A genetic engineering method for creating early-flowering and yield-increasing rice

Abstract

The application discloses a genetic engineering method for creating early-flowering and yield-increasing rice, and belongs to the technical field of plant genetic engineering. EFHP The PACMAD group-specific MADS gene is identified by a comparative genomics method EFHP The gene is introduced into a main rice variety and overexpressed, so that the flowering time of the rice can be significantly regulated, the rice plants can bloom about 7 days in advance, the biomass and yield are increased, and the yield per plant is increased by 23-35%. EFHP The gene has a double function of simultaneously regulating the flowering time and yield of the rice, solves the technical problem that early flowering and yield increase are difficult to be simultaneously improved, and provides new gene resources and breeding materials for rice molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a method utilizing maize-derived... EFHP Methods for regulating flowering time and yield in rice through gene regulation, particularly involving overexpression in rice varieties. EFHP Genes are used to achieve early flowering and increased yield. Background Technology

[0002] The Poaceae family is an important family within the monocotyledonous class, containing approximately 10,000 species widely distributed throughout the world. Poaceae can be divided into three groups: the basal group, the BOP group, and the PACMAD group. The BOP group includes crops such as rice and wheat, which prefer cooler regions; the PACMAD group includes crops such as maize, sorghum, and millet, which prefer warmer and more humid regions. The genes selected by the PACMAD group during its evolutionary adaptation to tropical environments differ from those of the BOP group. Studying these differences helps in understanding the molecular mechanisms of crop adaptive evolution.

[0003] Rice is the staple crop for more than half of the world's population, and its yield and quality are directly related to national food security. Under the multiple pressures of shrinking arable land, intensified climate change, and population growth, traditional rice breeding models face bottlenecks such as the difficulty in synergistically improving high yield and stress tolerance, and diminishing returns on trait improvement. Against the backdrop of global warming, rising temperatures will affect the seed filling of gramineous plants, leading to reduced crop yields. While early flowering can shorten the growth cycle, allowing crops to avoid high-temperature periods during grain filling, the shortened growth cycle may still affect crop yield. How to simultaneously achieve early flowering and increased yield is a core objective of ensuring food supply, and the synergistic improvement of these traits has become a research hotspot in the field of rice breeding.

[0004] Currently, some research has been conducted on genes that regulate early flowering, biomass, and yield in rice. For example, yield can be increased by editing or overexpressing photosynthesis-related genes and yield-limiting genes. However, these studies often suffer from limitations such as singular trait improvement and poor synergistic effects. No research has yet reported on genes from maize... EFHP Genes can synergistically regulate flowering time, biomass, and yield traits in rice. The main cultivated rice variety, Huanghuazhan, is widely promoted in my country and exhibits excellent resistance to rice blast and bacterial blight, along with a compact plant type and wide adaptability. However, it also has drawbacks such as a relatively long growth period, limited biomass, and insufficient potential for yield improvement; its entire growth period is 4 days later than the control variety, Yuexiangzhan. Achieving early flowering and yield-increasing modification of the Huanghuazhan variety will provide a demonstration for early flowering and yield-increasing breeding of main gramineous crop varieties, possessing significant agricultural production application value. Summary of the Invention

[0005] The purpose of this invention is to provide a method for regulating the flowering time and yield of rice by introducing and overexpressing maize-derived... EFHP Genes can be used to achieve synergistic improvement in rice, promoting early flowering and increased yield.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a genetic engineering method for creating early-flowering and high-yielding rice, using maize-derived... EFHP Genes are introduced into rice plants and overexpressed therein. EFHP The nucleotide sequence of the gene is shown in SEQ ID NO:1, wherein... EFHP Overexpression of the gene allows rice plants to simultaneously achieve earlier flowering time and increased yield.

[0007] Furthermore, the rice mentioned is a major variety that has been widely cultivated.

[0008] Furthermore, the aforementioned EFHP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0009] The present invention also provides a transgenic rice plant whose genome integrates the aforementioned EFHP Genes, and the stated EFHP The gene was overexpressed in the rice plants.

[0010] The present invention also provides a method for preparing the transgenic rice plant, comprising the following steps: (1) Constructing a structure containing the above EFHP Recombinant gene expression vectors; (2) The recombinant expression vector was introduced into rice recipient cells; (3) Cultivate the recipient cells to obtain transgenic rice plants.

[0011] The present invention also provides an application of the nucleotide sequence shown in SEQ ID NO:1 in regulating the flowering time and yield of rice.

[0012] The beneficial effects of this invention are as follows: (1) This invention uses comparative genomics to identify the MADS gene specific to the PACMAD group, and names it Early flower and high production. EFHP Through gene conversion technology, EFHP When the gene was introduced into rice, the plants exhibited earlier flowering and increased biomass yield compared to the wild type.

[0013] (2) This invention confirms that the corn source is EFHP Overexpression of the gene in rice can simultaneously regulate flowering time and yield, solving the technical problem of the difficulty in synergistically improving early flowering and increased yield in existing technologies.

[0014] (3) The method of the present invention has significant effects on the main rice varieties, which can advance the flowering time by about 7 days and increase the yield per plant by 23-35%, and has important application value.

[0015] (4) The method of the present invention is simple to operate and the genetically stable transgenic plants obtained provide new gene resources and breeding materials for rice molecular breeding. Attached Figure Description

[0016] Figure 1 The main cultivated varieties are Huang Huazhan (WT) and OE. EFHP -7 and OE EFHP -9 strains of T3 generation plants EFHP A bar chart showing the relative expression levels of genes; Figure 2 For Huang Huazhan (WT), OE EFHP- 7 and OE EFHP -9 strains of T3 generation plants flowering time statistical violin graph; Figure 3 For Huang Huazhan (WT), OE EFHP -7 and OE EFHP Phenotypic photographs of T3 generation plants of line -9 at 98 days of growth, scale bar = 30 cm; Figure 4 For Huang Huazhan (WT), OE EFHP -7 and OE EFHP -9 strains of T3 generation plants statistical bar chart; Figure 5 For Huang Huazhan (WT), OE EFHP -7 and OE EFHP -9 strain T3 generation plant average yield statistics bar chart, N=30. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0018] Example 1: PACMAD Ancestor EFHP Analysis of the evolutionary process of genes To screen for dominant genes in the evolution of the PACMAD taxa, 46 high-quality Poaceae genomes were collected from plant genome databases such as Gramene and Phytozome. Comparative genomic analysis was then performed to identify genes specific to the PACMAD taxa. EFHP Genes. Analysis results indicate that... EFHP The gene is specific to the PACMAD group and may be involved in regulating the flowering time, biomass, and yield of the PACMAD group.

[0019] To further clarify EFHP In the classification and potential functions of the MADS-box transcription factor family, we identified and found... EFHP The gene belongs to the SRF group and contains a K-box domain; its overall characteristics conform to the Mα subtype in Type I. Phylogenetic analysis shows that it is related to... EFHP Two branches with adjacent evolutionary positions EFHP The genes did not exhibit branch-specific clustering in any of the Poaceae species, suggesting that... EFHP The branch specificity may arise after the differentiation of PACMAD and BOP. Further collinearity analysis indicates that in the genome of the BOP branch, EFHP No collinearity was detected in the 10 neighboring genes on each side of PACMAD, indicating that... EFHP The specific distribution in the PACMAD branch is likely related to events of loss or gain of local genomic fragments.

[0020] EFHP The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.

[0021] Example 2: EFHP Preparation and identification of rice plants with gene overexpression 1. Construction of overexpression vectors a) Extract total DNA from maize KN5585 leaves.

[0022] b) Using the DNA obtained in step a as a template, PCR amplification was performed using primers XF657-9-F1 (5'-agctcggtacccgggTACTCAAGGACGTGTTTGACATGGCT-3', SEQ ID NO:3) and XF657-9-R1 (5'-TCGCCATCATCATGGATCCCATCATGCGATACTACT-3', SEQ ID NO:4) to obtain the amplification product.

[0023] c) Total RNA was extracted from maize KN5585 immature embryos and reverse transcribed to obtain cDNA.

[0024] d) Using the cDNA obtained in step c as a template, PCR amplification was performed using primers OE_9_F (5'-ATGATGGCGAGGAGGGACAG-3', SEQ ID NO:5) and OE_9_R (5'-CCACCATCCAGATCCAGAGCT-3', SEQ ID NO:6) to obtain the amplification product.

[0025] e) Using the amplification product obtained in step d as a template, PCR amplification was performed using primers XF657-9-F2 (5'-GGATCCATGATGATGGCGAGGAGGGAC-3', SEQ ID NO:7) and XF657-9-R2 (5'-tcatcatcgtccttgtagtCGGATcgCCACCATCCAGATCCAGAGCTTT-3', SEQ ID NO:8) to obtain the amplification product.

[0026] f) The amplification products recovered in steps b and e, together with the BamHI and SalI digested vector XF657, were subjected to homologous recombination reaction, and the recombinant products were transformed into DH5α Escherichia coli competent cells.

[0027] g) E. coli were screened by spreading them on Kana-resistant LB solid medium and sequenced to confirm that the target fragment was ligated into the XF657 vector.

[0028] 2. Obtaining recombinant Agrobacterium The recombinant plasmid XF657-NP-EFHP was electroporated into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105-XF657-NP-EFHP.

[0029] 3. Preparation of transgenic plants Embryogenic callus tissue of Huanghuazhan rice was soaked and infected with Agrobacterium tumefaciens EHA105-XF657-NP-EFHP, and then co-cultured, screened, differentiated and rooted in sequence to obtain 26 T0 generation regenerated plants.

[0030] 4. Identification of transgenic plants Leaves of T0 generation plants were collected, and genomic DNA was extracted. PCR was performed using primers XF657-9-F (5'-GTCGTCACTATCGCCTGTCA-3', SEQ ID NO:9) and XF657-9-R (5'-CCAAGTCCACCGTCAGCAT-3', SEQ ID NO:10). Plants with a 350 bp fragment amplified were considered positive.

[0031] Positive T0 generation transgenic plants are self-pollinated, and their seeds are harvested and cultivated into T1 generation plants. The identification method for positive T1 generation plants is the same as above.

[0032] Positive T1 generation transgenic plants are self-pollinated, and their seeds are harvested and cultivated into T2 generation plants. The identification method for positive T2 generation plants is the same as above.

[0033] If all T2 generation plants obtained through self-pollination are transgenic plants, then the T1 generation plant is a homozygous transgenic line, yielding an OE (Organic Evolution). EFHP -7、OE EFHP -9 strains.

[0034] 5. Expression level detection Total RNA was extracted from leaves of T3 generation plants and reverse transcribed to obtain cDNA. Using rice... Actin The gene is an internal reference (primers ACTIN-qRT-F: 5'-TGAGCATCTTGTCTCGCACAGA-3', SEQ ID NO:11; ACTIN-qRT-R: 5'-CGCCAACCTCGTCGTCATCA-3', SEQ ID NO:12). EFHP The primers for gene detection were 9_Qpcr_F1 (5'-ATGGAGGACAAGCGGAGGAAGG-3', SEQ ID NO:13) and 9_Qpcr_R1 (5'-TCGGTGGCGTCAAGATCGTCTA-3', SEQ ID NO:14). The results showed ( Figure 1 ), OE EFHP -7 and OE EFHP -9 EFHP The expression levels were 2366 times and 2680 times that of the wild type, respectively.

[0035] 6. Identification of agronomic traits Test plants: 30 wild-type (WT) rice plants, OE EFHP -7 lineage T2 generation plants (30 plants), OE EFHP -9 line T2 generation plants (30 plants).

[0036] The tested plants were planted at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Changping District, Beijing. In the early heading stage of the rice plants, the heading time of the earliest tiller was recorded as the heading time of that plant. The heading times of wild-type and OE rice were statistically analyzed separately. EFHP -7 and OE EFHP -9 Flowering period of the plants. Overexpression in the tested plants. EFHP See photos of the gene and its flowering period and results. Figure 2 , Figure 3 The wild type has a flowering time of approximately 102 days, OE EFHP -7 and OE EFHP The flowering period of the -9 plant is about 95 days, which can be as early as one week earlier.

[0037] During the late heading stage of rice plants, plant height was measured as an average from the soil surface to the tip of the highest leaf in each clump. After heading, the average height from the soil surface to the top of the highest panicle (excluding the awn) was measured. The plant height of each tested plant was recorded and statistically analyzed. The statistical results of plant height are shown below. Figure 4 Compared to wild-type rice plants, OE EFHP -7 and OE EFHP -9 plants exhibited an increased plant height phenotype. The average plant height of the wild type was 96.82 cm, OE EFHP The average plant height of the -7 variety was 104.8 cm. EFHP The average plant height of the -9 variety was 101.9 cm. See individual plant grain photos and average yield statistics for details. Figure 5 Wild-type plants accounted for an average yield of 21.8g per plant, with an OE of [missing information]. EFHP -7 and OE EFHP The yields per plant for -9 were 26.95g and 29.45g, respectively, OE EFHP The two overexpressed lines were produced at higher yields than the wild-type line.

Claims

1. A genetic engineering method for creating early-flowering and high-yielding rice, characterized in that: Corn-derived EFHP Genes are introduced into rice plants and overexpressed therein. EFHP The nucleotide sequence of the gene is shown in SEQ ID NO:1, wherein... EFHP Overexpression of the gene allows rice plants to simultaneously achieve earlier flowering time and increased yield.

2. The method according to claim 1, characterized in that, The EFHP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. A transgenic rice plant, characterized in that, Its genome integrates the contents described in claim 1 EFHP Genes, and the stated EFHP The gene was overexpressed in the rice plants.

4. A method for preparing the transgenic rice plant of claim 3, characterized in that, Includes the following steps: (1) Constructing a structure containing the contents of claim 1 EFHP Recombinant gene expression vectors; (2) The recombinant expression vector was introduced into rice recipient cells; (3) Cultivate the recipient cells to obtain transgenic rice plants.

5. The application of a nucleotide sequence shown in SEQ ID NO:1 in regulating flowering time and yield in rice.

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