Application of OsBB4 gene in illumination-dependent regulation of growth traits and yield traits of rice

Abstract

The invention particularly relates to application of an OsBB4 gene in illumination-dependent regulation of rice growth traits and yield traits. Wild type rice and OsBB4 silent homozygous mutant seeds are subjected to germination accelerating culture under the natural light condition and the weak light condition respectively, then seedlings are transplanted to an outdoor field for growth, photographing is carried out in the mature period, plant types are recorded, and the number of grains per ear and the thousand seed weight are counted. Results show that after natural light germination accelerating culture, the plant height, the number of grains per ear and the thousand seed weight of the mutant in the seedling stage and the mature stage are obviously lower than those of a wild type; after weak light germination accelerating culture, the plant heights of the two types have no significant difference in the mature period, but the number of grains per ear and thousand seed weight of the mutant are significantly higher than those of the wild type. According to the invention, the functional characteristics of the OsBB4 gene are clarified, a strategy for improving the gene to restore the plant type is provided aiming at the characteristic that the OsBB4 silent homozygous mutant responds to weak light, and finally the purpose of increasing the rice yield by increasing the number of grains per ear and the thousand seed weight is achieved. The invention provides important experimental basis and technical reference for rice high-yield cultivation optimization and molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving OsBB4 Application of genes in light-dependent regulation of growth and yield traits in rice. Background Technology

[0002] Rice is an important food crop globally, with my country's rice planting area reaching nearly 30 million hectares. 2 The annual output is approximately 210 million tons. In recent years, rice has made significant progress in variety improvement, production efficiency, and planting technology. However, it also faces challenges such as greenhouse climate, water shortage, reduction of arable land, and frequent pests and diseases. Seeking to increase the introduction and creation of high-quality germplasm resources and enrich their genetic diversity is particularly important for improving the breeding of high-quality, high-yield, and disease-resistant new varieties.

[0003] Currently, rice breeding goals mainly focus on high yield, high quality, and multiple resistances. Utilizing related genes to innovate germplasm resources or improve new varieties is an important aspect of bio-breeding. We need to explore more potential gene targets to improve crops. Summary of the Invention

[0004] The purpose of this invention is to provide OsBB4 Application of genes in the light-dependent regulation of growth and yield traits in rice. OsBB4 The gene-silenced rice's response to low light conditions can be used to control the rice's growth from dwarfism to a normal, ideal plant type by regulating the light intensity during the seedling stage, thereby increasing the number of grains per panicle and the thousand-grain weight, and ultimately achieving increased yield. The process is as follows: Figure 1 As shown.

[0005] The technical solution adopted in this invention is as follows: OsBB4 The application of genes in regulating growth and yield traits in rice, the aforementioned OsBB4 The CDS 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; the regulation is light-dependent; the growth traits include plant height; the yield traits include grains per ear and thousand-grain weight. Furthermore, suppressing or silencing the aforementioned OsBB4 Genes, combined with natural light germination culture, can reduce rice plant height, number of grains per panicle, and thousand-grain weight; Furthermore, suppressing or silencing the aforementioned OsBB4 Genes, combined with low-light germination culture, can increase the number of grains per panicle and the weight of a thousand grains in rice; Furthermore, the light intensity of the weak light is: 5000~8000 Lux from 8:00 to 18:00, and 18:00~22:000 Lux.

[0006] A method for increasing rice yield includes the following steps: (1) Obtain OsBB4 Rice materials in which gene function is suppressed or silenced; OsBB4 The CDS 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; (2) OsBB4 Rice materials with suppressed or silenced gene function were cultured under low light conditions to induce germination. (3) Transplant rice seedlings that have been germinated under low light conditions to the field for growth; Furthermore, the light intensity of the weak light is: 5000~8000 Lux from 8:00 to 18:00, and 18:00~22:000 Lux.

[0007] A sort of OsBB4 Mutant rice with weakened gene function, wherein the mutant rice is derived from wild-type rice. OsBB4 Gene suppression or silencing is obtained; the aforementioned OsBB4 The CDS 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. Compared with wild-type rice, the mutant rice, after being germinated and cultured under low light conditions and then transplanted to the field, has a higher number of grains per panicle and a higher thousand-grain weight.

[0008] OsBB4 The application of genes as molecular markers in screening low-light-tolerant rice germplasm resources, the OsBB4 The CDS 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.

[0009] A method for screening or creating rice varieties tolerant to low light conditions includes the following steps: (1) with OsBB4 Genes were used as selection markers to obtain rice materials with reduced expression levels or weakened functional activity. (2) The rice material obtained in step (1) was subjected to germination culture under low light conditions; (3) Transplant the rice seedlings that have been germinated in weak light conditions in step (2) to the field for growth, conduct yield trait identification, and screen out plants with increased number of grains per panicle and thousand-grain weight. Furthermore, the light intensity of the weak light is: 5000~8000 Lux from 8:00 to 18:00, and 18:00~22:000 Lux.

[0010] The main advantages of this invention are: This invention provides a new strategy and method for optimizing rice plant type and improving varieties, focusing in particular on the application of dwarfing genes in high-yield breeding with ideal plant type. It constructs a high-yield breeding model that can effectively coordinate yield components such as the number of grains per panicle and the weight of a thousand grains, providing an innovative path for breeding high-yield and widely adaptable rice varieties. Attached Figure Description

[0011] Figure 1 This is a flowchart illustrating the technical process of the present invention.

[0012] Figure 2 The construction map of the RNA interference plasmid pTCK303-OsBB4-RNAi.

[0013] Figure 3 PCR identification of some transgenic positive plants. Lane M, DL500 DNA Marker; Lane 1, negative control (ddH2O); Lane 2, RNA interference plasmid pTCK303-OsBB4-RNAi; Lanes 3-11, transgenic positive plants.

[0014] Figure 4 For some transgenic positive plants OsBB4 Gene expression level detection. WT represents wild-type Nipponbare rice; 3-11 represent transgenic positive plants.

[0015] Figure 5 for OsBB4 Growth phenotypes of silent homozygous mutant and wild-type rice seedlings after 10 days of germination culture under natural light conditions.

[0016] Figure 6 for OsBB4 Growth phenotypes of silent homozygous mutant and wild-type rice seedlings after 10 days of germination culture under low light conditions.

[0017] Figure 7 for OsBB4 Plant height of silent homozygous mutant and wild-type rice seedlings after 10 days of germination culture under natural light and low light conditions.

[0018] Figure 8 for OsBB4 The plant height of silent homozygous mutants and wild-type rice after germination culture for 10 days under natural light and low light conditions and transplanted to outdoor fields to grow to maturity.

[0019] Figure 9 for OsBB4 The number of grains per panicle in silent homozygous mutants and wild-type rice after germination culture for 10 days under natural light and low light conditions and transplanting to outdoor fields to grow to maturity.

[0020] Figure 10 for OsBB4The thousand-grain weight of silent homozygous mutants and wild-type rice after germination culture for 10 days under natural light and low light conditions and transplanting to outdoor fields for growth to maturity.

[0021] Figure 11 for OsBB4 Phenotypic characteristics of silent homozygous mutants and wild-type rice after germination culture under natural light for 10 days and transplanting to outdoor fields for growth to maturity.

[0022] Figure 12 for OsBB4 Phenotypic characteristics of silent homozygous mutants and wild-type rice after germination culture under low light conditions for 10 days and transplanting to outdoor fields for growth to maturity. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below. It should be noted that the embodiments described herein are merely illustrative examples, intended to aid in understanding the present invention, and not to cover all possible implementation methods. Based on the technical concept of the present invention, any equivalent substitutions or obvious modifications obtained by those skilled in the art without creative effort should fall within the protection scope of the present invention.

[0024] Example 1: Based on rice genome database OsBB4 Based on the CDS sequence of the gene (LOC_Os01g03340.1) (its nucleotide sequence is SEQ ID NO.1, and the amino acid sequence encoding the protein is SEQ ID NO.2), primer pairs for amplifying the RNAi fragment were designed: Os4F1: 5'-GGATCCCTAGTTCTCCGCTCGGGGTTT-3', Os4R1: 5'-GGTACCATGAGCAACACCACCATGGCTA-3'; Os4F2: 5'-GAGCTCATGAGCAACACCACCATGGCTATT-3', Os4R2: 5'-ACTAGTCTAGTTCTCCGCTCGGGGTTTGC-3'.

[0025] Total RNA was extracted from leaves of wild-type rice Nipponbare using the CTAB method and cDNA was obtained via reverse transcription. PCR amplification was performed using positive primers Os4F1 / Os4R1 and negative primers Os4F2 / Os4R2 to obtain a positive RNAi fragment (RNAi-S1) containing BamHI and KpnI restriction sites, and an antisense RNAi fragment (RNAi-S2) containing SacI and SpeI restriction sites. Subsequently, the RNAi-S1 fragment and the pTCK303 plasmid were double-digested with BamHI and KpnI. The digestion products were ligated with T4 DNA ligase to construct the recombinant plasmid pTCK303-RNAi-S1. Further double-digestion of the RNAi-S2 fragment and the recombinant plasmid pTCK303-RNAi-S1 with SacI and SpeI was performed. The digestion products were ligated with T4 DNA ligase to obtain the final RNAi-S2 fragment. OsBB4 The RNA interference plasmid pTCK303-RNAi-S1-RNAi-S2, also known as pTCK303-OsBB4-RNAi, is a gene that... Figure 2 ).

[0026] Agrobacterium-mediated transformation of rice embryogenic callus (refer to: Wang Yuzhen, Luo Jinglan, Xu Jin, Liu Xiangling. Agrobacterium-mediated genetic transformation and plant regeneration in rice [J]. Journal of North China Agricultural Sciences, 2005, 20(2): 8-11.) was used to introduce the constructed RNA interference plasmid pTCK303-OsBB4-RNAi into wild-type rice Nipponbare recipient material, resulting in 58 transgenic regenerated plants. Using the genomic DNA of the regenerated plants as templates, PCR amplification was performed using specific primers hpF (5'-GAAGATGTTGGCGACCTCGTATT-3') and hpR (5'-GATATGTCCTGCGGGTAAATAGC-3'). The regenerated plants were then identified as transgenic, and 30 positive plants were finally screened. Figure 3 , Figure 4 Positive plants were continuously self-pollinated, and selection was carried out step by step in the F2 and F3 generations to finally obtain... OsBB4 Silent homozygous mutant.

[0027] Example 2: Glass petri dishes were washed, sterilized at high temperature, and lined with sterile qualitative filter paper. Distilled water was added to moisten the filter paper. Wild-type rice Nipponbare and... OsBB4Silent homozygous mutant rice seeds were placed in petri dishes and germinated under two light conditions: outdoor natural light and indoor low light (5100 Lux light intensity from 8:00 to 18:00, 0 Lux light intensity from 18:00 to 22:00; white light source). During germination, the indoor temperature was controlled at 25-29℃, and the humidity was maintained at 75%. After 10 days of germination, the seedlings were transplanted to the outdoor field and managed with standard water and fertilizer under natural light. At maturity, the plant morphology was photographed, and the number of grains per panicle and the thousand-grain weight were recorded.

[0028] The results show that ( Figures 5 to 12 Germination and cultivation under natural light for 10 days. OsBB4 The plant height of the silent homozygous mutant rice seedlings was significantly lower than that of wild-type rice; and after 10 days of germination culture under low light conditions, OsBB4 There was no significant difference in plant height between the silent homozygous mutant rice and the wild-type rice; after germination culture under natural light for 10 days, the seedlings were transplanted to outdoor fields to grow to maturity. OsBB4 The plant height of rice seedlings from the silent homozygous mutant was significantly shorter than that of wild-type rice. OsBB4 The number of grains per panicle and the thousand-grain weight of the silent homozygous mutant rice seedlings were also significantly lower than those of wild-type rice; after germination culture under low light conditions for 10 days, they were transplanted to outdoor fields to grow to maturity. OsBB4 There was no significant difference in plant height between the silent homozygous mutant rice and the wild-type rice, but OsBB4 The number of grains per panicle and the thousand-grain weight of the silent homozygous mutant rice seedlings were significantly higher than those of wild-type rice.

Claims

1. OsBB4 The application of genes in regulating growth and yield traits in rice is characterized by: The OsBB4 The CDS sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2; The regulation is light-dependent; the growth traits include plant height; the yield traits include grains per ear and thousand-grain weight.

2. The application according to claim 1, characterized in that: inhibiting or silencing the OsBB4 gene, combined with natural light incubation, can reduce the plant height, grain number per spike, and thousand-grain weight of rice.

3. The application according to claim 1, characterized in that: inhibiting or silencing the OsBB4 gene, combined with low light germination culture, can increase the number of grains per panicle and thousand-grain weight of rice.

4. Use according to claim 3, characterized in that: The light intensity of the weak light is: 5000~8000 Lux from 8:00 to 18:00, and 0 Lux from 18:00 to 22:

00.

5. A method for increasing rice yield, characterized in that: Includes the following steps: (1) obtaining OsBB4 a rice material in which a gene function is suppressed or silenced; The OsBB4 The CDS 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; (2) OsBB4 Rice materials with suppressed or silenced gene function were germinated under low light conditions. (3) Rice seedlings that have been germinated under low light conditions will be transplanted to the field for growth.

6. The method according to claim 5, characterized in that: The light intensity of the weak light is: 5000~8000 Lux from 8:00 to 18:00, and 0 Lux from 18:00 to 22:

00.

7. A kind OsBB4 Rice mutants with weakened gene function are characterized by: The mutant rice is a variety of wild-type rice. OsBB4 Gene suppression or silencing is obtained; the aforementioned OsBB4 The CDS 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; Compared with wild-type rice, the mutant rice, after being germinated and cultured under low light conditions and then transplanted to the field, has a higher number of grains per panicle and a higher thousand-grain weight.

8. OsBB4 The application of genes as molecular markers in screening low-light-tolerant rice germplasm resources is characterized by: The OsBB4 The CDS 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.

9. A method for screening or creating rice varieties tolerant to low light conditions, characterized in that: Includes the following steps: (1) with OsBB4 Genes were used as selection markers to obtain rice materials with reduced expression levels or weakened functional activity. (2) The rice material obtained in step (1) was subjected to germination culture under low light conditions; (3) Rice seedlings that have been germinated under low light conditions will be transplanted to the field for growth, and yield traits will be identified to screen out plants with increased number of grains per panicle and thousand-grain weight.

10. The method according to claim 9, characterized in that: The light intensity of the weak light is: 5000~8000 Lux from 8:00 to 18:00, and 0 Lux from 18:00 to 22:00.

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