Brassica napus bnbzip16 gene and application thereof

Abstract

The present application relates to the technical field of oil crops, and particularly relates to a Brassica napus BnbZIP16 gene and application thereof. The present application finds a key gene for regulating oil metabolism of Brassica napus through transcriptome data analysis of different oil content Brassica napus seeds, and the key gene is named as BnbZIP16 gene. The expression amount of the BnbZIP16 gene is negatively correlated with the oil content of Brassica napus seeds. More specifically, the BnbZIP16 gene or the coded protein thereof provided by the present application provides an effective implementation approach for regulating seed oil metabolism of Brassica napus, improving the oil content of Brassica napus, constructing a high-oil-content Brassica napus variety, and improving the seed fatty acid type of Brassica napus, and provides a new idea for breeding of Brassica napus.

Description

Technical Field

[0001] This invention relates to the field of oilseed crop technology, and in particular to the BnbZIP16 gene of Brassica napus and its application. Background Technology

[0002] Seed oil content is an important agronomic trait of oilseed crops. Humans can absorb up to 99% of rapeseed oil, but our current understanding of the lipid metabolism network is still limited. Effectively increasing seed fat content is one of the main goals of breeding rapeseed and other oilseed crops. Screening for genes that regulate seed oil accumulation and continuously improving the lipid metabolism network in seeds can provide important theoretical guidance for molecular breeding for oilseed improvement.

[0003] Transcriptional regulation plays an important role in lipid metabolism. The transcription factors reported to be involved in lipid metabolism mainly belong to the following gene families: AP2 / EREP (Baud and Lepiniec, 2009), B3 (Mu et al., 2013), NF-Y (Mu et al., 2013), MYB (Stracke et al., 2001), WRKY (Song et al., 2020), and bZIP (Bai et al., 2021).

[0004] The bZIP family comprises 10 subfamilies in Arabidopsis thaliana. Currently, members of subfamilie A, such as ABI5 (Li et al., 2022) and bZIP67 (Mendes et al., 2013), are mainly reported to be involved in regulating lipid metabolism. In Arabidopsis thaliana, the main function of bZIP16 is to integrate light and hormone signals to promote seed germination and cell elongation (Hsieh et al., 2012), but there are no reports of its involvement in lipid metabolism. Summary of the Invention

[0005] The present invention is based on the discovery of a gene BnaC04g09600D in transcriptome data of rapeseed seeds with different oil contents. The expression level of this gene is negatively correlated with the oil content, and the gene has the highest amino acid sequence similarity to bZIP16, a member of the G subfamily of the bZIP family. Therefore, it is named BnbZIP16.

[0006] bZIP transcription factors are the most widespread and conserved class of transcription factors in eukaryotes. The G subfamily of bZIP proteins contains three conserved, proline-rich domains at the N-terminus, including GBF1 (bZIP41), GBF2 (bZIP54), GBF3 (bZIP55), bZIP16, and bZIP68. Research on the G subfamily has primarily focused on its role in ultraviolet and blue light signal transduction and the regulation of light-responsive promoters; there are no reports on its involvement in lipid metabolism.

[0007] Based on this, the present invention provides the BnbZIP16 gene of Brassica napus and its application.

[0008] More specifically, the first aspect of the present invention provides the use of the BnbZIP16 gene or its encoded protein in any of the following:

[0009] (1) Regulating oil metabolism in rapeseed seeds;

[0010] (2) Increase the oil content of Brassica napus;

[0011] (3) Construct high-oil-content rapeseed varieties;

[0012] (4) Improve the fatty acid type of Brassica napus seeds;

[0013] The CDS region of the BnbZIP16 gene is obtained by amplification of the nucleotide sequence as shown in SEQ ID NO.10-11; or the CDS region of the BnbZIP16 gene has more than 80% homology with the sequence obtained by amplification of the primers shown in SEQ ID NO.10-11.

[0014] Those skilled in the art will understand that the gene sequence of the BnbZIP16 gene described in this invention is not completely identical in different rapeseed varieties. Therefore, CDS sequences that have more than 80% homology with the sequences amplified by the primers shown in SEQ ID NO.10-11 are all within the scope of protection of this invention.

[0015] In the application provided by this invention, the BnbZIP16 gene negatively regulates oil metabolism in Brassica napus seeds.

[0016] To achieve the above-mentioned application provided by the present invention, the oil content of rapeseed seeds is increased by inhibiting the expression of the BnbZIP16 gene in plants.

[0017] Alternatively, the content of fatty acids C16:0, C18:0, C18:1, C20:0 and / or C20:1 in rapeseed seeds can be increased by inhibiting the expression of the BnbZIP16 gene in plants.

[0018] In specific experiments, the inventors detected three paralogous genes of BnbZIP16 in Brassica napus: BnaA05g08520D, BnaA03g56410D, and BnaA04g20520D.

[0019] Therefore, in the applications provided by this invention, the gene sequence of the BnbZIP16 gene is as shown in SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.

[0020] The CDS sequence of the BnbZIP16 gene is shown in SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0021] The amino acid sequence of the protein encoded by the BnbZIP16 gene is shown in SEQ ID NO.2.

[0022] Secondly, the present invention also provides a gRNA for knocking out the rapeseed BnbZIP16 gene, the sequence of which is shown in SEQ ID NO.12-13.

[0023] Thirdly, the present invention provides a method for constructing rapeseed varieties with high oil content by partially or completely inactivating the function of the BnbZIP16 gene in the target rapeseed variety.

[0024] In the method provided by this invention, the BnbZIP16 gene in the target rapeseed variety is knocked out using the gRNA shown in SEQ ID NO.12-13 based on the CRISPR-Cas9 system.

[0025] The beneficial effects of this invention are as follows:

[0026] This invention screened a key lipid metabolism gene, BnaC04g09600D, from transcriptome data of seeds with different oil contents in Brassica napus, and named it BnbZIP16. The expression level of this gene is negatively correlated with oil content. Experimental results showed that, in terms of total fatty acid content, overexpressing BnbZIP16 reduced the content by 15%-17.9% compared to the wild type, while knocking out BnbZIP16 increased it by 12.9%-19.7% compared to the wild type. Regarding fatty acid composition, the BnbZIP16 knockout material showed varying degrees of increase in C16:0, C18:0, C18:1, C20:0, and C20:1.

[0027] This invention specifically provides the gene sequence, CDS sequence, and amino acid sequence of the encoded protein of the BnbZIP16 gene, and also provides the gRNA sequence used to create BnbZIP16 knockout materials via CRISPR-Cas9. Based on the information provided by this invention, the regulation or improvement of oil metabolism in Brassica napus seeds can be achieved, providing new ideas for Brassica napus breeding. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 The image shows the pGWC-BnbZIP16 plasmid obtained by ligating the BnbZIP16 gene into the entry vector, as provided in Example 1 of this invention.

[0030] Figure 2 The plasmid map of pHZM137-BnbZIP16 obtained by ligating the BnbZIP16 gene into a plant expression vector, as provided in Example 1 of this invention.

[0031] Figure 3 This is a map of the pCBC-DT1T2 vector plasmid used in Example 2 of the present invention.

[0032] Figure 4 The image shows the plasmid pattern of the BnbZIP16 knockout vector pHSE401-giBnbZIP16 provided in Example 2 of this invention.

[0033] Figure 5 This image shows the total fatty acid content of seeds from control rapeseed, BnbZIP16 gene-edited rapeseed, and BnbZIP16 overexpressing rapeseed, measured using a GC-QQQ instrument in Example 4 of this invention. OE represents BnbZIP16 overexpressing rapeseed, KO represents BnbZIP16 gene-edited rapeseed, and WT represents the control wild rapeseed Westar. Statistical tests were performed using t.test, *, 0.01. <p<0.05;**,0.005<p<0.01;***,0.001<p<0.005;****,p<0.0001。

[0034] Figure 6This image shows the fatty acid composition of control rapeseed, BnbZIP16 gene-edited rapeseed, and BnbZIP16 overexpressing rapeseed seeds, as measured using a GC-QQQ instrument in Example 4 of this invention. OE represents BnbZIP16 overexpressing rapeseed, KO represents BnbZIP16 gene-edited rapeseed, and WT represents the control wild rapeseed Westar. Statistical tests were performed using t.test, *, 0.01. <p<0.05;**,0.005<p<0.01;***,0.001<p<0.005;****,p<0.0001。 Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] The M1, M2, M3 and M4 culture media used in this invention are conventional M1, M2, M3 and M4 culture media.

[0037] Example 1: Cloning and Overexpression Vector Construction of the BnbZIP16 Gene

[0038] This embodiment provides the cloning and overexpression vector construction process of the BnbZIP16 gene, as follows:

[0039] Fresh Westar seeds of Brassica napus were flash-frozen in liquid nitrogen, thoroughly ground in a mortar, and 50-100 mg of powder were extracted. RNA was extracted using the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit, and cDNA was obtained using the Total Gold DNA Reverse Transcription Kit. BnbZIP16 (BnaC04g09600D) was searched in the Brassica napus genome database (https: / / www.genoscope.cns.fr / brassicanapus / ) to find the CDS sequence of BnbZIP16 (SEQ ID NO.1). Then, primers with adapters and stop codons removed were designed based on the CDS sequence: forward: agcaggctttgact ttATGGCTAGCAATGAGATGGAG (SEQ ID NO.10); reverse: tgggtctagagactttccCGTT GAGTTTTTGTAGGTATCATCAA (SEQ ID NO.11). The CDS sequence of BnbZIP16 was cloned.

[0040] PCR amplification was performed using KOD high-fidelity enzyme. The PCR product was then ligated into the AhdI-digested entry vector pGWC using an in-fusion system, named pGWC-BnbZIP16. Figure 1 The bacteria were transformed into Escherichia coli DH5α, and positive clones were screened by PCR and sequenced. The sequence is shown in SEQ ID NO.1.

[0041] The next step is to construct it into the plant overexpression vector pHZM137 using the Gateway system, and name it pHZM137-BnbZIP16. Figure 2 ).

[0042] Based on the gene sequence of BnbZ IP16 (BnaC04g09600D) in the European rapeseed genome database (https: / / www.genoscope.cns.fr / brassicanapus / ), as shown in SEQ ID NO.3, and combined with the cloned CDS sequence SEQ ID NO.1, it was determined that the BnbZIP16 gene sequence contains 12 exons and 11 introns.

[0043] In the European Rapeseed Genome Database, whole-genome sequence alignment using the CDS sequence of BnbZIP16 revealed three paralogous genes: BnaA05g08520D, BnaA03g56410D, and BnaA04g20520D, with CDS sequences SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, and gene sequences SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively. Nucleotide and amino acid sequences of the BnbZIP16 paralogous genes were aligned using the MegAlign software in DNASTAR (Table 1). The results showed that BnbZIP16 and BnaA05g08520D exhibited the highest sequence similarity at the nucleotide and amino acid levels, respectively, at 97.6% and 98.2%.

[0044] Table 1. Similarity among family members of BnbZIP16(BnaC04g09600D)

[0045]

[0046] Example 2: Construction of the BnbZIP16 gene knockout vector

[0047] This embodiment provides a method for constructing a knockout vector for the BnbZIP16 gene, as follows: Search and screen for target sites for BnbZIP16 gene editing on the CRISPR-GE website (http: / / skl.scau.edu.cn / home / ), and select two target sites: gRNA1 (SEQ ID NO.12): ccattcacaaccctggttac and gRNA2 (SEQ ID NO.13): ttgcctatcaaaagatcaag.

[0048] Primers were designed as follows: forward: ATAATGGTCTCGATTGcattcacaaccctggttacGTTTTAGAGCTAGAAATAGC (SEQ ID NO.14); reverse: ATTATGGTCTCGAAACGTAACCAGGGTTGTGAATGCAATCTCTTAGTCGACTCTAC (SEQ ID NO.15). PCR was performed using pCBC-DT1T2 as a template. The pCBC-DT1T2 vector plasmid image is shown below. Figure 3 The vector was constructed using T4 ligase and then inserted into the pHSE401 vector (provided by the laboratory of Chen Qijun, China Agricultural University) which had been digested with BsaI, forming a dual-target knockout vector named pHSE401-giBnbZIP16. Figure 4 ).

[0049] Sequencing was performed using U626-IDF (SEQ ID NO.16):TGTCCCAGGATTAGAATGATTAGGC and U629-IDF (SEQ ID NO.17):TTAATCCAAACTACTGCAGCCTGAC. The correctly sequenced plasmids were then transformed into GV3101 Agrobacterium for the transformation of Brassica napus.

[0050] Example 3: Genetic transformation of the BnbZIP16 gene and screening of positive transgenic lines

[0051] This embodiment provides the genetic transformation of the BnbZIP16 gene and the screening of positive transgenic lines, with the following steps:

[0052] (1) Disinfection: Plump rapeseed Westar seeds were soaked in 75% alcohol for 1 min, disinfected with 0.15% mercuric chloride solution for 12 min, and washed with sterile ddH2O 5-6 times before being sown on MO medium and cultured in the dark at 22℃ for 6-7 days.

[0053] (2) Infection: Agrobacterium containing plasmids pHZM137-BnbZIP16 or pHSE401-giBnbZIP16 was activated and shaken. The cells were collected by centrifugation at 4000 r / min and suspended in suspension DM and poured into empty culture dishes for later use. The hypocotyls of the seedlings were cut with sterile forceps and scalpels, each 0.8-1.0 cm in length. The cut explants were placed in a dish containing bacterial suspension and immersed for 10 min. The infected explants were dried with sterile filter paper and transferred to M1 medium and incubated in the dark at 22℃ for 48 h.

[0054] (3) Callus induction: The co-cultured hypocotyl explants were transferred to M2 medium for callus induction and cultured under light for 20 days.

[0055] (4) Bud regeneration: Transfer the explants that are growing normally and have swollen ends to the differentiation medium M3 and culture them under light. Subculture them every 20 days until green buds appear.

[0056] (5) Rooting: The differentiated green shoots are transferred to M4 medium for growth. Subculture is carried out every 20 days until roots grow. After roots grow, the shoots are transferred to flower pots to keep them moist and harden off. After hardening off, the shoots are transplanted into large pots for cultivation.

[0057] Hygromycin B (Hyg, Roche, 200 μl / L) was added to the M2, M3 and M4 culture media for screening. After hardening, the overexpression materials were sprayed with herbicide (Basta, Coolaber, 3 ml / L) for screening. DNA was extracted from the leaves of the knockout materials and sequenced to determine whether the knockout was successful.

[0058] Example 4: Determination of fatty acid content in Brassica napus seeds

[0059] This embodiment provides a method for determining the fatty acid content of rapeseed seeds, as follows: Harvested control rapeseed (WT), BnbZIP16 gene-edited and overexpressing rapeseed seeds were dried in an oven at 55℃ and thoroughly ground. Approximately 100 mg was weighed and added to 3 ml of 7.5% KOH-CH3OH (with C17:0 standard added as an internal control; heptadecanoic acid, stock solution concentration 20-30 mg / ml). The mixture was incubated in a 70℃ water bath for 4 hours, inverting and mixing several times during incubation. Then, 2 ml of HCl-CH3OH (V / V, 1:1) solution and 2 ml of 14% BF3-CH3OH solution were added, and the mixture was incubated in a 70℃ water bath for 1.5 hours. Finally, 1 ml of 0.9% NaCl and 4 ml of n-hexane were added, and the mixture was thoroughly shaken and centrifuged at 4,000 rpm for 8 minutes. The upper organic phase was transferred to a sample vial, diluted 10-fold with n-hexane, and then subjected to GC analysis.

[0060] The samples were analyzed using a gas chromatography-triple quadrupole tandem mass spectrometer (GC-QQQ, Agilent 7890A-7001B). The chromatographic column was HP-FFAP (30 mm × 0.25 mm ID, 0.25 μm; Agilent). Qualitative and quantitative analysis of the samples was performed using the Masshunter workstation software. The NIST database was used for qualitative identification. Specific method parameters are shown in Table 2.

[0061] Table 2 Operating conditions for gas chromatography-triple quadrupole tandem mass spectrometry

[0062]

[0063] The total fatty acid content was calculated by comparing the peak areas corresponding to different fatty acids with the peak area of ​​the C17:0 internal standard based on the gas chromatography analysis results. Figure 5 ), and the content of each fatty acid component ( Figure 6 (Where WT is the control rapeseed; OE-3, OE-4, and OE-5 are overexpressing rapeseed seeds; and KO-24, KO-28, and KO-34 are gene-edited rapeseed seeds.)

[0064] The experimental results show that, in terms of total fatty acid content, the overexpression of BnbZIP16 in rapeseed materials decreased by 15%-17.9% compared to the wild type, while the knockout of BnbZIP16 in rapeseed materials increased by 12.9%-19.7% compared to the wild type. Regarding fatty acid composition, the knockout materials showed varying degrees of increase in C16:0, C18:0, C18:1, C20:0, and C20:1. Therefore, this invention provides a method for significantly increasing the oil content of Brassica napus seeds.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. BnbZIP16 The application of a gene or its encoded protein in the negative regulation of oil accumulation in rapeseed seeds is characterized by: The BnbZIP16 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

2. BnbZIP16 The application of genes or their encoded proteins in increasing the oil content of rapeseed is characterized by: By inhibiting BnbZIP16 The expression level of the gene increases the oil content of Brassica napus seeds; BnbZIP16 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

3. BnbZIP16 The application of genes or their encoded proteins in the construction of high-oil-content Brassica napus varieties is characterized by: By inhibiting BnbZIP16 The expression level of the gene increases the oil content of Brassica napus seeds; BnbZIP16 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

4. BnbZIP16 The application of genes or their encoded proteins in improving the fatty acid type of rapeseed seeds is characterized by: By inhibiting BnbZIP16 The expression level of the gene increases the content of fatty acids C16:0, C18:0, C18:1, C20:0 and / or C20:1 in rapeseed seeds; BnbZIP16 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

5. The application according to any one of claims 1-4, characterized in that, The BnbZIP16 The gene sequence is shown in SEQ ID NO.

3.

6. The application according to any one of claims 1-4, characterized in that, The BnbZIP16 The CDS sequence of the gene is shown in SEQ ID NO.

1.

7. A method for constructing rapeseed varieties with high oil content, characterized in that, The target rapeseed variety BnbZIP16 Gene function is partially or completely inactivated; The target rapeseed variety is Brassica napus; The BnbZIP16 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

8. The method according to claim 7, characterized in that, The BnbZIP16 The CDS sequence of the gene is shown in SEQ ID NO.

1.

9. The method according to claim 7, characterized in that, Based on the CRISPR-Cas9 system, gRNA sequences as shown in SEQ ID NO. 12-13 were used to target the rapeseed varieties. BnbZIP16 Genes are knocked out.

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