Exogenous gene significantly increasing linoleic acid content in Brassica napus and its application

Abstract

The present invention relates to the field of plant hybrid breeding, and more specifically to an exogenous gene that significantly increases the linoleic acid content in Brassica napus and its application. The exogenous gene, BcaFAD2.B1, has a nucleotide sequence as shown in SEQ ID NO: 1 and an amino acid sequence as shown in SEQ ID NO: 2. The present invention can rapidly identify exogenously infiltrated new Brassica napus high-linoleic acid materials, thereby assisting in rapeseed variety improvement and providing technical support for the production of polyunsaturated fatty acid plant oils, with significant research significance for human health.

Description

Technical Field

[0001] The present invention relates to the field of plant hybrid breeding, and in particular to an exogenous gene that significantly increases the linoleic acid content of Brassica napus and an application thereof. Background Art

[0002] Brassica napus is an important oilseed crop cultivated worldwide. However, it is also a young allotetraploid species with a relatively short history of domestication and cultivation, resulting in limited genetic diversity. Among the six cultivated Brassica cultivars, in addition to B. napus, B. rapa, B. juncea, and B. aethiopica are also widely cultivated as oilseed crops. These three cultivars each share a subgenome with B. napus, yet exhibit significant differences, making them crucial germplasm resources for improving B. napus.

[0003] To broaden its genetic base, the C. c Genomic composition and A of Brassica rapa cultivars r The genomic components were introduced into Brassica napus, and a strain HLL45 was cultivated in which the linoleic acid content was increased due to the introduction of the exogenous fragments. The linoleic acid content exceeded 30%, ranging from 32% to 34%.

[0004] Linoleic acid, an ω-6 fatty acid and a type of unsaturated fatty acid, is widely found in various plant oils and fats. Linoleic acid is involved in the synthesis of various lipids in the human body, but the human body cannot synthesize linoleic acid itself and must obtain it from food. Furthermore, increased linoleic acid intake may protect against cardiovascular disease. Conventional Brassica napus rapeseed (BR) has a low linoleic acid content in nature, typically between 10% and 20%. High-linoleic acid rapeseed material has great potential for improving human health and serving as an intermediate for the artificial synthesis of DHA and DPA. However, current improvements in BR are primarily focused on increasing oleic acid content and improving quality, while research on the creation of high-linoleic acid materials is rare. Therefore, this study, which significantly increased linoleic acid content in BR through exogenous gene introduction, has important production value for breeding high-linoleic acid rapeseed varieties and could be applied to future breeding practices for high-linoleic acid varieties. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an application of introducing the exogenous gene BcaFAD2.B1 of Brassica juncea through interspecific hybridization to significantly increase the linoleic acid content of Brassica rapa. Based on hybridization and self-pollination, a large number of new Brassica rapa high-linoleic acid inbred lines with exogenous introgression are created, and related genes are identified and experimentally verified based on genomic technology.

[0006] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0007] The present invention provides an exogenous gene BcaFAD2.B1 for significantly increasing the linoleic acid content of Brassica napus, and the nucleotide sequence thereof is shown in SEQ ID NO:1.

[0008] Furthermore, the amino acid sequence of the protein encoded by the above-mentioned exogenous gene BcaFAD2.B1 is shown in SEQ ID NO:2.

[0009] The present invention also provides an application of the exogenous gene BcaFAD2.B1 in cultivating new varieties of Brassica napus.

[0010] The present invention also provides a molecular marker for increasing the linoleic acid content in Brassica napus, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0011] The present invention also provides an application of the molecular marker in improving the linoleic acid content of Brassica napus and assisting breeding.

[0012] The present invention also provides a primer pair F1 / R1 for obtaining the above-mentioned molecular marker, characterized in that: the primer pair F1 / R1 is:

[0013] F1: CGGCAGACCTTACCCCGAG,

[0014] R1:TATCGGCTTTATCGCCTTCGT.

[0015] The present invention also provides a kit for identifying high linoleic acid Brassica napus varieties, the kit comprising the above primer pair F1 / R1 and the marker primer pair CF / CR, wherein CF1: TCTCGGGAAGACCTTACGAC,

[0016] CR1: AAGTGATCAACACGAGGAAAC.

[0017] The present invention also provides a method for identifying high-linoleic acid Brassica napus varieties using the above kit, comprising the following steps:

[0018] 1) Extracting DNA from the rapeseed variety to be tested;

[0019] 2) using the DNA as a template, performing PCR amplification using the primer pair and the labeled primer pair in the kit according to claim 7;

[0020] 3) Electrophoresis:

[0021] When the primer pair F1 / R1 can amplify the target band, but the primer pair CF / CR cannot amplify the target band, it indicates that the variety contains the gene BcaFAD2.B1 and has the characteristics of high linoleic acid content;

[0022] Alternatively, when the target band cannot be amplified using primer pair F1 / R1, and the target band appears when amplified using CF / CR, it indicates that the variety does not contain the gene BcaFAD2.B1 and its linoleic acid content level is low.

[0023] Beneficial effects of the present invention:

[0024] The present invention can promote the use of mustard penetration to increase the high linoleic acid content of rapeseed, quickly identify new cabbage-type rapeseed high linoleic acid materials infiltrated exogenously, thereby assisting in rapeseed variety improvement, and providing technical support for the production of polyunsaturated fatty acid vegetable oils, which has important research significance for human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 5. Fatty acid composition distribution of HLL45 and conventional Brassica napus. NY7 is Ningyou 7, and HLL45 is a new Brassica napus variety. Fatty acid composition analysis results were obtained by planting them in Wuhan, Hubei (WH) and Hezheng, Gansu (HZ) in 2017 and 2018, respectively. LEI represents linoleic acid content, LEN represents linolenic acid content, OLE represents oleic acid content, and EA represents erucic acid content.

[0026] Figure 2 It is the synthetic source pedigree of the new Brassica napus HLL45;

[0027] Figure 3 It is an exogenous introgression of 6 Mb of E. japonica at the end of chromosome C5 in HLL45;

[0028] Figure 4 This is the phylogenetic tree of FAD2 gene;

[0029] Figure 5 The effect of C5 structural variation on fatty acid composition;

[0030] Figure 6 The heat map of FAD2 transcriptome expression in three generations of HLL45 and its parents;

[0031] Figure 7 RT-PCR results of BcaFAD2.B1 in HLL45 and its parents;

[0032] Figure 8 This is the gel image of the amplification of exogenous gene molecular markers in HLL45 and three common Brassica napus varieties;

[0033] Figure 9 This is the gel image for molecular marker verification;

[0034] Figure 10 This is a gel image showing the predicted molecular markers in the population. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.

[0036] Example 1 Creation and screening of high linoleic acid materials

[0037] Through interspecific hybridization supplemented by molecular marker-assisted selection, a new Brassica napus inbred line with the introduction of genomic components of E. napus and Brassica rapa was created. After years of trait investigation and screening, a new Brassica napus germplasm resource HLL45 with a special fatty acid composition was obtained.

[0038] The test found that the total content of linoleic acid and linolenic acid in the new Brassica napus germplasm resource HLL45 is about 42% to 45%, while the total content of linoleic acid and linolenic acid in conventional Brassica napus is about 23% on average. The total content of linoleic acid (LEI) and linolenic acid (LEN) in conventional Brassica napus NY7 is about 24.87%. Compared with conventional Brassica napus NY7, the total content of linoleic acid and linolenic acid in the new Brassica napus line HLL45 increased by about 18 percentage points, mainly contributed by linoleic acid ( Figure 1 HLL45 has stable traits and has been self-pollinated for more than 10 generations. During the creation process, five representative parents were mainly included, namely two mustard varieties C4012 and 10167 (Brassica carinata, BBCC), two cabbage varieties DLZ and SQB (Brassica rapa, AA), and the rapeseed parent HS3 (Brassica napus, AACC). Figure 2 ).

[0039] Example 2 Identification of exogenous infiltrated fragments and exogenous gene FAD2

[0040] To explore the causes of fatty acid trait variation in HLL45, a denovo genome assembly of HLL45 was performed using Illumina sequencing, ONT sequencing, and Hi-C sequencing. The assembled HLL45 genome was then compared with the published conventional rapeseed Darmor-bzh and E. coli genome C4012. At the same time, FAD2 was identified and established in HS3 and the two E. coli parents. Comparison revealed that there was an exogenous introgression of approximately 6 Mb at the end of chromosome C5 of HLL45, originating from chromosomes 50.33 to 55.94 Mb ( Figure 3 ), which contains the key gene FAD2 (BcaFAD2.B1) for synthesizing linoleic acid and linolenic acid, and the nucleotide sequence of BcaFAD2.B1 is shown in SEQ ID NO: 1:

[0041]

[0042] The amino acid sequence of the protein encoded by BcaFAD2.B1 is shown in SEQ ID NO: 2:

[0043] MGAGGRMQVSPSPKKSETDTLKRVPCETPPFTVGELKKAIPPHCFKRSIPRSFSYLIWDIIVASCFYYVATTYFPLLPHPLSYIAWPLYWACQGCVLTGVWVIAHECGHHAFSDYQWLDDTVGLIFHSFLLVPYFSWKYSHRRHHSNTGSLERDEVFVPKKKSDIKWYGKYLNNPLGRTVMLTVQFTLGWPL YLAFNVSGRPYPEGFACHFHPNAPIYNDRERLQIYVSDAGILAVCYGLYRYAAAQGVASMVSLYGVPLLIVNAFLVLITYLQHTHPSLPHYDSSEWDWLRGALATVDRDYGILNKVFHNITDTHVAHHLFSTMPHYHAMEATKAIKPILGDYYQFDGTPWVKAMWREAKCIYVEPDRQGEKKGVFWYNNKL.

[0044] Based on the tree construction results, it was found that the FAD2 of HLL45 and FAD2 of E. japonica clustered into one cluster, while the FAD2 of Brassica napus branched far away. The results showed that the FAD2 of HLL45 originated from the exogenous introgression of B1 of E. japonica ( Figure 4 ).

[0045] Example 3 Exogenous infiltration significantly increases linoleic acid content

[0046] HLL45, which harbors an introgressed structural variation (SV) in C5, was crossed with G3D001, which did not harbor the SV. This F1 hybrid was then self-pollinated to produce an F2 population of 280 offspring, designated the HG DH population. To verify whether the 6-Mb SV at the terminal end of C5 in HLL45 was the cause of its fatty acid variation, strains with and without the C5 structural variation were identified within the HG DH population based on coverage, and the differences in fatty acid composition between these strains were investigated.

[0047] The study found that there were significant changes in fatty acid composition between the materials with and without structural variation at C5 in the HG group, mainly in oleic acid and linoleic acid ( Figure 5 ), while linolenic acid did not change significantly.

[0048] The results showed that the infiltration of exogenous BcaFAD2.B1 from E. coli into C5 significantly increased the linoleic acid content and decreased the oleic acid content.

[0049] Example 4 Analysis of transcription levels of exogenous genes

[0050] To explore the impact of this structural variation on the FAD2 gene in the C5 variation region, the expression patterns of all homologous genes of FAD2 in the new Brassica napus and its Brassica napus parent HS3 were analyzed using third-generation transcriptome data and RT-PCR with seeds 25 days after pollination as the research material.

[0051] The exogenous gene BcaFAD2.B1 (BnaHLLC05G045860) in HLL45 is only expressed in the seeds of HLL45, but not in its parent Brassica napus HS3 ( Figure 6 and Figure 7 ), the results showed that the infiltration and expression of the exogenous gene BcaFAD2.B1 in HLL45 was the direct cause of the increase in its linoleic acid.

[0052] Example 5

[0053] Screening of molecular markers for increasing linoleic acid content in Brassica napus

[0054] In order to perform molecular marker-assisted selection of exogenous genes, the exogenous gene BnaHLLC05G045860 (BcaFAD2.B1) in HLL45 was compared with the homologous genes of conventional Brassica napus to identify their specific sequences.

[0055] Comparison revealed that the specific sequence of the exogenous gene in HLL45 is 424 bp in total, located between 597 bp and 1020 bp of the gene sequence. Its nucleotide sequence is shown in SEQ ID NO: 3:

[0056] Cggcagaccttaccccgaggggttcgcctgccatttccacccgaacgctcccatctacaacgaccgtgaacgcctccagatatacgtctccgacgctggcatcctc gccgtctgctacggtctctaccgttacgcggccgcgcagggagtggcctcgatggtctccctctacggagttccgcttctgatagtcaacgcgttcctcgtcttga tcacttacttgcagcacacgcatccttcgctgcctcactacgactcgtctgagtgggattggttgaggggagcgttggccaccgttgacagagaactacggaatctt gaacaaggtcttccacaacatcacggacacgcacgtggcgcatcatctgttctccacgatgccgcattatcacgcgatggaggctacgaaggcgataaagccgata.

[0057] Example 6 Molecular marker design and experimental verification of exogenous genes

[0058] To quickly identify exogenously introgressed high-linoleic acid materials, we performed a multiple sequence alignment of the exogenously introduced genes on the C5 chromosome of HLL45 and the FAD2 gene of conventional Brassica napus without exogenous introduction, and designed a pair of exogenous gene-specific molecular marker primers F1 / R1:

[0059] F1: CGGCAGACCTTACCCCGAG,

[0060] R1:TATCGGCTTTATCGCCTTCGT.

[0061] The exogenous gene primers were used to amplify the DNA of E. napus (10167), the new type of Brassica napus HLL45 and two conventional Brassica napus types (HS3 and Tapidor). The exogenous primers could amplify bands in E. napus and the new type of Brassica napus HLL45, but no bands could be amplified in conventional Brassica napus without exogenous introduction ( Figure 8 ).

[0062] Example 7

[0063] A kit for identifying high-linoleic acid Brassica napus varieties, comprising a primer pair F1 / R1 and a labeled primer pair CF / CR, wherein:

[0064] F1: CGGCAGACCTTACCCCGAG,

[0065] R1:TATCGGCTTTATCGCCTTCGT;

[0066] CF1:TCTCGGGAAGACCTTACGAC,

[0067] CR1: AAGTGATCAACACGAGGAAAC.

[0068] The method for identifying high-linoleic acid Brassica napus varieties using the above kit comprises the following steps:

[0069] 1) Extracting DNA from the rapeseed variety to be tested;

[0070] 2) Using the above DNA as a template, perform PCR amplification using the primer pair and the labeled primer pair in the kit;

[0071] 3) Electrophoresis:

[0072] When the primer pair F1 / R1 can amplify the target band, but the primer pair CF / CR cannot amplify the target band, it indicates that the variety contains the gene BcaFAD2.B1 and has the characteristics of high linoleic acid content;

[0073] Alternatively, when the target band cannot be amplified using primer pair F1 / R1, and the target band appears when amplified using CF / CR, it indicates that the variety does not contain the gene BcaFAD2.B1 and its linoleic acid content level is low.

[0074] To verify the effectiveness of the designed kit, F1 / R1 and CF / CR were selected to amplify four materials with different linoleic acid content in the HG population (two each of high and low linoleic acid content materials).

[0075] In the case of materials with high linoleic acid content, F1 / R1 could amplify the band, while CF / CR could not. In the case of materials with low linoleic acid content, F1 / R1 could not amplify the band, while CF / CR could amplify the band ( Figure 9 The results showed that the exogenous gene primer pair can be used to identify exogenous genes that significantly increase the linoleic acid content in Brassica napus, and can be used for the efficient identification of subsequent hybrid breeding of high linoleic acid varieties.

[0076] Example 6 Application Case of Kit Detection for Identifying High Linoleic Acid Brassica Napus Varieties

[0077] To verify the effectiveness of the designed kit, F1 / R1 and CF / CR were used to amplify 10 materials with different linoleic acid content in the HG population (five each of high and low linoleic acid content materials).

[0078] The materials that can amplify the bands with F1 / R1 but not with CF / CR are all high linoleic acid content materials; the materials that can amplify the bands with F1 / R1 but not with CF / CR are all low linoleic acid content materials ( Figure 10 The results showed that the designed kit can be used for the detection of high linoleic acid varieties in subsequent hybrid breeding.

[0079] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A foreign gene from Ethiopian mustard BcaFAD2.B1 The application of the method in cultivating new germplasm with high linoleic acid content of Brassica napus is characterized by: The application is to transfer the exogenous gene BcaFAD2.B1 By introducing Brassica napus into the hybridization, the linoleic acid content of the existing Brassica napus varieties is increased, and a new Brassica napus germplasm with high linoleic acid content is obtained; wherein the exogenous gene BcaFAD2.B1 The nucleotide sequence is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The new germplasm is subjected to assisted selection of an exogenous gene molecular marker, wherein the nucleotide sequence of the exogenous gene molecular marker is shown in SEQ ID NO:

3.

3. The use according to claim 2, characterized in that: The amplification primer pair F1 / R1 of the exogenous gene molecular marker is: F1: CGGCAGACCTTACCCCGAG, R1:TATCGGCTTTATCGCCTTCGT.

4. The use according to claim 1, characterized in that: Identifying the new Brassica napus germplasm with high linoleic acid content, the identification method comprises the following steps: 1) Extract DNA from the rapeseed variety to be tested; 2) Using the above DNA as a template, PCR amplification was performed using the amplification primer pair F1 / R1 and the control labeled primer pair CF1 / CR1; wherein the primer pair F1 / R1 is: F1: CGGCAGACCTTACCCCGAG, R1:TATCGGCTTTATCGCCTTCGT; The control labeled primer pair CF1 / CR1 is: CF1:TCTCGGGAAGACCTTACGAC, CR1: AAGTGATCAACACGAGGAAAC; 3) Electrophoresis: When the exogenous gene primer pair F1 / R1 can amplify the target band, and the control primer pair CF1 and CR1 cannot amplify the target band, it means that the rapeseed variety to be tested contains the exogenous gene that increases the linoleic acid content. BcaFAD2.B1 , and has the characteristics of high linoleic acid content; Alternatively, when the target band cannot be amplified using the exogenous gene primer pair F1 / R1, and the target band is amplified by the control primer pair CF1 and CR1, it indicates that the rapeseed variety to be tested does not contain the exogenous gene that increases the linoleic acid content. BcaFAD2.B1 , its linoleic acid content level is at normal levels.

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