Application of osSPMS2 gene in improving rice yield

Abstract

The application belongs to the technical field of biotechnology and genetic engineering, and particularly relates to OsSPMS2 Application of a gene in increasing rice yield. OsSPMS2 By targeting editing a target site sequence as shown in SEQ ID NO. 3 in a rice OsSPMS2 gene, a rice mutant with a nucleotide sequence as shown in SEQ ID NO. 8 or SEQ ID NO. 9 in a Nipponbare background is obtained, and a rice mutant with a nucleotide sequence as shown in SEQ ID NO. 10 or SEQ ID NO. 11 in a Sugun 118 background is obtained. OsSPMS2 The application effectively realizes the increase of rice grain weight and yield and has potential application value in increasing rice grain weight and yield.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and genetic engineering technology, and relates to... OsSPMS2 Application of genes in increasing rice yield. Background Technology

[0002] Rice is an important food crop, with more than half of the world's population relying on it as their staple food. According to statistics on global hunger compiled by the Food and Agriculture Organization of the United Nations (https: / / www.fao.org / hunger / en / ), more than 700 million people worldwide were still suffering from hunger in 2022. Increasing rice yield is a crucial measure to address the food shortage crisis and ensure food security. Rice yield per plant is determined by three factors: the number of panicles per plant, the number of grains per panicle, and grain weight. Grain weight is a key factor controlling rice yield, and increasing grain weight is one of the effective ways to improve rice yield. Grain weight is a complex quantitative trait controlled by multiple genes and has always been a focus of genetic research and breeding.

[0003] Polyamines are a class of organic compounds ubiquitous in nature, characterized by having multiple amino groups and being aliphatic or aromatic compounds. In plants, polyamines mainly include putrescine (Put), spermidine (Spd), and spermine (Spm). Polyamines play important roles in regulating plant growth and development, responding to stress, and cell division and differentiation. Research has found that spermine synthase genes... OsSPMS1 Negative regulation of seed germination and rice yield, overexpression OsSPMS1 This not only significantly reduces rice plant height, grain weight, and yield, but also significantly inhibits seed germination. Knocking out polyamine oxidase... OsPAO5 The gene significantly improves grain weight, grain number, and yield potential, and promotes mesocotyl elongation in rice, increasing seedling emergence rate. Recent studies have shown that overexpression of the thermal spermamine synthase gene... OsACL5 This leads to leaf curling, smaller grains, reduced grain weight, and decreased yield in rice, while the knockout mutant exhibits the opposite phenotype, indicating that... OsACL5 These are important genes that regulate rice growth and development. Therefore, genes related to the polyamine synthesis pathway are involved in regulating rice grain weight and yield traits.

[0004] With the continuous development of biotechnology, an increasing number of new genes promoting superior grain weight and yield have been located and cloned. However, how to introduce these superior grain weight genes to rapidly improve rice grain weight and yield has become a major challenge in the breeding process. Traditional breeding methods mainly involve introducing superior genes into different varieties through hybridization and backcrossing. However, traditional breeding methods are time-consuming and labor-intensive, with long breeding cycles, and the introduction of target genes may lead to changes in other agronomic traits. In recent years, with the discovery and widespread application of CRISPR / Cas9 technology, genes related to rice grain weight and yield can be edited in a targeted manner, which helps to rapidly breed rice varieties with enhanced grain weight and yield. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides... OsSPMS2 The application of genes in increasing rice yield OsSPMS2 Gene mutations can effectively increase rice grain weight and yield.

[0006] The technical solution provided by this invention is as follows:

[0007] This invention provides OsSPMS2 The application of genes in increasing rice yield, mutation OsSPMS2 The gene increases rice grain length and improves the thousand-grain weight; OsSPMS2 The nucleotide coding sequence of the gene is shown in SEQ ID NO. 1.

[0008] This invention also provides mutations OsSPMS2 The application of gene lines in increasing rice yield, using mutations OsSPMS2 The gene-modified strains increase rice grain length and improve the thousand-grain weight; OsSPMS2 The nucleotide coding sequence of the gene is shown in SEQ ID NO. 1.

[0009] Furthermore, the aforementioned OsSPMS2 The amino acid sequence of the gene’s protein is shown in SEQ ID NO. 2.

[0010] Furthermore, the mutation OsSPMS2 The gene strain is OsSPMS2 Non-frameshift homozygous mutant of the gene.

[0011] This invention also provides mutations OsSPMS2 The application of gene kits in increasing rice yield, mutation OsSPMS2 The gene kit contains OsSPMS2 CRISPR / Cas9-gRNA expression vector targeting specific genes; OsSPMS2 The nucleotide coding sequence of the gene is shown in SEQ ID NO. 1.

[0012] Furthermore, using mutations OsSPMS2 Gene-based kits can increase rice grain length and improve the thousand-grain weight of rice.

[0013] Furthermore, the target sequence of the gRNA is shown in SEQ ID NO.3.

[0014] Furthermore, mutations OsSPMS2 The genetic approach includes the following steps:

[0015] According to rice OsSPMS2 CRISPR / Cas9 editing vectors were constructed using the target site sequence shown in SEQ ID NO.3 of the gene;

[0016] The CRISPR / Cas9 editing vector was transformed into Agrobacterium tumefaciens, which then infected recipient rice callus tissue, and positive plants were obtained by screening.

[0017] The selected positive plants were planted into lines, and homozygous non-frameshift mutant plants were obtained through sequencing identification, which resulted in rice plants with enhanced grain length, grain weight and yield phenotypes.

[0018] Furthermore, according to rice OsSPMS2 The gene target site sequence is used to construct a CRISPR / Cas9 editing vector, including:

[0019] Use restriction endonucleases Aar I digest the intermediate vector SK-gRNA with enzyme I to generate sticky ends, and add adapter sequences to the sticky ends to form a leading sequence as shown in SEQ ID NO.4 and a trailing sequence as shown in SEQ ID NO.5.

[0020] Furthermore, the recipient rice is the japonica rice variety Nipponbare and Suken 118.

[0021] Beneficial effects

[0022] The present invention provides OsSPMS2 The application of genes in increasing rice yield OsSPMS2 Genes and their encoded proteins can regulate rice grain weight and yield. This invention utilizes CRISPR / Cas9 technology to target and edit this gene to rapidly obtain rice plants with significantly enhanced grain weight and yield, which has broad application prospects. Attached Figure Description

[0023] Figure 1 Wild-type Nipponbare (NIP) and rice provided for embodiments of the present invention OsSPMS2 CRISPR / Cas9 mutant gene nosspms2-4 , nosspms2-5 Sequencing maps near the target site and NIP, nosspms2-4and nosspms2-5 A comparison diagram of amino acid sequences;

[0024] Figure 2 Wild-type Suken 118 (SK118) and rice provided for embodiments of the present invention OsSPMS2 CRISPR / Cas9 mutant gene sosspms2-3 , sosspms2-4 Sequencing maps near the target site and SK118, sosspms2-3 and sosspms2-4 A comparison diagram of amino acid sequences;

[0025] Figure 3 NIP and rice mutants nosspms2-4 , nosspms2-5 Comparison of grain shape and statistical analysis of thousand-grain weight and yield per plant;

[0026] Figure 4 For SK118 and rice mutant sosspms2-3 , sosspms2-4 The comparison of grain shape and statistical analysis of thousand-grain weight and yield per plant were conducted. Detailed Implementation

[0027] The technical solutions of the present invention will be further described below with reference to specific embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the examples are all conventional conditions; the materials and reagents used are all commercially available unless otherwise specified; the recipient rice materials are Nipponbare and Suken 118.

[0029] Example 1

[0030] This embodiment uses CRISPR / Cas9 technology to... OsSPMS2 Gene editing was performed to obtain mutant materials. The gene editing method used was the CRISPR / Cas9 system provided by Wang Kejian's research group at the China National Rice Research Institute.

[0031] 1.1 Obtained from the Rice Gene Database (http: / / rice.plantbiology.msu.edu / ) OsSPMS2 The cDNA sequence of the gene, the nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0032] 1.2 Based on rice OsSPMS2The gene coding region-specific target sequence is shown in SEQ ID NO.3, specifically 5'-CAGCGGCTTCCCGTTCCCGG-3', and was designed and constructed. OsSPMS2 Primers for gene editing vectors. Based on restriction endonucleases. Aar The sticky ends of the intermediate vector SK-gRNA are generated by enzyme I digestion. A GGCA adapter sequence is added to the leading primer, as shown in SEQ ID NO.4, specifically: 5'-GGCACAGCGGCTTCCCGTTCCCGG-3'; and an AAAC adapter sequence is added to the trailing primer, as shown in SEQ ID NO.5, specifically: 5'-AAACCCGGGAACGGGAAGCCGCTG-3'.

[0033] 1.3 The intermediate vector SK-gRNA was subjected to restriction endonuclease at 37°C. Aar After overnight digestion with enzyme I, gel recovery was performed. The enzyme digestion system is shown in Table 1.

[0034] Table 1

[0035]

[0036] 1.4 Mix 20 μl each of the pre- and post-introductions from step 1.2 (pre- and post-introduction concentrations of 100 μM) together, denature at 100 °C for 5 minutes, and cool to room temperature to form a fragment with sticky ends.

[0037] 1.5 Using T4 DNA ligase (New England Biolabs), the fragment with sticky ends generated in step 1.4 was ligated with the enzyme-digested SK-gRNA from step 1.3 for 1 hour at room temperature. The ligation system is shown in Table 2.

[0038] Table 2

[0039]

[0040] 1.6 The ligation product from step 1.5 was heat-transformed into Escherichia coli DH5α competent cells, plated on ampicillin-resistant (Amp) plates, cultured overnight at 37°C, and single clones were picked and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing to obtain the intermediate vector SK-gRNA-OsSPMS2 carrying the target fragment.

[0041] 1.7 The intermediate vector SK-gRNA-OsSPMS2 from step 1.6 was used... Kpn I and Bgl II was used for enzyme digestion and fragment recovery, and the final vector pC1300-Cas9 was used... Kpn I andBamH I was subjected to enzyme digestion and recovery; the enzyme digestion system is shown in Table 3. Utilizing... BamH I and Bgl II. The isosinetase characteristics of both fragments led to the ligation of the fragments with the final vector, using the ligation system shown in Table 2. The ligation product was heat-shock transformed into *E. coli* DH5α competent cells, plated on kanamycin-resistant (Kan) plates, and incubated overnight at 37°C. Single colonies were picked and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing analysis. OsSPMS2 Gene editing vector pC1300-Cas9-gRNA- OsSPMS2 .

[0042] The SK-gRNA vector enzyme digestion system is shown in Table 3 below:

[0043] Table 3

[0044]

[0045] The pC1300-Cas9 vector digestion system is shown in Table 4 below:

[0046] Table 4

[0047]

[0048] 1.8 The vector pC1300-Cas9-gRNA successfully constructed in step 1.7 was then... OsSPMS2 Agrobacterium EHA105 competent cells were transformed by heat shock, plated on kanamycin-resistant (Kan) plates, and incubated at 28°C for at least 30 hours. Single clones were picked, plasmids were extracted, and positive clones were identified.

[0049] 1.9 Select mature embryo-induced callus tissues from conventional japonica rice varieties Nipponbare and Suken 118, and infect the rice callus tissues with the positive clones obtained in step 1.8. After Agrobacterium co-culture, screening of positive callus, callus differentiation and seedling emergence, seedling rooting, and hardening, the desired callus tissues are obtained. OsSPMS2 Mutant plant.

[0050] Example 2

[0051] This embodiment involves rice. OsSPMS2 Identification of homozygous non-frameshift mutant plants includes the following steps:

[0052] 2.1 DNA was extracted from the leaves of T1 generation mutant plants, primers were designed for PCR amplification, and the amplified products were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with... OsSPMS2 The original gene sequence (SEQ ID NO. 1) was compared to determine the characteristics of each strain. OsSPMS2 Gene editing status. Primer sequences are shown below:

[0053] osspms2 -F: 5'-ACTTCGAGGAGGAGCAGGAT -3' (Sequence shown in SEQ ID NO.6)

[0054] osspms2 -R: 5'-CCTGGCCACATCGGGTTATT -3' (Sequence shown in SEQ ID NO.7)

[0055] 2.2 Simultaneously, hygromycin marker detection was performed to screen for lines that do not contain the hygromycin resistance marker gene. The primer sequences for hygromycin marker detection are shown below:

[0056] Hm-F: 5'-GGACTTCGGGGCAGTCCT-3'

[0057] Hm-R: 5'-CGATGTAGGAGGGCGTGG-3'

[0058] 2.3 Through the above screening, two homozygous nonframeshift mutants with different mutation types were obtained from the backgrounds of Nipponbare and Suken 118.

[0059] like Figure 1 As shown, this is a Nipponbare (NIP) image and a Nipponbare background image provided in an embodiment of the present invention. OsSPMS2 Sequencing map of the mutation site in the gene mutant. Two images with a Japanese background. OsSPMS2 The mutants are those with the deletion of three bases "GGG". nosspms2-4 The mutant in which “AACGGGAAGCCGCTG” is replaced with “CTT” nosspms2-5 .in, nosspms2- 4 The gene sequence is shown in SEQ ID NO.8. nosspms2-5 The gene sequence is shown in SEQ ID NO. 9. Two OsSPMS2 All mutants resulted in changes in amino acids. nosspms2-4 Only one amino acid is missing. nosspms2-5 This resulted in the loss of 4 amino acids.

[0060] like Figure 2 As shown, this is an embodiment of the present invention, illustrating the Suken 118 (SK118) and Suken 118 background. OsSPMS2 Sequencing map of the mutation sites in the gene mutant. Background of two Suken 118 plants. OsSPMS2 The mutants are those with the deletion of three bases "GGG". sosspms2-3 And mutants lacking 6 bases "GCCGGG" sosspms2-4 .in, sosspms2- 3The gene sequence is shown in SEQ ID NO.10. sosspms2-4 The gene sequence is shown in SEQ ID NO.11. Two OsSPMS2 All mutants resulted in changes in amino acids. sosspms2-3 Only one amino acid is missing. sosspms2-4 This resulted in the loss of two amino acids.

[0061] Example 3

[0062] This embodiment involves rice. OsSPMS2 The statistical analysis of grain weight and yield of mutant plants is performed using the following steps:

[0063] Wild-type rice varieties NIP and SK118, as well as those identified as homozygous in two different backgrounds, were tested. OsSPMS2 Non-frameshift mutants were planted into lines, and after maturity, wild-type (NIP / SK118) and mutant ( nosspms2-4, nosspms2-5, sosspms2-3 and sosspms2-4 Mature seeds were photographed, observed, and measured to compare the morphology of rice grains, and further statistical analysis was conducted on the thousand-grain weight and yield per plant.

[0064] like Figure 3 As shown, two NIP and NIP background OsSPMS2 nonframeshift mutant nosspms2-4, nosspms2- 5 Significant differences exist in grain length. Mutants nosspms2-4 and nosspms2-5 The grain length was significantly larger than that of wild-type NIP. Compared with NIP, the mutant lines... nosspms2-4 and nosspms2-5 The particle lengths increased by 2.12% and 2.25%, respectively. Further statistical analysis showed that, compared with NIP, nosspms2-4 and nosspms2-5 The thousand-grain weight increased by 7.76% and 2.95% respectively, and the yield per plant increased by 13.57% and 10.96% respectively.

[0065] like Figure 4 As shown, SK118 and two in the background of SK118 OsSPMS2 nonframeshift mutant sosspms2-3, sosspms2-4 Significant differences exist in grain length. Mutants sosspms2-3 and sosspms2-4 The grain length was significantly larger than that of wild-type SK118. Compared with SK118, the mutant lines... sosspms2-3 and sosspms2-4 The particle length increased by 2.27% and 2.00%, respectively. Further statistical analysis showed that, compared with SK118, sosspms2-3 and sosspms2-4The thousand-grain weight increased by 6.73% and 9.00% respectively, and the yield per plant increased by 9.32% and 12.69% respectively.

[0066] The above results confirm that the editor OsSPMS2 The non-frameshift mutant obtained from the gene can significantly increase rice grain weight and yield. Therefore, this invention provides an important genetic resource for breeding high-yielding rice varieties and provides a breeding method for increasing rice grain weight and yield.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Mutations OsSPMS2 Use of strains of genes in increasing yield of rice, characterized in that, Using mutations OsSPMS2 The strain of the gene increases the grain length of rice and improves the thousand-grain weight of rice; the OsSPMS2 The nucleotide coding sequence of the gene is shown as SEQ ID NO.

1. The mutations OsSPMS2 Strains of the gene are OsSPMS2 Non-frame shift homozygous mutants of the gene; mutation OsSPMS2 a method of a gene comprising the steps of: According to the rice OsSPMS2 The target site sequence of SEQ ID NO. 3 in the gene is constructed into a CRISPR / Cas9 editing vector; The CRISPR / Cas9 editing vector is transformed into Agrobacterium, the receptor rice callus is infected, and positive plants are screened; The screened positive plants are planted into a plant line, identified by sequencing, and a homozygous non-frame shift mutant plant is obtained, i.e. a rice plant with the phenotype of increased grain length, grain weight and yield; The receptor rice is japonica variety Nipponbare and Sukeng 118; Nipponbare background OsSPMS2 The nucleotide sequence of the mutant is shown as SEQ ID NO. 8 or SEQ ID NO. 9 in the Shukeng 118 background OsSPMS2 The nucleotide sequence of the mutant is shown as SEQ ID NO. 10 or SEQ ID NO.

11.

2. Use according to claim 1, characterized in that, The OsSPMS2 The amino acid sequence of the protein of the gene is shown in SEQ ID NO.

2.

3. mutation OsSPMS2 The use of the kit of genes in increasing the yield of rice is characterized in that, mutation OsSPMS2 The kit of genes comprises a CRISPR / Cas9-gRNA expression vector targeting the OsSPMS2 gene; said CRISPR / Cas9-gRNA expression vector targeting the OsSPMS2 nucleotide coding sequence of the gene is shown in SEQ ID NO. 1; Using mutations OsSPMS2 The kit of genes increases grain length and thousand-grain weight of rice. mutation OsSPMS2 a method of a gene comprising the steps of: According to the rice OsSPMS2 The target site sequence of SEQ ID NO. 3 in the gene is constructed into a CRISPR / Cas9 editing vector; The CRISPR / Cas9 editing vector is transformed into Agrobacterium, the receptor rice callus is infected, and positive plants are screened; The screened positive plants are planted into a plant line, identified by sequencing, and a homozygous non-frame shift mutant plant is obtained, i.e. a rice plant with the phenotype of increased grain length, grain weight and yield; The receptor rice is japonica variety Nipponbare and Sukeng 118; Nipponbare background OsSPMS2 The nucleotide sequence of the mutant is shown as SEQ ID NO. 8 or SEQ ID NO. 9 in the Shukeng 118 background OsSPMS2 The nucleotide sequence of the mutant is shown as SEQ ID NO. 10 or SEQ ID NO.

11.

4. Use according to claim 1 or 3, characterized in that, According to the rice OsSPMS2 The target site sequence of the gene constructs a CRISPR / Cas9 editing vector, comprising: Use of restriction enzymes Aar I The intermediate vector SK-gRNA is cut with the enzyme, and a sticky end is produced to which a linker sequence is added to form a forward primer sequence as shown in SEQ ID NO. 4 and a reverse primer sequence as shown in SEQ ID NO. 5.

Images