Gene related to sweet potato vine length and application of gene in cultivation of short-vine sweet potatoes

Abstract

The invention discloses a gene related to sweet potato vine length and application of the gene in cultivation of short-vine sweet potatoes, and belongs to the technical field of gene engineering. The gene code is an amino acid sequence as shown in SEQ ID NO. 2. By improving the expression of the gene, the vine length of a sweet potato plant can be shortened, the stem internode of the sweet potato is shortened, and the leaves are denser. The gene is expected to regulate and control the vine length and / or internode length of sweet potatoes and improve germplasm resources of sweet potatoes, and has high economic and application values.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology breeding, specifically involving genes related to sweet potato vine length and their application in the cultivation of short-vine sweet potatoes. Background Technology

[0002] sweet potato( Ipomoea batatas Sweet potatoes are an important food, feed, industrial raw material, and energy crop in my country. Leafy sweet potatoes are a type of sweet potato variety used for their fresh, tender stems and leaves as vegetables, and their stem and leaf yields are higher than those of ordinary sweet potatoes. Short vines and multi-branching are the most basic biological traits of leafy sweet potatoes. In traditional breeding practices, when using conventional hybridization breeding techniques to directionally improve sweet potato traits, because sweet potatoes are allohexaploid plants with a complex genetic background and wide segregation of traits in hybrid offspring, multiple generations of self-pollination purification and field phenotypic identification are required to screen out stable lines that combine the characteristics of short vines and multi-branching with excellent stem and leaf qualities (such as tenderness, crude fiber content, and vitamin content). This process often takes 5-10 years or even longer, and the long breeding cycle and low efficiency significantly restrict the updating and iteration of leafy sweet potato varieties. In contrast, transgenic technology, through the precise introduction or editing of target genes, can achieve targeted improvement of traits related to short vines, multiple branches, and stem and leaf quality, thereby effectively shortening the breeding cycle and providing technical support for the rapid cultivation of superior leafy sweet potato varieties.

[0003] Currently, there are no reports on genes and molecular markers that control sweet potato vine length. Breeding short-vine sweet potatoes mainly relies on conventional hybridization breeding, which takes a long time, up to 5-10 years. This invention uses a transgenic technology to breed short-vine sweet potatoes, combined with a rapid transgenic method, which greatly shortens the breeding time, and genetically stable short-vine sweet potatoes can be obtained in 1-2 years. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides a gene related to sweet potato vine length and its application in the cultivation of short-vine sweet potatoes.

[0005] On one hand, the present invention provides a protein that regulates the length of sweet potato vines and / or the length of internodes, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0006] On the other hand, the present invention provides a gene that regulates the length of sweet potato vines and / or the length of internodes, characterized in that the gene encodes a protein as shown in SEQ ID NO.2.

[0007] Preferably, the sequence of the gene is the nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO.1.

[0008] On the other hand, the present invention provides an expression cassette, recombinant vector, recombinant cell, transgenic plant tissue or transgenic plant encoding the aforementioned protein.

[0009] On the other hand, the present invention provides an expression cassette, recombinant vector, recombinant cell, transgenic plant tissue or transgenic plant for expressing the aforementioned gene.

[0010] On the other hand, the present invention provides the application of the aforementioned gene or protein in regulating sweet potato vine length and / or internode length.

[0011] Furthermore, the regulation of sweet potato vine length is achieved by increasing the expression level of the aforementioned gene or protein to shorten the vine length; the regulation of sweet potato internode length is achieved by increasing the expression level of the aforementioned gene or protein to shorten the internode length of the sweet potato stem.

[0012] On the other hand, the present invention provides the use of the gene or the protein in the cultivation of short-vine sweet potatoes and / or sweet potatoes with shortened internodes.

[0013] Furthermore, the cultivation of short-vine sweet potatoes is achieved by overexpressing the aforementioned genes or proteins; the cultivation of sweet potatoes with shortened internodes is achieved by overexpressing the aforementioned genes or proteins.

[0014] Furthermore, in the aforementioned applications, the aforementioned gene is linked to a vector to construct an overexpression vector; the overexpression vector is then transferred into sweet potato using Agrobacterium-mediated transformation, and cultured to obtain sweet potato plants with elevated expression levels of the aforementioned gene.

[0015] On the other hand, the present invention also provides a method for cultivating short-vine sweet potatoes and / or sweet potatoes with shortened internodes, characterized by including the step of overexpressing the aforementioned gene or the aforementioned protein in the sweet potato.

[0016] Preferably, the overexpression of the aforementioned gene or protein in sweet potato includes the following steps: The aforementioned gene, or a recombinant vector or recombinant cell containing the aforementioned gene, is introduced into sweet potato cells, tissues, or organs, and then the transformed sweet potato cells, tissues, or organs are cultured to obtain sweet potatoes transgenic with the aforementioned gene.

[0017] On the other hand, the present invention also provides the application of the aforementioned gene in the identification of short-vine sweet potatoes and / or sweet potatoes with shortened internodes.

[0018] On the other hand, the present invention also provides a method for identifying short-vine sweet potatoes and / or sweet potatoes with shortened internodes, characterized by detecting the expression level of the aforementioned gene, and when the expression level of the gene is significantly higher than that of conventional sweet potato varieties, the sweet potato is determined to have the characteristics of short vines and / or shortened internodes.

[0019] The present invention has the following advantages and effects compared with the prior art: 1) This invention provides a new protein and its encoding gene, which has the function of regulating sweet potato vine length and sweet potato stem internode length.

[0020] 2) This invention provides a new method for regulating sweet potato vine length and / or internode length, namely, shortening the vine length and / or shortening the internode length of sweet potato stem segments by increasing the expression level of the aforementioned genes or proteins.

[0021] 3) This invention provides a new method for cultivating short-vine sweet potatoes and / or sweet potatoes with shortened internodes, namely, cultivating short-vine sweet potatoes and / or sweet potatoes with shortened internodes by overexpressing the aforementioned genes or proteins, thereby improving the yield and harvesting efficiency of tender stems and leaves, reducing harvesting costs and increasing economic benefits through plant variety improvement. Attached Figure Description

[0022] The method of the present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Figure 1 The diagram shows the construction of the overexpression vector for the target gene.

[0024] Figure 2 The image shows the PCR detection of transgenic plants. The leftmost column is the marker, WT represents wild-type sweet potato, and #1-#7 represent transgenic sweet potatoes.

[0025] Figure 3 The results of RT-qPCR quantification of the target gene in transgenic plants are shown. Different letters indicate significant differences between groups.

[0026] Figure 4 The figures show the phenotypic analysis of sweet potatoes transgenic with the target gene and wild-type sweet potatoes. Figure A is the whole plant control of potted plants (1 week of growth), Figure B is the main vine control of #1 transgenic plant and WT (4 weeks of growth), Figure C is the main vine control of #3 transgenic plant and WT (4 weeks of growth), and Figure D is the main vine control of #4 transgenic plant and WT (4 weeks of growth).

[0027] Figure 5 The figures show the leaf density of transgenic sweet potatoes and wild-type sweet potatoes (after 4 weeks of growth). Figure A is the main vine leaf density control between transgenic plant #1 and WT, Figure B is the main vine leaf density control between transgenic plant #3 and WT, and Figure C is the main vine leaf density control between transgenic plant #4 and WT. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] The gene nucleotide sequence in the example is shown in SEQ ID NO.1: ATGGATATGGATATGGATATAGTTGGGAATTATTATTCTGGGAATTTTCTAAGTGCTGCTGCGGCGGCGTCGAGTTTTTGGTCGCCGGAAATGGGTGCAGTAGTGTCTTCGCCACTGTCTTCTTCTGATACTGGGAGTTGCAGCGCTACTATGAAAGCGAATTTGTCGGATGAAGAGGTGTTGTTGGCTTCTAATAATCCGAAGAAACGCGCTGGGAGGAAGAAGTTTCGGGAGACTCGACACCCGGTGTACCGGGGAGTGAGGAGGAGGAACTCCGGGAAGTGGGTGTGTGAGGTGAGGGAGCCCAACAAGAAGTCCAGGATATGGCTGGGAACTTTCCCCACGGCTGAAATGGCGGCTAGAGCTCATGACGTGGCCGCCATCGCTCTCAGAGGCCGCTCCGCCTGTCTCAACTTCGCCGACTCGGCTTGGAGGCTTCCATTCCCGGCGTCCGCCGACCCCAAGGACATCCAGAAAGCTGCGGCGGAGGCGGCGGAGGCTTTCCGTCCAGTGGCACTGCCAGCAAACCAAAACCAAGCCCAACGGATTATTCTAGAAGCTGAAGAAGAAGAAGAAGAAGAGTGCAACAGTAGTATGAAAGAGGAACAAGTGTCGACAACCAACGAAAACGTGTTCTTCATGGACGAGGAAGCGTTTTTCGATATGCCCGGATTGCTTGCTGACATGGCTCAAGCCCTGATGCTACCTCCACCTCAATGCGCACTAGTGGACCGTTCCAATGACGTGGAGCTTGATGCTGACGTGTCACTCTGGTCTTTCTCCATTTAA The protein amino acid sequence is shown in SEQ ID NO.2: MDMDMDIVGNYYSGNFLSAAAAASSFWSPEMGAVVSSPLSSSDTGSCSATMKANLSDEEVLLASNNPKKRAGRKKFRETRHPVYRGVRRRNSGKWVCEVREPKNKSRIWLGTFPTAEMAARAHDVAAIALRG RSACLNFADSAWRLPFPASADPKDIQKAAAEAAEAFRPVALPANQNQAQRIILEAEEEEEECNSSMKEEQVSTTNENVFFMDEEAFFDMPGLLADMAQALMLPPPQCALVDRSNDVELDADVSLWSFSI.

[0031] Example 1: Construction of gene overexpression vector A target gene overexpression vector was constructed using the CDS sequence (SEQ ID NO.1) of the target gene as a template. Homologous recombination was used to construct the pEGOEP35S-G418-Gene overexpression vector (Wuhan Aidijing Biotechnology). The obtained recombinant vector was then transformed into Agrobacterium competent cells LBA4404. A schematic diagram of the vector is shown below. Figure 1 As shown.

[0032] Example 2: Obtaining and Identifying Transgenic Sweet Potatoes 1. Specific procedures for transferring overexpression vectors into Agrobacterium: (1) Take Agrobacterium competent cells LBA4404 out of the -80℃ freezer and thaw them slowly in an ice box; (2) Take 30 μL of Agrobacterium competent cells in a clean bench and add them to a 1.5 mL centrifuge tube. Then add 3 μL of the recombinant vector constructed in Example 1, gently aspirate and mix, place in an ice box and incubate on ice for 5 min. (3) After ice bath for 5 minutes, place the centrifuge tube in liquid nitrogen for 5 minutes, then quickly place the centrifuge tube in a 37°C water bath for 5 minutes, and then ice bath for 5 minutes. (4) In a clean bench, take 600 μL of non-resistant LB liquid culture medium and add it to the above 1.5 mL centrifuge tube, and place it in a shaker at 28℃ and 200 rpm for 3-4 h. (5) Centrifuge the cultured bacterial solution at 8000 rpm for 1 min at room temperature, collect Agrobacterium, discard 500 μL of supernatant, gently aspirate and resuspend the cells in the remaining 100 μL of supernatant, and then evenly spread the resuspended bacterial solution on LB solid resistance medium containing 50 μg / L kanamycin and 20 μg / L rifampin. Then invert the medium and incubate it in a 28℃ incubator for about 2 days. (6) Perform PCR detection on the single clonal spots that grow on the culture medium; (7) Use a sterile toothpick to pick up a positive single clone and inoculate it into a 50 mL centrifuge tube containing 20 mL of liquid LB medium. Shake at 28 °C and 200 rpm until turbid. Then use the glycerol preservation method to preserve the bacterial solution at -80 °C for genetic transformation.

[0033] 2. The specific process for converting the overexpression vector into sweet potato: (1) Agrobacterium containing the overexpression vector was inoculated into LB medium containing Kan and Rif and incubated overnight at 28°C until the OD600 was 0.8~1.0.

[0034] (2) Take 25-30 cm long sweet potato stem segments of Fucaishu No. 18, cut off the leaves, petioles and possible adventitious roots of the 3-4 stem nodes at the base, and make holes in the stem nodes.

[0035] (3) Immerse the treated sweet potato stem sections in the bacterial solution for 8-12 hours. During this time, gently shake the solution to ensure better inoculation.

[0036] (4) Place the infected stem segments into Erlenmeyer flasks filled with distilled water according to their morphological orientation. Incubate in the dark for 1 day.

[0037] (5) Use cefotaxime with a working solution concentration of 250 mg / L to inhibit the bacteria in the stem segments. The sweet potato stem segments and nodes are subjected to antibacterial treatment for 1 h, during which time they are gently shaken.

[0038] (6) Rinse the stem segments twice with distilled water, transplant the sweet potato seedlings into pots containing nutrient soil, and carry out routine management while waiting for the sweet potato seedlings to produce tubers.

[0039] (7) Sprouting treatment was performed on the transgenic tubers that formed tubers. Leaves from the sprouts were quick-frozen in liquid nitrogen and placed in a -80℃ freezer for DNA and RNA extraction.

[0040] 3. Verification of genetically modified sweet potatoes PCR was used to detect whether the transgenic plants obtained contained the transgenic sequence. Vector-specific primers 35S-F: CCTAACAGAACTCGCCGTAAA and 35S-R: GAAGGGTCTTGCGAAGGATAG were used. Results are shown below. Figure 2All seven transformed lines (#1-#7) amplified specific bands of the expected size (approximately 500 bp), confirming the successful transgenicity of these lines. Further, we selected the PCR-positive lines (#1, #3, #4) for transcriptional analysis. The expression levels of the target gene in the transgenic plants were detected using qRT-PCR: F: GTCTTCGCCACTGTCTTCTT and R: GCGCGTTTCTTCGGATTATTAG. Sweet potato EF1α was used as an internal reference gene: qEF1α-F: TGCCTTGTGGAAGTTTGA and qEF1α-R: GGAGTATTTGGGAGTGGTG. The relative expression levels of the target gene were calculated using the 2^(-ΔΔCt) method.

[0041] The results showed that the expression levels of the target gene were significantly upregulated in strains #1, #3, and #4. Figure 3 We subsequently selected overexpression lines #1, #3, and #4 for phenotypic experiments.

[0042] Example 3: Phenotypic Analysis of Transgenic Sweet Potatoes Transgenic sweet potatoes #1, #3, and #4 were planted in the same substrate as wild-type sweet potatoes and placed in the same growing environment (20,000 lx light, 16h light / 8h dark, 28℃, 75% humidity) under the same cultivation and management measures. Their growth phenotypes were photographed and recorded (comparing the stem length of the same part of the sweet potato). It can be seen that the transgenic plants have shorter main vines than the wild-type plants (CK: 22.24 ± 0.47 cm, #1: 10.42 ± 0.28 cm, #3: 16.83 ± 0.35 cm, #4: 9.01 ± 0.32 cm, values ​​are presented as mean ± standard error). Figure 4 The plant shape is more upright. Figure 4 BD is Figure 5 (Photo of leaves removed). Furthermore, compared to wild-type plants, the internodes of the stems in transgenic plants were significantly shortened ( Figure 4-5 ), with denser leaves ( Figure 5 ).

[0043] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A gene that regulates sweet potato vine length and / or internode length, characterized in that, The gene encodes the amino acid sequence shown in SEQ ID NO.

2.

2. A protein that regulates sweet potato vine length and / or internode length, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. An expression cassette, recombinant vector, recombinant cell, transgenic plant tissue, or transgenic plant containing the gene described in claim 1.

4. The use of the gene of claim 1 or the protein of claim 2 in regulating sweet potato vine length and / or internode length.

5. The application according to claim 4, characterized in that, The regulation of sweet potato vine length is achieved by increasing the expression level of the gene or protein to shorten the vine length; the regulation of sweet potato internode length is achieved by increasing the expression level of the gene or protein to shorten the internode length of the sweet potato stem.

6. The use of the gene of claim 1 or the protein of claim 2 in the cultivation of short-vine sweet potatoes and / or sweet potatoes with shortened internodes.

7. The application according to claim 6, characterized in that, The cultivation of short-vine sweet potatoes is achieved by overexpressing the gene or the protein; the cultivation of sweet potatoes with shortened internodes is achieved by overexpressing the gene or the protein.

8. A method for cultivating short-vine sweet potatoes and / or sweet potatoes with shortened internodes, characterized in that, The method includes the step of overexpressing the gene of claim 1 or the protein of claim 2 in sweet potato.

9. The method according to claim 8, characterized in that, Overexpression of the gene or protein in sweet potato includes the following steps: Genes, or recombinant vectors or recombinant cells containing genes, are introduced into sweet potato cells, tissues or organs, and then the transformed sweet potato cells, tissues or organs are cultured to obtain transgenic sweet potatoes.

10. A method for identifying short-vine sweet potatoes and / or sweet potatoes with shortened internodes, characterized in that, This includes detecting the expression level of the gene described in claim 1. When the expression level of the gene is significantly higher than that of conventional sweet potato varieties, it is determined that the sweet potato has the characteristics of short vines and / or shortened internodes.

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