Brassica napus BnaKIX8 gene for regulating and controlling silique size and application of brassica napus BnaKIX8 gene

Abstract

The invention discloses a brassica napus BnaKIX8 gene for regulating and controlling the pod size and application of the brassica napus BnaKIX8 gene. According to the invention, the BnaKIX8 gene is obtained by cloning from brassica napus, and the BnaKIX8 gene is constructed into a plant overexpression vector and transformed into arabidopsis Col-0 for functional verification. Experimental results show that compared with a wild type, the BnaKIX8 gene overexpressed plant silique length is remarkably shortened, the number of seeds is reduced, the seed size and thousand seed weight are remarkably increased, and it is indicated that the BnaKIX8 gene plays an important role in regulating plant organ development, especially in the aspects of silique and seed morphology.

Description

Technical Field

[0001] The present invention relates to the field of gene function, and more specifically to a method for regulating silique size in Brassica napus. BnaKIX8 Genes and their applications. Background Art

[0002] The size and morphology of plant organs largely determine the yield and quality of crops. Therefore, the study of the regulatory mechanism of organ development is not only of great theoretical significance, but also provides key genetic resources for molecular breeding of high-yield crop varieties. In recent years, with the development of genomics and molecular biology, the genes related to regulating plant organ size have been gradually analyzed, including KIX (KINASE-INDUCIBLE DOMAIN INTERACTING) family genes have attracted widespread attention due to their functions in plant cell cycle, organ development and hormone signaling pathways.

[0003] The KIX (KINASE-INDUCIBLE DOMAIN INTERACTING) domain mediates the interaction with the transcription factor activation domain, and participates in regulating cell division and differentiation in plants, thereby affecting the organ development and final morphology of plants. KIX8 As a key member of this family, it has significant phenotypic regulatory effects in a variety of plants, including Arabidopsis, soybean, tomato, and Medicago truncatula. KIX8 Loss-of-function mutants exhibit large leaves and seeds; in tomatoes KIX8 The mutant produced larger fruits with thicker skin; gmKIX8-1 The mutant seeds and leaves were significantly larger; in Medicago truncatula, KIX8 Loss of function also leads to increased leaf and seed size, whereas ectopic expression of this gene in Arabidopsis thaliana results in decreased cotyledon area and seed weight. KIX8 Genes involved in regulating plant organ size are common across multiple species.

[0004] Brassica napus L. is a globally important oilseed crop and a major cultivated variety in China, enjoying widespread cultivation and high economic value. However, limited understanding of the genetic mechanisms regulating its yield traits means that there is still significant room for improvement in its yield. In particular, key agronomic traits such as silique length, seed number, and 1000-seed weight lack clear molecular regulatory mechanisms, significantly limiting the development of new high-yield, high-quality varieties.

[0005] although KIX8 Significant progress has been made in the study of genes in model plants, but there has been no clear report on their role in Brassica napus. KIX8 The functions of homologous genes, especially their mechanisms in regulating silique development and yield traits, remain largely unknown. BnaKIX8 Genes not only help to deeply understand the regulatory mechanism of organ development, but also provide new genetic resources and theoretical basis for the molecular breeding of high-yield rapeseed varieties. Summary of the Invention

[0006] The present invention aims to provide a method for preparing Brassica napus L. BnaKIX8 Genes and their application in regulating the size of plant organs, especially in the development of siliques, solve the problem of lack of functional analysis and utilization of key regulatory genes of rapeseed yield traits in existing technologies, thereby providing a theoretical basis and genetic resource support for the molecular design breeding of high-yield rapeseed varieties.

[0007] To achieve the above objectives, the present invention provides the following technical solutions: The invention provides a kind of Brassica napus BnaKIX8 Brassica napus BnaKIX8 The gene is a nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence having at least 95% homology thereto, and the Brassica napus BnaKIX8 The gene has the function of regulating the size of siliques in plants.

[0008] On the other hand, the present invention also provides the above-mentioned Brassica napus BnaKIX8 The protein encoded by the gene has an amino acid sequence shown in SEQ ID NO: 2 and has the function of regulating plant silique length, seed size and 1000-seed weight.

[0009] On the other hand, the present invention also provides a transgenic plant cell, wherein the exogenously expressed Brassica napus BnaKIX8 gene, and the gene is under the regulation of a functional promoter.

[0010] In another aspect, the present invention further provides a transgenic plant comprising the transgenic plant cell described above. In one embodiment, the plant is Arabidopsis thaliana or Brassica napus.

[0011] On the other hand, the present invention also provides a method for regulating the size of plant siliques, the method comprising: BnaKIX8 The gene is introduced into plant cells to obtain transgenic plants with shortened siliques, larger seeds and increased 1000-grain weight. In one embodiment, the gene is expressed by constructing an overexpression vector pCambia1302-BnaKIX8The Agrobacterium is introduced into plant cells through Agrobacterium-mediated transformation. In one embodiment, the Agrobacterium is GV3101, and the plant is Arabidopsis Col-0 or Brassica napus.

[0012] On the other hand, the present invention also provides Brassica napus BnaKIX8 Application of genes in regulating plant silique size, preparation of Brassica napus containing BnaKIX8 Genetically modified plants, relative to wild-type plants, in which Brassica napus BnaKIX8 The expression of genes increased; among them, Brassica napus BnaKIX8 The base sequence is shown in SEQ ID NO: 1. In one embodiment, the plant is Brassica napus or Arabidopsis thaliana.

[0013] The present invention is obtained by cloning from Brassica napus BnaKIX8 The gene was constructed into a plant overexpression vector and transformed into Arabidopsis Col-0 for functional verification. The experimental results showed that compared with the wild type, BnaKIX8 The silique length of the gene-overexpressing plants was significantly shortened, the number of seeds was reduced, while the seed size and 1000-seed weight were significantly increased, indicating that BnaKIX8 Genes play an important role in regulating plant organ development, especially the size of siliques and seeds.

[0014] This invention is the first to functionally verify the BnaKIX8 The role of genes in plant organ size regulation has expanded our understanding of KIX The understanding of the biological functions of family genes provides a theoretical basis and genetic resources for high-yield molecular breeding of crops such as rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] tú1 Brassica napus BnaKIX8 Gene cloning PCR followed by electrophoresis detection (M: DL2000 DNA Marker; 1: BnaKIX8 PCR product); tú2 Recombinant overexpression vector pCambia1302-BnaKIX8 Build processes; tú3 A is pCambia1302 vector Nco Ⅰ Single enzyme digestion electrophoresis (M: DNA Ladder (0.1-10 kb)), 3B is the overexpression vector pCambia1302-BnaKIX8 Electrophoresis detection after bacterial solution PCR (M: DL2000 DNA Marker; 1-7 are transformed single colonies); tú4A is the resistance screening of transgenic Arabidopsis thaliana; 4B is the PCR identification of positive transgenic Arabidopsis seedlings (DL2000 DNA Marker, 1-6 are the screened positive seedlings, 7 is the wild-type control); tú5 The figure shows the phenotypic comparison between wild-type and overexpressing Arabidopsis T1-1 and T1-6. tú6 Comparison of silique traits between wild type and overexpressed Arabidopsis thaliana; tú7 Comparison of seed size between wild-type and gene-overexpressing Arabidopsis; tú8 for wild-type and overexpressed Arabidopsis BnaKIX8 Comparison of expression levels. DETAILED DESCRIPTION

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0017] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0019] Example 1 is Brassica napus BnaKIX8 Gene cloning 1 Materials and Methods 1.1 Materials Seeds of the spring Brassica napus L. cultivar Westar were collected from the greenhouse of the Provincial Key Laboratory of Growth and Development Regulation, Yichun University. The greenhouse culture conditions were 28°C during the day with 16 h of light and 18°C ​​at night with 8 h of light. RNA was extracted from leaves after seedling emergence.

[0020] 1.2 Experimental reagents and instruments Plant total RNA extraction kit (Beijing Tiangen), plasmid mini-extraction kit (Beijing Tiangen), agarose gel recovery kit (Beijing Tiangen), pMD19-T vector (Baoriyi Biotechnology Co., Ltd.), PrimeSTAR® Max DNA Polymerase (Baoriyi Biotechnology Co., Ltd.), PCR instrument (eppendorf), gel imaging system (Gel DOC 1000), electrophoresis instrument (Junyi JY600c), and competent Escherichia coli DH5α were homemade by strains preserved in our laboratory.

[0021] 1.3 Methods 1.3.1 Total RNA extraction Select fresh Brassica napus leaves and take an appropriate amount. Wrap the leaves in tin foil and quickly place them on ice. Add liquid nitrogen to a pre-sterilized mortar and pestle, then quickly add the prepared leaves and grind them into a powder. Add liquid nitrogen and grind three times to ensure the leaves are thoroughly ground. Total RNA is extracted from the ground rapeseed powder according to the TIANGEN RNAsimple product instructions. The extracted RNA samples are tested for purity by electrophoresis gel.

[0022] 1.3.2 BnaKIX8 Gene cloning The RNA obtained above was reverse transcribed into cDNA, and the gene was cloned by RT-PCR. Primers were designed using Primer 5.0 software (Table 1). RT-PCR amplification conditions were as follows: 98°C initial denaturation for 1 minute, 98°C denaturation for 10 seconds, 56°C annealing for 15 seconds, and 68°C extension for 1 minute, for 35 cycles; final extension at 68°C for 5 minutes. PCR products were examined by 1% agarose gel electrophoresis. Bands matching the target fragment size were recovered and excised. The recovered products were ligated with the pMD-19T vector overnight at 16°C and transformed into competent Escherichia coli DH5α cells. The cells were cultured in a culture medium containing resistance for 14–16 hours. Positive colonies were selected for colony PCR analysis. Colonies that detected the target band by electrophoresis were saved and sent to Shanghai Biotechnology for sequencing.

[0023] surface Primer sequence information 2 Results and Analysis BnaKIX8 Gene cloning: Using the reverse transcribed cDNA of total RNA from Brassica napus Westar leaves as a template, after PCR amplification and gel electrophoresis, the amplified result was about 970 bp ( tú1 The PCR product was excised and recovered, TA-ligated, and transformed. The positive clone was sent to Shanghai Bioengineering for sequencing. The results showed that the obtained sequence was 972 bp in size, as shown in the sequence list SEQ No. 1. The sequencing results were compared with the amplified sequence and the fragments were consistent, successfully verifying the clone. BnaKIX8 Genetic accuracy.

[0024] Example 2 is Brassica napus BnaKIX8 Construction of gene overexpression vector and transformation of Arabidopsis thaliana 1 Materials and Methods 1.1 Test materials Arabidopsis thaliana wild type Columbia seeds (stored by the Yichun University Laboratory).

[0025] 1.2 Main reagents and instruments Plant genomic DNA extraction kit, (Shanghai Biotech) seamless cloning kit (Beijing Tiangen), Nco Ⅰ restriction enzymes (NEB Biotechnology Co., Ltd.), PrimeSTAR ® Max DNA Polymerase (Bao Ri Yi Biotechnology Co., Ltd.), SYBR ® Premix Ex Taq™ II (Tli RNaseH Plus) (Baori Biotechnology Co., Ltd.), 2× EsTaq MasterMix (Kangwei Century Biotechnology Co., Ltd.), Silwet L-77, sodium hypochlorite, isopropanol, sucrose, kanamycin (Kan), rifampicin (Rif), hygromycin (Hyg), centrifuge (Centrifuge 5418), PCR instrument (Eppendorf), StepOnePlus Real-Time PCR instrument, shaker (ZWY-100H), gel imaging system (Gel DOC1000), electrophoresis apparatus (Junyi JY600c), constant temperature water bath (HHS-20-4), and ice machine; primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. (Table 1).

[0026] 1.3 Host bacteria and vectors Escherichia coli DH5α competent cells (preserved by the laboratory of Yichun University), Agrobacterium tumefaciens GV3101 competent cells (self-made), and pCambia1302 (hereinafter referred to as p1302 for convenience).

[0027] 1.4 BnaKIX8 Construction and identification of gene plant overexpression vectors 1.4.1 Acquisition of target genes according to BnaKIX8 The full-length CDS sequence of the gene was used to design specific primers (1302-BnaKIX8-F and 1302-BnaKIX8-R, Table 1) to contain the target gene. BnaKIX8 PCR amplification was performed using the T vector as a template; PCR amplification system: Primer-F 2 μL, Primer-R 2 μL, cDNA 3 μL, PrimeSTAR Max 25 μL, ddH2O 18 μL.

[0028] 1.4.2 Construction and identification of overexpression vectors Build via seamless cloning p1302-BnaKIX8 , Schematic diagram of overexpression destination vector ( tú2 ).use Nco I pair of carriers p1302 Perform single enzyme digestion, enzyme digestion system: pCambia1302 plasmid 15 μl, rCutsmart buffer 5 μl, Nco I 1 μl, ddH2O 29 μl, after the band size was verified by electrophoresis, the gel was cut and recovered, and then EasyGeno Single Assembly Mix was used for connection at 50℃ for 15min. BnaKIX8 Gene ligation into vector p1302 The ligation products were transformed into DH5α Escherichia coli competent cells and screened with solid culture medium (LB+Kan). Then, single colonies on the culture medium were randomly picked for colony PCR using primers (1302-BnaKIX8-F and 1302-BnaKIX8-R, Table 1). The plasmids with correct amplified bands were extracted and sent to Shanghai Biotechnology for sequencing verification.

[0029] 1.5 Genetic transformation of Arabidopsis plants Freeze-thaw method p1302-BnaKIX8 The recombinants were introduced into Agrobacterium competent cells and then screened. Monoclonal colonies on the culture medium were picked and added to liquid culture medium (LB+Kan+Rif), and shaken in a 28°C shaker for 36-48 h. The shaken bacterial solution was added to a centrifuge tube and centrifuged at 6000 rpm at 4°C for 5 min. The supernatant was aspirated with a pipette, taking care not to aspirate the bacteria. 15 ml of buffer was added to the centrifuge tube, and centrifuged at 5000 rpm at 4°C for 5 min. Buffer was added again until the OD600 of the bacterial solution was about 0.8, and then 1 μl of Silwet-L77 was added and mixed by inversion. The inflorescence was soaked in the above solution for 20 seconds. The inflorescence was infected 2-3 times during the entire growth period. After the infection was completed, it was placed in a light-proof plastic box for dark culture. After 24 h, it was placed in a light culture room for culture. The seeds were harvested after the pods matured and recorded as the T1 generation.

[0030] 1.6 Obtaining transgenic positive plants The harvested seeds were spread on selective medium (1 / 2 MS + 30 μg / ml hygromycin (Hyg)), and then placed in a 4°C refrigerator for vernalization for 2-3 days. Then, they were moved to a well-lit incubator and cultured until seedlings grew. Positive seedlings survived, while non-positive seedlings died. Positive seedlings were transplanted to nutrient-rich soil, and the seeds were harvested after the pods matured, which were recorded as the T2 generation.

[0031] 1.7 Detection of gene expression levels by qRT-PCR Two overexpression proteins synthesized by reverse transcription BnaKIX8The cDNA of the transgenic Arabidopsis positive plants (T1-1, T1-6) and wild type Col-0 were used as templates. BnaKIX8 Sequence design of qPCR primers (Table 1). The reaction system was 10 µL: 2×SYBR Green Mix 5 µL, upstream and downstream primers 0.4 µL each, 50×ROX Dye 0.2 µL, cDNA 1 µL, ddH2O 3 µL. Reaction program: 95°C 30 s; 95°C 5 s, 55°C 30 s, 72°C 1 min 40 cycles; 95°C 15 s, 60°C 1 min, 95°C 15 s. Each sample was replicated three times using 2 －∆∆CT The relative expression levels were calculated by the method, and the expression levels of the wild type were used as the reference standard. Graphpad10.1 software was used for significant difference analysis and plotting.

[0032] 1.8 Seed trait analysis and data processing Seed traits such as seed size, silique length and width, number of seeds in silique, and 1000-seed weight were analyzed during the maturity stage of Arabidopsis thaliana.

[0033] Arabidopsis seeds and siliques were photographed using a stereoscope, and the images were measured using ImageJ software. Graphpad 10.1 and SPSS 27 software were used to analyze the significant differences in the data and plot them.

[0034] 2 Results and Analysis 2.1 BnaKIX8 Obtaining the target fragment Select wild-type Arabidopsis plants that grow well in soil. BnaKIX8 The T vector was used as a template to amplify BnaKIX8 The gene fragment band size is about 720 bp, which is consistent with the band size of the clone in Example 1.

[0035] 2.2 Plant overexpression vectors p1302-BnaKIX8 Construction Extract the plasmid of p1302 overexpression strain and use Nco I performed single enzyme digestion on the vector p1302 and recovered the target band ( tú3 A, and then connected with the target gene fragment using seamless cloning ligase. After successful connection, it was transformed into DH5α Escherichia coli and screened with culture medium. Single colonies were picked and colony PCR was performed using primers (1302-BnaKIX8-F and 1302-BnaKIX8P-R, Table 1). The bright band size was about 720 bp ( tú3 B), and BnaKIX8 The gene fragment sizes match, indicating BnaKIX8 The gene has been ligated into the p1302 vector.

[0036] 2.3 Agrobacterium-mediated genetic transformation of plants and acquisition of transformed Arabidopsis plants The recombinant plasmid was transformed into Agrobacterium GV3101 using the freeze-thaw method. Specific primers (1302-BnaKIX8-F and 1302-BnaKIX8-R) were used for bacterial testing, and the target band size was consistent with expectations. Transgenic T1 seeds were screened using a screening medium (1 / 2 MS + 30 μg / ml hygromycin (Hyg)). Positive plants had dark green leaves, long hypocotyls, and relatively long roots. Non-transgenic plants had yellow leaves and were unable to grow normally. tú4 A). After the T1 generation positive plants grew up, DNA was extracted and PCR identification was performed using the hygromycin Hyg1022 universal primer. Six randomly selected resistant plants all amplified the target band of approximately 1000 bp, while the wild-type control did not amplify the target band ( tú4 B), indicating the gene BnaKIX8 It has been successfully integrated into the Arabidopsis genome, and two transgenic plants (T1-1 and T1-6) were randomly selected for phenotypic analysis. 2.4 Overexpression BnaKIX8 Analysis of transgenic Arabidopsis expression levels Overexpression of 2 BnaKIX8 RNA was extracted from the positive transgenic Arabidopsis plants (T1-1, T1-6) and qRT-PCR analysis was performed. The results showed that compared with wild-type Arabidopsis plants, the two transgenic plants had BnaKIX8 The expression levels were significantly upregulated ( tú8 ).

[0037] 2.5 Overexpression BnaKIX8 Phenotypic identification of transgenic Arabidopsis During the flowering and maturity stages of the overexpressing plants, we conducted phenotypic observations on two of the overexpressing plants (T1-1 and T1-6). We found that the rosette leaves of the two overexpressing Arabidopsis plants were wrinkled and smaller, and the flowering period was earlier than that of the wild type ( tú5 A). In terms of plant shape, T1-1 is taller than the wild type, while T1-1 and T1-6 are significantly shorter ( tú5 B) The number of primary branches in both overexpressing plants remained unchanged. We focused on analyzing seed size, 1000-seed weight, silique length, and seed number in siliques.

[0038] 2.6 Overexpression BnaKIX8 Analysis of transgenic Arabidopsis silique traits When the overexpression plants T1-1 and T1-6 were about to mature, the siliques 3-10 positions above the base were taken to measure the length and width of the siliques and the number of seeds in the siliques. BnaKIX8 The silique lengths of the overexpression lines were 9.82±0.23 mm and 7.64±0.45 mm, which were significantly shorter than the wild type (14.40±0.82 mm). Only T1-6 had a significantly smaller silique width than the wild type ( tú6 In addition, the number of seeds in the wild-type silique was 59.00±1.58, while the number of seeds in the T1-1 and T1-6 siliques was 17.00±3.53 and 6.60±1.14, respectively, which were significantly reduced. The silique area / seed number of T1-1 and T1-6 were 0.51±0.08 mm, respectively. 2 , 0.88±0.06 mm 2 , compared with 0.22±0.02 mm of the wild type 2 Both significantly increased ( tú6 B). At the same time, it was observed that the number of shrunken pods in the overexpression strain increased, indicating that the overexpression BnaKIX8 This will result in a decrease in seed density.

[0039] 2.7 Overexpression BnaKIX8 Analysis of transgenic Arabidopsis seed traits Observation under a stereomicroscope revealed that overexpression in Arabidopsis BnaKIX8 gene, the seed size is larger than that of the wild type ( tú7 The 1,000-grain weight and size (area) of Arabidopsis seeds were calculated, and it was found that the average seed size (area) of the wild-type strain was 0.12±0.01 mm. 2 , the average thousand-grain weight was 20.10±0.55 mg; BnaKIX8 The average seed size (area) of the overexpression lines was 0.17±0.02 mm 2 , 0.16±0.01 mm 2 , the average thousand-grain weights were 29.79±0.89 mg and 23.74±0.34 mg respectively. Statistical analysis showed that compared with the wild-type strain, BnaKIX8 The seed area and 1000-grain weight of the overexpression strains were significantly increased. BnaKIX8 The effect of increasing seed size and weight may be due to reducing seed density.

Claims

1. Brassica napus BnaKIX8 A gene characterized by Brassica napus BnaKIX8 The gene is a nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence having at least 95% homology thereto, and the gene has the function of regulating silique size in plants.

2. Brassica napus according to claim 1 BnaKIX8 A gene characterized by The protein encoded by the gene has an amino acid sequence shown in SEQ ID NO: 2, and has the function of regulating plant silique length, seed size and 1000-seed weight.

3. A transgenic plant cell, characterized in that: The plant cell exogenously expresses the Brassica napus protein according to claim 1 or 2 BnaKIX8 gene, and the gene is under the control of a functional promoter.

4. A transgenic plant, characterized in that: The plant comprises the transgenic plant cell according to claim 3. The transgenic plant according to claim 4 , wherein the plant is Arabidopsis thaliana or Brassica napus.

6. A method for regulating the size of plant siliques, characterized in that: The method comprises the steps of: BnaKIX8 The gene is introduced into plant cells to obtain transgenic plants with shortened silique length, larger seeds and increased thousand-grain weight.

7. The method according to claim 7, wherein the gene is expressed by constructing an overexpression vector pCambina1302- BnaKIX8 And introduced into plant cells through Agrobacterium-mediated transformation. 8 . The method according to claim 8 , wherein the Agrobacterium is GV3101, and the plant is Arabidopsis thaliana Col-0 or Brassica napus.

9. The Brassica napus of claim 1 BnaKIX8 The application of a gene in regulating the size of plant siliques is characterized in that: Preparation of Brassica napus BnaKIX8 Genetically modified plants, relative to wild-type plants, in which the Brassica napus BnaKIX8 The expression of the gene is increased; wherein the Brassica napus BnaKIX8 The base sequence is shown in SEQ ID NO:

1.

10. The use according to claim 7, characterized in that The plant is Brassica napus or Arabidopsis thaliana.

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