Application of b. subtilis protease SBT1.4 in regulating yield traits of rapeseed

Abstract

This invention discloses the application of SBT1.4 protease in regulating rapeseed yield traits. The SBT1.4 gene is expressed at the highest level in leaves; the SBT1.4 protein is located in the cell membrane. Overexpression of the SBT1.4 protease encoding gene in rapeseed reduces the number of siliques per plant and decreases aboveground biomass. SBT1.4 mutant plants increase the number of siliques per plant, increase aboveground biomass, and increase yield. Therefore, SBT1.4 protease can be used to regulate the number of siliques, biomass, and yield of rapeseed. Plants with increased silique number, increased biomass, and increased yield can be obtained by mutating or interfering with the SBT1.4 protease encoding gene to render the SBT1.4 protease inactive or reduce its activity. This is of great significance for improving crop yield.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of Bacillus subtilis protease SBT1.4 in regulating rapeseed yield traits. Background Technology

[0002] Rapeseed (Brassica napus) is the world's fifth largest crop and an important oilseed crop in my country. However, due to my country's large population and limited planting area, increasing rapeseed yield is of paramount importance in addressing the shortage of vegetable oil resources and ensuring the security of my country's oilseed supply. Rapeseed yield is mainly composed of the number of effective siliques per plant, the number of seeds per silique, and the thousand-seed weight. Plant biomass can also serve as an indicator of survival ability; plants with large initial biomass and rapid growth have stronger environmental adaptability. Therefore, research on the biomass accumulation mechanism of rapeseed is also helpful in increasing rapeseed yield.

[0003] Substance proteases (SBTs) are the second largest family of serine proteases, involved in plant embryogenesis, cuticle and epidermal formation, seed differentiation and development, organ abscission, programmed cell death, and plant responses to biotic and abiotic environments. Numerous SBT gene family members have been identified in species such as Arabidopsis, grape, and tomato. In Arabidopsis, the SBT gene family is one of the largest known protease gene families, comprising 56 members, numbered SBT1-6; in grape, there are 80 SBT members divided into 8 subfamilies; and in tomato, there are 15 SBT members divided into 5 subfamilies. Among the Arabidopsis SBT members, AtSBT1.4 is a senescence-related substance protease that participates in ABA signaling and drought tolerance regulation through interaction with OPEN STOMATA1. During Arabidopsis senescence, it downregulates branching number and silique yield. Loss-of-function mutants exhibit increased inflorescence branching, silique number, and seed yield. The increase in seed number is accompanied by a decrease in seed size, and an increase in the number of siliques per plant, leading to increased yield. However, SBT has not yet been systematically studied in rapeseed. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide an application of rapeseed subtilisin SBT1.4 in regulating rapeseed yield traits; a second objective of the present invention is to provide a reagent for reducing the activity of rapeseed subtilisin SBT1.4 by mutating or interfering with the SBT1.4 protease encoding gene, and its application in preparing a method to increase rapeseed yield; a third objective of the present invention is to provide a method for increasing rapeseed yield.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] 1. Application of Brassica subtilis protease SBT1.4 in regulating rapeseed yield traits, the nucleotide sequence encoding the Brassica subtilis protease SBT1.4 is shown in SEQ ID NO.3.

[0007] Preferably, the rapeseed yield traits are the number of pods per plant, the dry weight of the aboveground parts, or the yield per plant.

[0008] 2. The application of reagents that mutate or interfere with the SBT1.4 protease encoding gene to reduce the activity of rapeseed subtilisin SBT1.4 in the preparation of rapeseed yield-enhancing agents, wherein the nucleotide sequence of rapeseed subtilisin SBT1.4 is shown in SEQ ID NO. 3.

[0009] Preferably, the rapeseed yield is the number of pods per plant, the dry weight of the above-ground parts, or the yield per plant.

[0010] 3. A method to increase rapeseed yield by mutating or interfering with the expression of the rapeseed subtilisin SBT1.4 gene, thereby obtaining rapeseed with increased yield.

[0011] In a preferred embodiment of the present invention, the method for interfering with the expression of the rapeseed subtilisin SBT1.4 gene involves expressing a specific fragment of the rapeseed subtilisin SBT1.4 gene to form a hairpin structure through reverse complementation, thereby hindering normal gene transcription and significantly downregulating the expression level.

[0012] Preferably, the nucleotide sequence of the specific fragment is shown in SEQ ID NO.4.

[0013] The beneficial effects of this invention are as follows: This invention discloses the application of Bacillus subtilis protease SBT 1.4 in regulating rapeseed yield traits. GUS histochemical staining revealed that BnaC01.SBT1.4 is expressed in most tissues, with the highest expression level in leaves and the lowest in siliques and seeds. A transient tobacco transformation method was used to investigate the localization of BnaC01.SBT1.4 protein to the cytoplasmic membrane. Agronomical trait studies of transgenic plants showed that, compared with control plants, the number of siliques per plant in overexpressing transgenic rapeseed (168±28, 180±13) was reduced by an average of 16.31% compared to the wild type (208±1), while the number of siliques per plant in interference transgenic rapeseed (395±68, 284±39) was increased by an average of 62.96% compared to the wild type (208±1). Overexpressing transgenic rapeseed... The aboveground dry weight of the wild type (36.75±0.77, 37.97±0.07g) was on average 9.55% lower than that of the wild type (41.30±0.94g), while the aboveground dry weight of the interfering transgenic rapeseed (62.52±10.32, 55.67±0.16g) was on average 43.08% higher than that of the wild type (41.30±0.94g). The length of the siliques of the interfering transgenic rapeseed (8.83±0.82, 8.92±0.79cm) was on average 17.75% lower than that of the wild type (10.79±0.05cm). The yield per plant of the interfering transgenic rapeseed (10.335±0.58, 13.56±1.68g) was on average 83.33% higher than that of the wild type (6.52±0.65g). Agronomic trait survey data showed that overexpression and interference with BnaC01.SBT1.4 could significantly alter the number of siliques, aboveground biomass, silique length, and plant yield. Therefore, mutating or interfering with the SBT1.4 protease-encoding gene in plants to render the SBT1.4 protease inactive or reduce its activity is of great significance for regulating the number of siliques, biomass, and yield per plant and can be used to increase rapeseed yield. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0015] Figure 1 The expression of the BnaC01.SBT1.4 promoter in Arabidopsis thaliana (ad represents the expression of the whole plant, roots and leaves of Arabidopsis thaliana at different stages of vegetative growth, eh represents the expression of the promoter in flowers, siliques, main stem and lateral branches after bolting, and the scale bar is 5 mm).

[0016] Figure 2 Subcellular localization of BnaC01.SBT1.4 (GFP represents green excitation light state, Bright represents bright field, Merge represents superposition state, scale bar length is 20 μm);

[0017] Figure 3 For the detection of transgenic rapeseed plants and gene expression levels (scale bar length is 10cm);

[0018] Figure 4 For the study of transgenic agronomic traits (a: plant height; b: height of the first effective branch; c: length of the main inflorescence; d: number of siliques in the main inflorescence; e: silique length; f: number of grains per silique; g: number of siliques per plant; h: aboveground dry weight of the plant; i: grain yield per plant). Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0020] The materials used in this invention are as follows: plant materials: wild-type Arabidopsis thaliana Col-0, BnaC01.SBT1.4 overexpressing transgenic rapeseed plants, BnaC01.SBT1.4 interference transgenic rapeseed plants, experimental strains: Escherichia coli DH5α, Agrobacterium tumefaciens GV3101, vectors: pEarleyGate101, pFGC5941M, pCAMBIA1305.1.

[0021] Example 1: Cloning and expression of the BnaC01.SBT1.4 gene

[0022] Total RNA was extracted from ZS11 leaves using the EZ-10 DNA away RNA Mini-Preps Kit instructions. The RNA quality and concentration were determined by agarose gel electrophoresis. cDNA was synthesized using the USEVERBRIGHT INC. reverse transcription kit.

[0023] Using the rapeseed genome website Brassica napus database ( http: / / www.genoscope.cns.fr / brassicanapus / The chromosomal location information of SBT family members was obtained, and primers were designed based on the ORF sequence of the BnaC01.SBT1.4 gene. The specific primers are as follows:

[0024] Upstream primer: 5'-caccatggccgccaagctctctctctcct-3' (SEQ ID NO.1);

[0025] Downstream primer: 5'-gaatgactgaactgacccctgactccat-3' (SEQ ID NO.2);

[0026] The extracted cDNA was used as a template for PCR amplification. The amplification product was sequenced to obtain a 2328bp cDNA sequence, as shown in SEQ ID NO.3.

[0027] Using the pENTR / D-TOPO Cloning Kit, the amplified BnaC01.SBT1.4 gene CDS sequence was recombined into the pENTR / D-TOPO entry vector via attL1 and attL2 sites. The vector was then transformed into competent DH5α cells of E. coli. After the cloned and propagated with the correct sequence, the plasmid was extracted using the EasyPure Plasmid MiniPrep Kit and subjected to a LR reaction with the pEarleyGate101 expression vector to insert the gene sequence between the attR1 and attR2 sites, forming the pEarleyGate101-BnaC01.SBT1.4 vector for overexpression and subcellular localization.

[0028] Using the constructed overexpression vector plasmid as a template, a BnaC01.SBT1.4 interference fragment (SEQ ID NO.4) containing restriction enzyme sites was cloned. The cloned interference fragment was ligated to the pEASY-Blunt simple cloning vector to obtain the T-BnaSBT1.4 plasmid with the correct sequence. The T-BnaC01.SBT1.4 plasmid and the empty vector pFGC594M were digested with restriction endonucleases AatII and NcoI, and the two linear fragments were ligated overnight with T4 ligase. The resulting fragments were transformed into E. coli. The samples were detected and sequenced using RNAiSBT1.4-R (SEQ ID NO.5)+RPAP2 (SEQ ID NO.6) and RNAiSBT1.4-F (SEQ ID NO.7)+F35S3ND (SEQ ID NO.8) to obtain the intermediate vector pFGC5941M-Bna.SBT1.4 with antisense strand insertion. The intermediate vectors pFGC5941M-Bna.SBT1.4 and T-Bna.SBT1.4 were double-digested with restriction endonucleases XbaI and SpeI, and then ligated overnight with T4 ligase. The resulting fragments were then transformed into *E. coli*. Sequencing was performed using RNAiSBT1.4-F (SEQ ID NO.7)+OCS5NDR (SEQ ID NO.9) and RNAiSBT1.4-R (SEQ ID NO.5)+FPAP2 (SEQ ID NO.10). The plasmid with correctly sequenced plasmids was identified as the interference expression vector for BnaC01.SBT1.4. The primer sequences are as follows:

[0029] The intermediate vector pFGC5941M-Bna.SBT1.4 with antisense strand insertion was obtained by detection and sequencing using RNAiSBT1.4-R (SEQ ID NO.5)+RPAP2 (SEQ ID NO.6) and RNAiSBT1.4-F (SEQ ID NO.7)+F35S3ND (SEQ ID NO.8). The intermediate vectors pFGC5941M-Bna.SBT1.4 and T-Bna.SBT1.4 were double-digested with restriction endonucleases XbaI and SpeI, and the two fragments were ligated overnight with T4 ligase. The resulting fragments were then transformed into *E. coli* and ligated using RNAiSBT1.4-F (SEQ ID NO.7)+OCS5NDR (SEQ ID NO.9) and RNAiSBT1.4-R (SEQ ID NO.5)+FPAP2 (SEQ ID NO.10).

[0030] RNAiSBT1.4-R: 5'-actagtccatggccaacggaaagagagatgacgtgg-3' (SEQ ID NO.5)

[0031] RPAP2: 5'-ggatccacctaagcatgcatttgaaaa-3' (SEQ ID NO.6)

[0032] RNAiSBT1.4-F: 5'-tctagagacgtcgtcttgatccggttccttctacat-3' (SEQ ID NO.7)

[0033] F35S3ND:5'-ggaagttcatttcatttggagag-3'(SEQ ID NO.8)

[0034] OCS5NDR: 5'-cgatcataggcgtctcgcatatctc-3' (SEQ ID NO.9)

[0035] FPAP2: 5'-atttaaatgacgtcaggtttacattcaagacaca-3' (SEQ ID NO.10)

[0036] Based on the promoter sequence of the BnaC01.SBT1.4 gene, specific primers were designed as follows:

[0037] Upstream primer for the BnaC01.SBT1.4 gene promoter: 5'-ccgagctatggcagcagct-3' (SEQ ID NO. 11);

[0038] The downstream primer of the BnaC01.SBT1.4 gene promoter is 5'-tgcaatggcggatggattgaagaag-3' (SEQ ID NO.12).

[0039] The 1501 bp upstream sequence of the promoter was amplified using SEQ ID NO.11 and SEQ ID NO.12, and the specific sequence is shown in SEQ ID NO.13. After amplification using recombinant primers, the sequence was inserted between the restriction enzyme sites BamH1 and Pst1 using recombination to form the fusion vector pCAMBIA1305.1-BnaC01.SBT1.4. The primers for the recombinant vector are as follows:

[0040] Recombinant upstream primer: 5'-gagctcggtacccggggatccccgagctatggcagcagct-3' (SEQ ID NO. 14)

[0041] Recombinant downstream primer: 5'-gccaagcttgcatgcctgcagtgcaatggcggatggatt-3' (SEQ ID NO. 15)

[0042] The obtained fusion vector was transformed into Agrobacterium GV3101 and then infected with wild-type Arabidopsis thaliana to obtain T3 generation transgenic plants. GUS histochemical staining was used to stain these plants, and the results are as follows: Figure 1 As shown, BnaC01.SBT1.4 in Arabidopsis thaliana exhibits constitutive expression characteristics, with expression occurring in various stages and parts of the plant, although the expression levels vary. During the vegetative growth stage, low levels of expression are observed in leaves and roots. During the reproductive growth stage, expression levels are higher in leaves, highest in stems and flowers, followed by siliques, and lowest in seeds.

[0043] To determine the intracellular expression location of BnaC01.SBT1.4 protein, based on the characteristics of the yellow protein marker gene YFP fused to the pEarleyGate101-BnaC01.SBT1.4 vector, the expression of BnaC01.SBT1.4 was determined by fluorescent protein localization using a transient tobacco transformation method. The results are as follows: Figure 2 As shown, the BnaC01.SBT1.4 protein is located in the cell membrane.

[0044] Example 2: Effects of BnaC01.SBT1.4 on rapeseed growth

[0045] The pEarleyGate101-BnaC01.SBT1.4 vector was transformed into engineered bacteria and then into rapeseed ZS11. The expression levels of the BnaC01.SBT1.4 gene in the transgenic rapeseed plants were then detected. The results are as follows: Figure 3As shown in the figure. The results showed that the expression level of the BnaC01.SBT1.4 gene was increased in overexpression plants, while the expression level of the BnaC01.SBT1.4 gene was decreased in interference expression plants.

[0046] Results of the agronomic trait study of transgenic plants as follows Figure 4 As shown. Analysis revealed that compared to the wild-type ZS11, the transgenic plants showed no significant differences in plant height, height of primary effective branches, and number of pods per silique. Regarding the number of pods per plant, the overexpression plants had 168±28 and 180±13 pods, respectively, a decrease of 16.31% compared to the wild-type's 208±1 pods; the interference plants had 395±68 and 284±39 pods, respectively, an increase of 62.96% compared to the wild-type. The aboveground dry weight of the overexpression transgenic rapeseed was 36.75±0.77 and 37.97±0.07 g, respectively, compared to the wild-type. The average dry weight of the affected plants was 41.30±0.94g, a decrease of 9.55%. The average dry weight of the aboveground parts of the affected plants increased by 43.08% compared to the wild type, reaching 62.52±10.32g and 55.67±0.16g respectively. The length of the siliques of the affected transgenic rapeseed was 8.83±0.82cm and 8.92±0.79cm, a decrease of 17.75% compared to the wild type's 10.79±0.05cm. The average yield per plant (10.335±0.58g and 13.56±1.68g) increased by 83.33% compared to the wild type (6.52±0.65g).

[0047] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. Use of Brassica napus subtilisin SBT1.4 for increasing yield traits in Brassica napus, characterized in that: The nucleotide sequence encoding the rapeseed subtilisin SBT1.4 is shown in SEQ ID NO.3; the rapeseed yield trait is the number of siliques per plant, aboveground dry weight, or yield per plant; the application is to improve rapeseed yield traits by interfering with the expression of the rapeseed subtilisin SBT1.4 gene.

2. A method of improving yield traits in Brassica napus, characterized in that: Rapeseed with improved yield traits was obtained by interfering with the expression of the subtilisin SBT1.4 gene in rapeseed. The nucleotide sequence of the subtilisin SBT1.4 gene is shown in SEQ ID NO.

3. The yield traits of rapeseed are the number of siliques per plant, aboveground dry weight, or yield per plant.

3. The method of claim 2, wherein: The method for interfering with the expression of the rapeseed subtilisin SBT1.4 gene involves expressing a specific fragment of the rapeseed subtilisin SBT1.4 gene and forming a hairpin structure through reverse complementation, thereby hindering normal gene transcription and significantly downregulating the expression level.

4. The method of claim 3, wherein: The nucleotide sequence of the specific fragment is shown in SEQ ID NO.4.

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