Rice trypsin inhibitor Osbbti13, gene encoding the same and application

Abstract

This invention discloses a rice trypsin inhibitor, OsBBTI13, its encoding gene, and its applications. The amino acid sequence of the rice trypsin inhibitor OsBBTI13 is shown in SEQ ID No. 1, and its encoding gene... OsBBTI13 The nucleotide sequence is shown in SEQ ID No. 2. The Japanese white rice... OsBBTI13 After gene interference expression, the transformed plants exhibited a shortened growth period under both long-day and short-day conditions, with a significant increase in yield under short-day conditions. Furthermore, the transformed plants showed better adaptability to low temperatures, exhibiting an earlier growth period at 20°C. Therefore, silencing gene expression... OsBBTI13 Genes can be used to breed new early-maturing, high-yielding, and cold-resistant rice varieties, which have good market prospects and economic value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and more specifically, to a rice trypsin inhibitor OsBBTI13, its encoding gene, and its applications. Background Technology

[0002] Photoperiod is an environmental signal that controls many developmental responses in animals, plants, and fungi. In most plants, the transition from vegetative growth to flowering is generally controlled by environmental factors, and photoperiod plays a crucial role in flowering, synchronizing the vitality of many species with seasonal changes. Molecular mechanisms underlying plant responses to photoperiod primarily include leaf light signaling, the entrainment of diurnal rhythms, and the generation of a movement signal that propagates throughout the plant. In photoperiod-controlled plants, flowering, tuber formation, and bud formation are only a small subset of many different responses. Comparisons of known molecular mechanisms controlling these responses reveal significant differences in regulatory mechanisms, despite commonalities. For most plants, photoperiod is critical to their flowering transition. Flowering is one of the most important processes influencing plant adaptability and productivity; elucidating its mechanisms could have a significant impact not only on the flowering and growth stages of rice but also on its morphology, final yield, and stress tolerance.

[0003] Rice is a short-day (SD) plant, meaning it accelerates flowering under SD conditions, mediating the day-day response. Similar to Arabidopsis, the genetic pathways of photoperiod in rice are conserved. The diversity of flowering time among rice varieties is mainly due to differences in flowering gene alleles and their interactions. Photoperiod regulation pathways are key to controlling rice flowering, with flowering genes Hd3a and RTF1 at their core, regulated by upstream Hd1-dependent, Ehd1-dependent, and Hd1 and Ehd1-independent pathways. In rice, Hd1 promotes flowering under SD conditions but inhibits it under LD conditions. OsGI transcription is controlled by a diurnal rhythm clock, exhibiting a rhythmic expression pattern that peaks at the end of the photoperiod, similar to Arabidopsis. Mutations in osgi affect not only Hd1 but also a large group of rice genes; approximately 75% of rice transcripts show altered mutation levels throughout the day. Ghd7 encodes a CCT (CO, CO-LIKE, and CAB1) domain protein, isolated by analyzing natural variations in flowering time. Ghd7 affects the levels of Ehd1 and Hd3a transcripts but not Hd1 mRNA levels. Under LD conditions, Ghd7 inhibits Ehd1, Hd3a, and RFT1, thus delaying flowering. In summary, two independent flowering pathways exist in rice: a conserved Hd1-Hd3a pathway and a unique Ghd7-Ehd1-Hd3a / RFT1 pathway. These two pathways integrate ambient photoperiod signals to control flowering. As a long-day plant, rice has few related regulatory pathways and genes involved in its flowering network. Current research indicates the existence of 91 Bowman-Birk genes in plants, mostly distributed in flowering plants such as rice, barley, maize, soybean, peanut, and Erythrina variegata. Major functional studies focus on insect resistance, plant growth and development, and plant responses to abiotic stress. This study, through prior gene information mining, successfully cloned the first rice Bowman-Birk family gene BBTI13. Functional validation of photoperiod regulation in rice has been completed. Results show that rice transformed with the RNAi-BBTI13 interference vector exhibits earlier flowering, a significantly shorter growth period, and improved cold resistance compared to the wild type. This will provide important theoretical guidance for the molecular design and genetic improvement of rice varieties. Summary of the Invention

[0004] The purpose of this invention is to provide a rice trypsin inhibitor OsBBTI13, its encoding gene, and its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The present invention first provides a rice trypsin inhibitor OsBBTI13, the amino acid sequence of which is shown in SEQ ID No. 1.

[0006] The present invention further provides a gene encoding the above-mentioned rice trypsin inhibitor OsBBTI13, wherein the gene is... OsBBTI13 , OsBBTI13 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0007] The present invention also provides a plant interference expression vector, wherein the plant interference expression vector comprises the above-mentioned... OsBBTI13 Gene; the starting vector backbone of the plant interference expression vector is pTCK303.

[0008] The present invention also provides an Agrobacterium strain comprising the above-described plant interference expression vector.

[0009] The present invention also provides the above-mentioned OsBBTI13 Application of genes in the construction of rice with earlier growth period, higher yield, and improved cold resistance.

[0010] This invention also provides a method for advancing the growth period of rice, increasing rice yield, and improving the cold resistance of rice, the method comprising: applying [method / method] to rice... OsBBTI13 Silencing genes, inhibiting OsBBTI13 Gene expression levels.

[0011] This invention also provides a kit for constructing rice with advanced growth period / increased yield / improved cold resistance, the kit containing rice that can silence [the seed]. OsBBTI13 Gene reagents to inhibit OsBBTI13 Gene expression levels.

[0012] The beneficial effects of this invention are as follows: This invention involves extracting total RNA from rice, reverse transcribing it into cDNA, and then using the cDNA as a template for PCR amplification to obtain the cDNA shown in SEQ ID No. 2. OsBBTI13 The CDS sequence of the gene, whose encoded amino acid sequence is shown in SEQ ID No. 1, was constructed in two steps, forward and reverse, into the downstream of the Ubiquitin promoter and the downstream of the Rice intron of the plant interference expression vector pTCK303. This was then transformed into rice, and positive transgenic rice plants were obtained through screening. The results showed that RNAi interference expression... OsBBTI13 The gene not only significantly promotes earlier heading and a shorter growth period in rice, but also ensures early maturity even under relatively low temperatures of 20℃. Since the growth period of rice is a crucial target trait in breeding, its regulation not only affects the variety's utilization of light and temperature resources but also its yield potential. Especially in specific ecological regions, the growth period of a variety directly determines its adaptability. Therefore, OsBBTI13Genes hold promise for solving the problem of early maturity and increased yield in rice, and may also address the issues of introduction and adaptation in various horticultural plants, medicinal plants, and economic crops. Attached Figure Description

[0013] Figure 1 pTCK303- is an RNAi interference expression vector BBTI13 The map.

[0014] Figure 2 RNAi interference expression OsBBTI13 Identification of transgenic plants. WT is wild-type rice, RNAi- OsBBTI13 -1 and RNAi- OsBBTI13 -2 is a transgenic positive line.

[0015] Figure 3 RNAi interference expression OsBBTI13 Phenotypic characteristics of transgenic plants under short-day conditions. WT is wild-type rice, RNAi- OsBBTI13 -1 and RNAi- OsBBTI13 -2 is a transgenic positive line.

[0016] Figure 4 RNAi interference expression OsBBTI13 Phenotypic characteristics of transgenic plants under long-day conditions. WT is wild-type rice, RNAi- OsBBTI13 -1 and RNAi- OsBBTI13 -2 is a transgenic positive line.

[0017] Figure 5 RNAi interference expression OsBBTI13 Comparison of heading time in transgenic rice materials. WT is wild-type rice, RNAi- OsBBTI13 -1 and RNAi- OsBBTI13 -2 represents a transgenic positive line. SD represents short-day conditions, and LD represents long-day conditions.

[0018] Figure 6 RNAi interference expression OsBBTI13 Comparison of grain number per panicle in transgenic rice materials. WT is wild-type rice, RNAi- OsBBTI13 -1 and RNAi- OsBBTI13 -2 represents a transgenic positive line. SD represents short-day conditions, and LD represents long-day conditions. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0020] Example 1 Rice OsBBTI13 Gene cloning and construction of plant interference expression vectors Based on homologous data from the rice genome database (http: / / rice.plantbiology.msu.edu / ) OsBBTI13 The gene (LOC_Os03g60840.1) was used as the basis for PCR amplification primers Bi13F / Bi13R. Total RNA was extracted from rice, reverse transcribed into cDNA, and then amplified by PCR using the cDNA as a template. The cDNA was then recovered, cloned into the pGXT intermediate vector, and pGXT- was obtained. BBTI13 Backbone vector transformant, select pGXT- BBTI13 A single positive colony was obtained and sent for sequencing analysis to obtain rice. OsBBTI13 The CDS sequence of the gene (SEQ ID No. 2) and the amino acid sequence of the protein it encodes are shown in SEQ ID No. 1. The nucleotide sequence of the primers Bi13F / Bi13R is as follows: Bi13F: 5'-ATGAGGAGCAGCTCGGCTCTGTTC-3', Bi13R: 5'-TCAGCGGGCGGCGGCGGTGCAGCG-3'.

[0021] Using two primer pairs, Bi13F1 / Bi13R1 and Bi13F2 / Bi13R2, and with rice pGXT-BBTI13 as a template, a PCR amplification system and reaction program were constructed using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (catalog number: P505-d1). The PCR products were purified, recovered, and cloned into the pGXT intermediate vector. After double digestion with BamHI / KpnI and SpeI / SacI, the products were sequentially constructed into the pTCK303 backbone vector containing the Ubiquitin promoter and then into the RiceIntron element. The final RNAi interference expression vector pTCK303- was obtained. BBTI13 ( Figure 1 Its full sequence is shown in SEQ ID No. 3. The nucleotide sequences of primers Bi13F1 / Bi13R1 and Bi13F2 / Bi13R2 are as follows: Bi13F1: 5'-GGTACCATGAGGAGCAGCTCGGCTCTGTTC-3', Bi13R1: 5'-GGATCCTCAGCGGGCGGCGGCGGTGCAGCG-3'; Bi13F2: 5'-ACTAGTATGAGGAGCAGCTCGGCTCTGTTC-3', Bi13R2: 5'-GAGCTCTCAGCGGGCGGCGGCGGTGCAGCG-3'.

[0022] Example 2: Transformation of transgenic rice plants Seed disinfection: After shelling mature seeds, transfer them to a 50 mL Erlenmeyer flask, rinse with 75% alcohol for 2 min, then treat with 2% sodium hypochlorite on a shaker at 80 rpm for 30 min, rinse with sterile water 5-8 times, and then transfer to a petri dish with high-pressure filter paper to dry.

[0023] Callus induction: Seeds were transferred to N6-C callus induction medium (containing N6 basal medium + 2 mg / L 2,4-D) (N6 basal medium was purchased from PhytoTechnology Laboratories™, catalog number: C167). 20-30 seeds were placed in each dish and incubated at 28°C in the dark for one month to grow embryogenic callus. N6 formulation: 4.1 g N6 basal medium, 1 mL Cu / Mo / Co, vitamins and glycine, KI, 100 mg inositol, 250 mg tyrosine, 2.6 g proline, and 35 g sucrose were added to a final volume of 1 L. The pH was adjusted to 5.6. 5.0 g / L gelling agent was added, and the mixture was autoclaved at 121°C for 15 min.

[0024] Agrobacterium infection: RNAi interference expression vector pTCK303- BBTI13 Agrobacterium was transformed, and positive transformants were obtained by PCR. Single colonies were picked and cultured in LB liquid medium containing 50 mg / ml rifampicin and 50 mg / ml kanamycin on a shaker at 250 rpm for 1-3 days. OD was then adjusted. 600 The readings were between 0.6 and 1.0; the cells were collected by centrifugation at 4000×g for 5 min and resuspended in N6-AS liquid medium (N6 basal medium + 150 uM acetylsylgenone final concentration) to obtain the Agrobacterium infection solution. The OD was adjusted. 600 Readings between 0.1 and 0.2 are kept for later use. Transfer 20 g of embryogenic callus tissue to a 50 mL centrifuge tube, add 50 mL of Agrobacterium infection solution, and incubate at 28 ℃ for 30 min to fully infect. Pour out the liquid culture, dry the embryogenic callus on filter paper, and transfer it to N6-AS solid medium. Co-culture at 26 ℃ in the dark for 3 days.

[0025] Screening for resistant callus: After co-culture, the infected callus tissue was transferred to an empty 250 mL Erlenmeyer flask, and carbenicillin at a final concentration of 250 mg / L was added. The flask was then incubated in the dark at 28°C for 30 min, washed 5-8 times with sterile water, and transferred to screening medium N6-S (N6 basal medium + 50 mg / mL hygromycin). The resistant callus was then grown in the dark at 28°C.

[0026] Regenerated plant growth: The resistant callus was transferred to N6-R (MS basal medium + 0.5 mg / L 6-BAP + 0.2 mg / L NAA) plant regeneration medium and cultured at 28℃ for 15-25 days. Then it was transferred to MS-RG (MS medium) rooting medium and cultured at 28℃ for plant growth. Finally, two resistant seedlings were obtained and named RNAi-BBTI13-1 and RNAi-BBTI13-2.

[0027] Example 3: PCR molecular detection and identification of transgenic positive lines 1) Rice leaves were ground with liquid nitrogen, and genomic DNA was extracted from the rice leaves using the CTAB method.

[0028] 2) PCR amplification system and PCR reaction procedure: Reaction system: Using Beijing Tiangen's Golden Easy PCR System, the PCR amplification mixing system is as follows: Template <1 μg hypF (10 μM) 2 μL hypR (10 μM) 2 μL 2×Reaction Mix 12.5 μL Golden DNA Polymerase 0.5 μL Add ddH2O to a final volume of 25 μL. PCR reaction cycle: 3) PCR products were detected by electrophoresis at 220V and running on 1.5% agarose gel.

[0029] 4) PCR test results (see) Figure 2 a) From the top view, the target fragment of the hygromycin gene of 845bp was amplified in two transgenic rice positive lines, RNAi-BBTI13-1 and RNAi-BBTI13-2; wild-type WT was the negative control and no amplification band was found.

[0030] The nucleotide sequences of primers hypoF / hypR are as follows: hygF: 5'-TACACAGCCATCGGTCCAGA-3', hygR: 5'-TAGGAGGGCGTGGATATGTC-3'.

[0031] Example 4: Quantitative real-time PCR expression identification of transgenic positive lines 1) Rice leaves were ground with liquid nitrogen, and total RNA was extracted from the rice leaves using a plant total RNA extraction kit (Beijing Tiangen, catalog number: DP432). The RNA was transcribed into cDNA using a Novozymes reverse transcription kit (HiScript® II Q Select RT SuperMix for qPCR) and then detected and analyzed by real-time PCR.

[0032] 2) Quantitative real-time PCR system and PCR reaction procedure: Two-step reaction system: The Real-time PCR system was prepared using the Taq Pro Universal SYBR qPCR Master Mix system from Novizan Nanjing as follows: Template <100 ng RB13F (10 μM) 1 μL RB13R (10 μM) 1 μL 2 × Taq Pro Universal SYBR qPCR Master Mix 5 μL Add ddH2O to a final volume of 10 μL PCR reaction conditions: The nucleotide sequences of primers RB13F / RB13R are as follows: RB13F: 5'-TTGGCCTTTGTTCTTCTCGC-3', RB13R: 5'-ACTTGACCGCAGCTCTTGCAC-3'.

[0033] 3) Analyze the Ct values ​​given by the ABI Life PCR Q3 software.

[0034] turn out( Figure 2 (b) The relative expression levels of the target genes in the two positive strains, RNAi-OsBBTI13-1 and RNAi-OsBBTI13-2, were significantly downregulated compared with the wild type, indicating a significant interference effect.

[0035] Example 5: Phenotypic Analysis of Transgenic Positive Lines The rice was grown in an artificial climate chamber from the time the seeds germinated. The long-day condition was 16 hours of light / 8 hours of darkness, and the growth temperature was 27°C. The short-day condition was 8 hours of light / 16 hours of light, and the growth temperature was 27°C. The hydroponic nutrient solution formula was based on the standard recommended by the International Rice Research Institute (IRRI) (1000 times dilution).

[0036] like Figure 3 and Figure 4 As shown, compared with wild-type Nipponbare rice, RNAi-OsBBTI13 transgenic plants exhibit an upright plant type under both long-day and short-day conditions.

[0037] like Figure 5 As shown, under both long-day and short-day conditions, the heading dates of the transgenic lines RNAi-BBTI13-1 and RNA-BBTI13-2 were significantly earlier than those of the wild-type Nipponbare rice. The average heading date of the transgenic lines was approximately 35 days under long-day conditions and approximately 55 days under short-day conditions.

[0038] like Figure 6 As shown, the transgenic lines exhibited an average decrease of approximately 69.9% in the number of grains per ear under long-day conditions, and an average increase of approximately 63.6% under short-day conditions. Therefore, RNAi interference expression... OsBBTI13 Genetically modified rice showed reduced yields under long-day conditions but increased yields under short-day conditions.

[0039] Obviously, the present invention has been described in detail above with general descriptions and specific embodiments. However, further modifications or improvements can be made to it, such as using the CRISP / Cas9 gene editing method to verify the knockout of the OsBBTI13 gene, which is obvious to those skilled in the art. Therefore, all such modifications and technical improvements made without departing from the spirit and design of the present invention are within the scope of protection claimed by the present invention.

Claims

1. The application of silencing the OsBBTI13 gene in constructing rice with advanced growth period / increased yield, characterized by: The nucleotide sequence of the OsBBTI13 gene is shown in SEQ ID No. 2; the advanced growth period refers to an earlier heading period; the increased yield refers to an increase in the number of grains per ear under short-day conditions.

2. A method for advancing the growth period of rice / increasing rice yield, characterized in that: The OsBBTI13 gene in rice was silenced to suppress its expression level; the nucleotide sequence of the OsBBTI13 gene is shown in SEQ ID No. 2; the advancement of rice growth period refers to advancing the heading period of rice; the improvement of rice yield refers to increasing the number of grains per panicle under short-day conditions.

3. A reagent kit for constructing rice with advanced growth period / increased yield, characterized in that: The kit contains a reagent that can silence the rice OsBBTI13 gene to inhibit the expression level of the OsBBTI13 gene; the nucleotide sequence of the OsBBTI13 gene is shown in SEQ ID No. 2; the advanced growth period refers to an earlier heading period; the increased yield refers to an increase in the number of grains per panicle under short-day conditions.

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