ O 0.025 g/L, divide into portions and store at -20°C for later use. 200 × AAI vitamins Inositol 20 g/L, thiamine hydrochloride 2 g/L, nicotinic acid 0.2 g/L, pyridoxine hydrochloride 0.2 g/L, divided into portions and stored at -20°C for later use. 200×AAI amino acids Glutamine 17.5 g/L, aspartic acid 5.3 g/L, arginine 3.48 g/L, glycine 0.15 g/L, divided and stored at -20°C for later use. 100 x Iron salt 600 ml 2.78 g of ferrous sulfate FeSO ₄ .7H ₂ O was dissolved in 300 ml of water, 3.73 g of Na ₂ EDTA.2H ₂ O was heated to about 70°C and dissolved in 300 ml of water, and the two solutions were mixed in equal amounts and stored at 4°C for later use. 2,4-D1 mg/ml Dissolve 100 mg of 2,4-D in a small amount of 1N sodium hydroxide, make up to 100 ml, and store at 4°C. 6-BA 1 mg/ml Dissolve 100 mg of 6-BA in a small amount of 1N sodium hydroxide, make up to 100 ml, and store at 4°C. NAA 1 mg/ml Dissolve 100 mg of NAA in a small amount of 1N sodium hydroxide, make up to 100 ml, and store at 4°C. IAA 1 mg/ml Dissolve 100 mg of IAA in a small amount of 1N sodium hydroxide, make up to 100 ml, and store at 4°C. Zeatin 1 mg/ml Dissolve 100 mg of zeatin in 95% ethanol, make up to 100 ml, and store at 4°C. Hygromycin 50 mg/ml Dissolve 5 g of hygromycin in 100 ml of water, filter through a 0.22 μm filter to sterilize, divide into 2 ml centrifuge tubes, and store at -20°C. Kanamycin 50 mg/ml Dissolve 5 g of kanamycin in 100 ml of water, filter through a 0.22 μm filter membrane for sterilization, divide into 2 ml centrifuge tubes, and store at -20°C. Carbenicillin 250 mg/mL Dissolve 5 g of carbenicillin in 20 ml of water, filter through a 0.22 μm filter membrane for sterilization, divide into 2 ml centrifuge tubes, and store at -20°C. 1N Sodium hydroxide Dissolve 5.6 g of sodium hydroxide in 100 ml of water and store at room temperature.

The specific operation process is as follows:

1. Induction of rice callus: For each transformation experiment, pick about 200 mature, plump, healthy and clean rice seeds, peel off the husk, place in a 100 ml sterile glass bottle, wash with 75% ethanol three times, add equal volumes of sterile water and 10% sodium hypochlorite NaClO, two drops of Tween 20, shake and sterilize for 20 minutes, wash with sterile water 3-5 times until the foam is washed off. Use sterile filter paper to absorb the moisture on the surface of the seeds, place them flat on the induction medium, and culture them in a 33°C constant temperature incubator in the dark for 3-4 weeks until callus grows from the embryo.

2. Subculture of rice callus: Select and peel off the healthy callus with vigorous growth, place it on a new induction medium and subculture it for 1-2 weeks to expand and maintain the vitality of the callus.

3. Infection of callus tissue: Take out the glycerol storage tube of Agrobacterium carrying the target gene vector from the -80℃ low-temperature refrigerator, take a small amount of Agrobacterium and streak it on a solid YEP medium plate containing 50 mg/L kanamycin, and culture it in the dark at 28℃ for 2-3 days. Pick a single colony and inoculate 5 ml of YEP liquid medium for dark culture at 28℃ overnight. Take a small amount of bacterial liquid to extract plasmid DNA for vector-specific PCR detection to confirm that the strain carries the target gene vector. At the same time, take part of the bacterial liquid to apply YEP medium plate for culture and observation (the remaining culture medium can be temporarily stored at -80℃ for direct use later). The Agrobacterium used for infection must be free of contamination by other bacteria. The whole plate must be smooth and free of granular or other colored fungi or molds to ensure the purity of the obtained Agrobacterium, and strict aseptic operation must be performed to ensure no contamination in subsequent work.

After confirming that the Agrobacterium plate is free of contamination, directly scrape the bacteria into a 100 ml Erlenmeyer flask containing 25 ml of suspension infection medium, and culture it on a 100-120 rpm rotary shaker at a constant temperature of 25°C for 2-3 hours. Take a sample to measure the OD ₆₀₀ value, and adjust the OD ₆₀₀ value of Agrobacterium to 0.1-0.2 with the suspension infection medium before using it for callus infection. Pick small granular healthy callus into a 250 ml sterile glass bottle, add Agrobacterium and shake it for 3-5 minutes, then use sterile filter paper to absorb the bacterial liquid on the surface of the callus, and place it on the filter paper on the surface of the co-culture medium. A sterile filter paper is placed on the co-culture medium in advance to prevent the growth of Agrobacterium, and co-culture is carried out in the dark at 28°C for 3 days.

4. Screening and identification of resistant callus: After 3 days of co-cultivation, transfer the rice callus to a sterilized 250 ml glass bottle, wash it several times with sterile water until the water is clear, and finally soak and wash it with carbenicillin at a final concentration of about 250 mg/L on a shaker at 100 rpm for 0.5 hours. Transfer the callus to a culture dish, use sterile filter paper to absorb the water on the surface of the callus, place it in a sterile clean bench to air dry for 2.5 hours, then divide the callus into small packages and place it on the screening medium, and culture it in a 33°C constant temperature incubator in the dark for 3-4 weeks.

The screening medium contains 400 mg/L carbenicillin to inhibit the growth of Agrobacterium, and 50 mg/L hygromycin to select transformed cells. Non-transformed cells stop growing and gradually die on the screening medium. After 3-4 weeks of culture, the successfully transformed cells will grow resistant callus. After the resistant callus continues to grow, an appropriate amount of samples are taken, genomic DNA is extracted, and PCR identification is performed using primers specific to the transformation vector. Each PCR-positive callus is an independent transformation event. If necessary, the edited DNA fragment can also be amplified by PCR using primers specific to the target gene, and sequenced directly or after cloning to analyze the gene editing effect.

5. Callus differentiation into seedlings: Transfer the resistant callus that is positive and grown by PCR into a culture bottle containing differentiation medium, maintain 16 hours of light per day, and culture at 28°C for 2-3 weeks. Green dots can be seen after differentiation. Some green dots can differentiate into seedlings and grow elongated after about 2 weeks.

6. Rooting culture: Transplant the differentiated seedlings with a height of about 2-5 cm and normal morphology into a culture bottle containing rooting medium. Incubate at 28°C for 1-2 weeks at 16 hours of light per day to strengthen the seedlings and until healthy roots are differentiated.

7. Acclimate and transplant the resistant regenerated plants: When the root system of the seedlings is about 3-4 cm long and relatively strong, open the sealing film of the culture bottle and add a small amount of distilled water. After 24 hours of hardening, carefully use tweezers to remove the plants to avoid damaging the stems and roots, rinse the culture medium attached to the roots with clean water, and transplant them to the seedling tray containing vermiculite nutrient soil, cover to maintain humidity, and cultivate them in a 28℃/24℃ artificial climate room or greenhouse with 16/8 hours of light until new leaves grow, then transplant the seedlings to larger flower pots and soak them in water for cultivation, and fertilize them appropriately until they bloom and bear fruit. During this period, the leaves of these T0 plants can be sampled to extract DNA for transgenic identification and gene editing analysis.

III. Gene Editing Identification of Transformation Events

Resistant regenerated plants must be tested by molecular biological methods to confirm whether the genetic transformation is successful. The commonly used simple and reliable detection method is PCR amplification. Sample a small amount of callus tissue of the resistant transformation event or young leaf tissue of the regenerated plant, extract genomic DNA for PCR amplification, design vector-specific DNA primers according to the DNA sequence of the transformation vector, and the plant that can obtain the specific amplified fragment is positive for transformation, and the target gene editing effect can be further tested. According to the target gene DNA sequence and the expected Cas9 cut point position, design PCR primers covering about 300 bp upstream and downstream of the cut site, amplify the target gene fragment of about 400-800 bp, and directly sequence it with the same upstream or downstream primers after purification. If the DNA fragment sequence of the regenerated plant is no different from the wild-type sequence, there is no gene editing; if the measured DNA sequence shows overlapping peaks near the expected Cas9 cut site, it means that the sequenced PCR fragment is a mixed template and the regenerated plant should be heterozygous editing; if the DNA fragment of the regenerated plant has a certain sequence difference from the wild type, it is homozygous gene editing. The PCR fragment of the target gene of the above-mentioned heterozygous edited plant can be further cloned using a PCR fragment cloning kit, and at least 3 clones can be selected for sequencing using vector-specific primers to confirm the specific editing changes in the target gene sequence.

The DNA sequence analysis and alignment, DNA vector cloning construction design, PCR primers and CRISPR gRNA design involved in the molecular biology experiments in the present invention were all completed using the DNA analysis software Geneious Prime (Biomatters Ltd.).

Rice resistant callus or leaves of regenerated T0 plants were sampled and genomic DNA was extracted using a rapid DNA extraction method based on SDS (Sodium dodecylsulphate), and Cas9 gene-specific PCR was performed to identify transformation events. The PCR reaction system used the Quick Taq HS DyeMix (DTM-101) kit (TOYOBO Life Science), and the 20-microliter PCR reaction system contained 10 µl 2x Quick Taq HS DyeMix, 1.0 µl 10 pmol/µl primer Cas9-F1 (SEQ ID NO: 10) and 1.0 µl 10 pmol/µl primer Cas9-R1 (SEQID NO: 11), 6.0 µl sterile water, and finally 2.0µl 50 ng/µl sample genomic DNA. The PCR reaction conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute for a total of 30 cycles, and finally extension at 72°C for 7 minutes, and maintained at 4°C. The PCR amplification products were separated by 1% agarose gel electrophoresis, and samples that successfully amplified the 540 bp long fragment (SEQ ID NO: 12) specific to the Cas9 gene were determined to be transgenic positive. Similar PCR analysis can also be performed using the vector hygromycin resistance gene HygR specific primers Hyg-F1 and Hyg-R1 (SEQ ID NO: 13, 14), and samples that successfully amplified the 402 bp long fragment (SEQ ID NO: 15) specific to the HygR gene were determined to be transgenic positive.

By similar PCR amplification method and using OsDTH7 (LOC_Os07g49460) specific primers OsDh7-F2 and OsDh7-R2 (SEQ ID NOs: 16, 17), a 1329 bp long target fragment near the OsDTH7-G1 gene editing site (SEQ ID NO: 18) can be amplified from Cas9 positive samples. Using OsDTH8 gene (LOC_Os08g07740) specific primers OsDh8-F1 and OsDh8-R1 (SEQ ID NOs: 19, 20), a 1329 bp long target fragment near the OsDTH8-G1 gene editing site (SEQ ID NO: 21) can be amplified. Using the OsGhd7 gene (LOC_Os07g15770) specific primers OsG7-F4 and OsG7-R4 (SEQ ID NOs: 22, 23), a target fragment of 826 bp near the gene editing site of OsGhd7-G3 or its complementary chain OsGhd7-G3r can be amplified (SEQ ID NO: 24). Using the OsHd1 gene (LOC_Os06g16370) specific primers OsHd1-F3 and OsHd1-R3 (SEQ ID NOs: 25, 26), a target fragment of 801 bp near the gene editing site of OsHd1-G1 can be amplified (SEQ ID NO: 27). These PCR fragments were sequenced directly in one direction using the above PCR primers. If there was an overlapping peak near the expected gene editing site, or even caused the downstream DNA to be unable to be accurately sequenced, it was determined that the DNA sequence editing occurred at the site. The PCR fragments of the edited samples were TOPO cloned into the pCR2.1 vector (Thermo Fisher Scientific), and three clones of each sample were sent for sequencing, so that the specific DNA editing sequence changes could be accurately analyzed.

The transformation efficiency of each gene editing vector for Koshihikari rice was not high. The callus tissue samples of the resistant events screened were sampled for the above-mentioned PCR analysis, and all were identified as real transformation events. Sequencing analysis of the target gene fragments confirmed that these events achieved gene editing, and the gene editing efficiency reached 100%. The analysis results are summarized in Table 2.

Table 2 Genetic transformation and editing efficiency of Koshihikari growth-stage regulatory genes

Experiment number Gene Editing Vectors Number of infected callus Resistance Events Conversion efficiency% PCR positive events Gene editing incidents 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 of gene editing events

The T0 plants that have achieved gene editing are transplanted to the greenhouse for cultivation, naturally self-pollinated and matured, and the T1 generation seeds are harvested and sown in the greenhouse in due time. When the seedlings grow to about three leaves, a small amount of leaf samples are taken to extract genomic DNA for detailed PCR detection and target gene sequencing analysis (Figure 3). Because rice self-pollination follows Mendelian genetic segregation, the gene editing vector fragment inserted into the genome and the edited target gene are generally not on the same chromosome, and the two are genetically separated independently. Some T1 plants will no longer contain any editing vector DNA and are non-transgenic offspring. Among them, the target gene of some plants remains edited. About 16 T1 plants may have one plant that does not carry the gene editing vector fragment and the target gene is homozygous edited. If stable non-transgenic homozygous edited plants can be obtained in the T1 generation, the analysis and identification experiment can be expanded after harvesting its T2 generation seeds. If non-transgenic homozygous edited plants are not obtained, it is necessary to plant the T2 generation seeds of heterozygous edited plants in the greenhouse for natural genetic segregation, and select non-transgenic homozygous edited T2 generation plants through similar analysis.

The isolated T1 progeny plants were first PCR amplified using the same Cas9 and HygR gene-specific primers as mentioned above to check whether the plants still carried the gene editing vector fragment, and non-transgenic progeny plants that could not be amplified were selected (Figure 3). Then, the same OsDTH7 gene-specific primers OsDh7-F2 and OsDh7-R2 were used to amplify the 1329 bp target fragment of the gene editing region (Figure 3), and the OsDh7-F2 primer was used for direct unidirectional sequencing analysis. The results were aligned with the wild-type sequence of the OsDTH7 gene (SEQ ID NO: 18), confirming the editing status of the OsDTH7 gene. The same OsDTH8 gene-specific primers OsDh8-F1 and OsDh8-R1 were used to amplify the 1329 bp target fragment of the gene editing region (Figure 3), and the OsDh8-F1 primer was used for direct unidirectional sequencing analysis. The results were aligned with the wild-type sequence of the OsDTH8 gene (SEQ ID NO: 21), confirming the editing status of the OsDTH8 gene. The same OsGhd7 gene-specific primers OsG7-F4 and OsG7-R4 were used to amplify the target fragment of 826 bp in the gene editing region (Figure 3), and the OsG7-F4 primer was used for direct unidirectional sequencing analysis. The results were aligned with the wild-type sequence of the OsGhd7 gene (SEQ ID NO: 24), confirming the editing status of the OsDTH8 gene. The same OsHd1 gene-specific primers OsHd1-F3 and OsHd1-R3 were used to amplify the target fragment of 801 bp in the gene editing region (Figure 3), and the OsHd1-F3 primer was used for direct unidirectional sequencing analysis. The results were aligned with the wild-type sequence of the OsHd1 gene (SEQ ID NO: 27), confirming the editing status of the OsHd1 gene.

Based on the results of PCR amplification and sequencing analysis of the target gene editing region DNA fragments, four independent families of non-transgenic, homozygous edited R55 plants, R55-1-2-9, R55-1-4-10, R55-1-5-5, and R55-1-6-17, were screened and obtained. The OsGhd7 and OsHd1 genes were edited (SEQ ID NOs: 28-31 and SEQ ID NOs: 32-35), and the differential sequences only occurred at the expected editing sites (Figure 4a). Six independent families of non-transgenic, homozygous edited R66 plants were obtained, and the OsGhd7 gene was edited (SEQ ID NOs: 36-41), and the differential sequences only occurred at the expected editing sites (Figure 4b). Five independent families of non-transgenic, homozygous edited R81 and R89 plants were obtained, and the OsDTH7 and OsDTH8 genes were edited (SEQ ID NOs: 42-46 and SEQ ID NO: 47-51), the difference sequence only occurs at the expected editing site (Figure 5). The target site editing of each gene editing family is summarized in Table 3, among which the complementary chain sequence is shown at the OsGhd7-G3r site of the R66 gene editing family. Because these DNA sequence editing only occurs in the protein coding region where the expected editing site is located, that is, they are all CRISPR-Cas9-mediated specific gene editing, causing frameshift mutations in each target gene, especially blocking the normal expression of each protein functional domain sequence, and effectively knocking out the function of each gene in the edited rice family.

Table 3 Changes in target gene sequences in gene-edited families during the reproductive period

5. Phenotypic analysis of offspring plants of gene editing events

Heilongjiang Province spans ten geographical latitudes (about 43.5-53.5 degrees north latitude), and the ecological environment is extremely complex and diverse. According to the annual accumulated temperature, it is divided into six accumulated temperature zones or ecological zones, from above 2700℃, 2500-2700℃, 2300-2500℃, 2100-2300℃, 2100-1900℃, to below 1900℃. Each zone has a reduced accumulated temperature of about 200℃ compared with the previous one, and the maturity period is also reduced by about 7 days accordingly. The rice experimental base of Kenfeng Seed Industry is located in Acheng District, Harbin, north of 45 degrees north latitude, and belongs to the first accumulated temperature zone. Non-transgenic homozygous edited materials are isolated and planted in a dedicated plot for observation. Each material is planted in a 5-meter-long single row or partition, with a plant spacing of 10 cm and about 45 plants per row. At the same time, wild-type parents are planted in the same area as a control to observe the comprehensive field performance and growth period changes. Generally, rice varieties suitable for planting in Acheng area will head and flower in late July to early August. The date when the top of the rice ears of about 10% of the plants in the whole area or row of each material are exposed to the leaf sheath is the beginning of heading period of the family, the date when the top of the rice ears of about 50% of the plants in the whole area or row are exposed to the leaf sheath is the heading period, and the date when the top of the rice ears of about 80% of the plants in the whole area or row are exposed to the leaf sheath is the full heading period. According to the planting date and the beginning of heading period of each material, the number of days required for heading and flowering is calculated and recorded, and compared with the control to determine whether the growth period is effectively shortened.

In 2021, non-transgenic, homozygous edited T2 segregating progeny of R55 were planted in a dedicated isolation plot at the Acheng District Rice Experimental Base in Harbin, and non-transgenic, homozygous edited T2 segregating progeny of R66, R81 and R89 were planted in 2022. Each family was planted in a single row of 5 meters long with a plant spacing of 10 cm, with a total of about 45 plants. At the same time, wild-type Koshihikari was planted as a control to observe the comprehensive field performance and growth period changes. The growth and development of each gene-edited family was normal, and they began to head in late July. The date when the top of the rice ears of about 10% of the plants in the whole row exposed the leaf sheath was recorded as the heading date of the family. The number of days required for heading and flowering was calculated based on the planting date and heading date of each material. It took about 100 days from planting to heading for each edited material (see Table 4). The main cultivation area of Koshihikari is the central and southern part of Japan, located at approximately 33-38 degrees north latitude. It has a long growing period and cannot be planted normally in Acheng District of Harbin. As a control, the wild type Koshihikari planted on April 18 did not start heading until after September 10. It takes 145 days from planting to heading, and there is not enough time to complete fruiting before the frost in early October.

Since all edited lines were able to head and flower in late July, the time required was about 45 days shorter than that of the wild-type control Koshihikari. The editing effect was extremely significant. In addition, it was not necessary to knock out all four growth-control genes DTH7, DTH8, Ghd7, and Hd1 at the same time. Only one Ghd7 gene, or two genes DTH7 and DTH8, needed to be knocked out to achieve similar effects (see Table 4), which was beyond expectations. Therefore, adverse side effects caused by knocking out too many genes could be avoided as much as possible.

All edited families have effectively shortened the growth period, and can be planted and matured in the first or even second accumulated temperature zone of Heilongjiang. However, while significantly shortening the growth period, the phenotypes of the edited combinations are obviously different. Some materials, such as the R55 family with edited Ghd7 and Hd1 genes, have uneven heading, small inflorescence, sparse fruit, and are very easy to lodging; the R66 family with only edited Ghd7 gene has small inflorescence and sparse fruit, which seriously affects the yield; while the R81 and R89 families with edited DTH7 and DTH8 genes have normal growth and development, which is the best editing combination at present, and no significant phenotypic defects are observed, and can be mature and harvested before frost in early October (Figure 6).

Table 4 Changes in flowering period of gene-edited families during the growth period

Gene-edited families Sowing time Heading stage Required days 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 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 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 100.4 Koshihikari 2022.4.18 2022.9.10 145 Immature

The above gene-edited lines do not exist in nature yet. They are novel germplasm resources created by gene editing methods. They can be directly used as growth-period improved lines for comprehensive trait evaluation, or as special materials for growth-period improved lines for conventional breeding improvement of other rice varieties. Based on the unique editing sequences of flowering control gene fragments of each line, special primers can be designed and specific PCR molecular markers can be developed to identify and track the offspring plants of each edited line.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and 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 fragment311 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 gene editing tools to simultaneously edit rice DTH7 and DTH8 genes in changing the flowering period of rice. 2. The use according to claim 1, characterized in that the gene editing tool includes the CRISPR/Cas9 system, and its derivative tools cytosine editor, adenine editor or guide editor, or a gene editing system based on different Cas12 enzymes, or other gene editing tools. 3. The use according to claim 2 is characterized in that the gene editing tool is a CRISPR/Cas9 system, and the gene editing transformation vector designed based on the CRISPR/Cas9 system contains three gene expression units ZmU6 pro:gRNA:AtU6-26 term, ZmUBI1 pro:SpCas9:PsE9 term and 35S pro:HYG:35S term, and a BsaI restriction site is left between ZmU6 pro and gRNA. After the vector is linearized, one or more gRNA sequences are inserted using DNA ligase or Gibson cloning method to form a gene editing vector for any target site of DTH7 and DTH8 genes. 4. The use according to claim 3 is characterized in that gRNA is designed for different regional identifiable sites of DTH7 and DTH8 genes, and multiple gRNAs are interactively connected through tRNA technology and then inserted between ZmU6 pro and the gRNA backbone of the gene editing transformation vector by cloning method. 5. The use according to claim 4 is characterized in that the designed gene editing transformation vector inserts a tRNA-OsDTH7-G1-gRNA-tRNA-OsDTH8-G1 fragment artificially synthesized in an interactive arrangement of tRNA and gRNA backbones between ZmU6 pro and gRNA, and the sequence of the fragment is shown in SEQ ID NO: 7. 6. The use according to any one of claims 3 to 5, characterized in that the genetic transformation method for changing the flowering period of rice is to introduce the designed gene editing transformation vector into rice cells through Agrobacterium-mediated introduction, and then screen to obtain non-transgenic, stably inherited homozygous edited rice lines. 7. The use according to claim 6, characterized in that the genetic transformation method comprises the following steps: S1: Recipient rice cells are genetically transformed to obtain regenerated plants; S2: Detect whether the gene editing of the regenerated plants is successful; S3: Screening for heritable, non-transgenic and stably edited offspring plants. 8. The application according to claim 7, characterized in that step S1 comprises: S1-1 Rice callus induction; S1-2 Subculture of rice callus; S1-3 callus infection; Screening and identification of S1-4 resistant callus; S1-5 callus differentiated into shoots; S1-6 rooting culture; S1-7 was acclimated and transplanted to obtain regenerated plants. 9. The application according to claim 8, characterized in that, step S2 comprises: S2-1 Collect resistant callus or young leaf tissue of regenerated plants, extract genomic DNA for PCR amplification; S2-2 Based on the sequences of the target genes DTH7 and DTH8 and the expected Cas9 cut-off point, PCR primers covering a certain length upstream and downstream of the cut-off site were designed to amplify the target gene fragments; S2-3 determines whether the gene editing is successful based on the difference between the amplified target gene fragment and the target gene fragment amplified in the plant that has not been genetically transformed. 10. Use of the offspring plants obtained according to any one of claims 7 to 9 in rice breeding. 11. The use according to claim 10, characterized in that the DTH7 gene 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 is respectively as shown in SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, and the DTH8 gene is respectively as shown in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51.