A ShN / AINV3.1 gene for regulating sugarcane germination rate and its application

Abstract

The present invention discloses a ShN / AINV3.1 gene for regulating sugarcane germination rate and its application. The nucleotide sequence of the sugarcane ShN / AINV3.1 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. Research in the present invention has found that overexpressing the ShN / AINV3.1 gene can significantly increase the germination rate of sugarcane, increasing the length of sugarcane sprouts within a given timeframe and accelerating the germination rate of sugarcane. This invention provides technical support for improving sugarcane varieties and promoting the development of the sugarcane industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a ShN / AINV3.1 gene for regulating the germination rate of sugarcane and an application thereof. Background Art

[0002] Sugarcane (Saccharum spp.) is the most important sugar crop in my country and the world, and is the main raw material for sugar production in my country. About 90% of the country's total sugar production comes from sugarcane.

[0003] In plants, sucrose invertase (EC 3.2.1.26, INV) catalyzes the irreversible hydrolysis of sucrose into glucose and fructose and is a key enzyme regulating sucrose metabolism. Furthermore, sucrose invertase has been shown to contribute to many aspects of plant growth and development, organogenesis, sugar transport, stress responses, carbon allocation, phloem unloading, source / sink regulation, and the regulation of sugar composition and levels in sink tissues.

[0004] Sucrose invertases are divided into two major categories according to the pH value: acid invertase subfamily (acid INV sub-family) and neutral / alkaline INV sub-family (i.e. N / AINV). Among them, acid invertase can be further divided into cell wall invertase (cell wall invertase, CWINV) and vacuole invertase (vacuole invertase, VINV; also known as soluble acid invertases, SAINV), while neutral / alkaline invertase is classified as cytoplasmic invertase.

[0005] In Arabidopsis, the expression of the AtINV gene is associated with the growth and development of floral organs and seeds. Increased cell wall invertase activity in Arabidopsis accelerates flowering and increases seed yield by nearly 30%. Inhibiting the expression of the N / AINV gene in tobacco leads to stunted pollen development and male sterility. Inhibiting INVINH1 in tomatoes increases INV activity, delays leaf senescence, and increases seed and fruit yield.

[0006] Studies have shown that GhVIN1-mediated hexose signaling may act as an early event to control gene expression and, in turn, regulate ovule epidermal cell differentiation. Silencing GhVIN1 also inhibits a set of regulatory genes through sugar signaling, thereby preventing cotton fiber initiation from the ovule epidermis. Using RNAi to knock down the cotton target gene GhVIN1 impaired male and female fertilization, with reduced seed coat GhVIN1 expression being the primary driver of reduced female flower fertility. Silencing the tomato SiN / AINV7 gene increases ROS content in pollen, reduces pollen viability, and results in parthenogenetic fruit formation. Mutating OsVIN2 in rice results in smaller grains, suggesting that OsVIN2 affects sucrose metabolism, thereby regulating grain size. Expression of the sorghum SbVIN1 gene begins early in seed development and reaches its highest level 29 days after pollination. Summary of the Invention

[0007] The purpose of the present invention is to deeply explore the gene function of ShN / AINV3.1 gene in sugarcane seed germination, so as to provide important guiding significance for breeding sugarcane varieties with high seed germination rate using modern biotechnology.

[0008] The present invention provides a ShN / AINV3.1 gene for regulating sugarcane germination rate, the nucleotide sequence of which is shown in SEQ ID NO.1, specifically:

[0009] ATGAAGCGGGTGTCGTCGCACGTCTCGCTGGCCTCGGAGGCGGAGATCAATCTCGATCTGTCGCGC

[0010] CTCATCATCGACAGGCCGCAGCGGTTCACGCTGGAGCGGAAGCGCTCCTTCGACGAGCAGTCGTG

[0011] GAGCGAGCTCTCGCACTCCCACTCCCACCGCAACAACGACGGCTTCGACAGCGTGCTGCAGTCGC

[0012] CCGCATTCCCGTCCGGCGGATTCGACTCGCCTTTCTCCATCGGCACGCATTTCGGCGGGGGCGGCCC

[0013] GCACCCGCTGGTCAACGAGGCGTGGGAGGCGCTCAGGAAATCCGTCGTCTACTTCCGGGAACAGC

[0014] CCGTCGGTACCGTCGCTGCCGTGGATCATGCGTCCGAGGAAGTGCTCAACTATGATCAGGTCTTTGT

[0015] GAGGGATTTTGTTCCGAGTGCATTGGCTTTTCTGATGAACAATGAGACTGACATAGTGAAGAATTTT

[0016] CTCTTGAAAACTCTTCACCTTCAGAGCTCTGAGAAAATGGTAGACCGGTTCAAGCTTGGAGCAGGA

[0017] GCGATGCCTGCCAGTTTCAAGGTGGACCGTAACAAAAACAGAAACACTGAAACCTTAGTTGCTGAT

[0018] TTTGGTGAGAGTGCAATCGGCAGGGTGGCACCGGTTGACTCTGGATTTTGGTGGATCATTCTCCTTC

[0019] GGGCGTATACAAAGTACACCGGAGATGTTAGTTTGTCGGAATCACCTGATTGCCAGAAGTGCATGA

[0020] GGTTGATACTGAATCTCTGCTTATCTGAAGGATTTGATACTTTTCCAACTCTGCTTTGCACAGATGGC

[0021] TGCTCAATGATTGATCGTCGAATGGGTATATATGGTTATCCCATTGAGATCCAAGCCCTATTCTATATG

[0022] GCATTAAGATGTGCTCTCCAAATGCTCAAGCCAGAGGGCGAAGGGAAGGATTTCATAGAGAAGATA

[0023] GGGCAACGGCTACATGCACTAACCTACCACATGAGGAACTACTTCTGGCTAGATTTTCACCAGCTGA

[0024] ATAACATATACAGATACAAAACAGAAGAGTATTCCCACACAGCTGTGAACAAGTTTAACGTCATTCC

[0025] GGATTCCATTCCTGATTGGGTTGTTTGATTTCATGCCATGCCGAGGAGGCTACTTCCTTGGCAATGTCA

[0026] GCCCTGCTATGATGGATTTCCGGTGGTTTGCCCTTGGCAATTGCATTGCCATTGTATCATCTCTAGCTA

[0027] CCCCAGAACAGTCAGTTGCTATAATGGATCTGATTGAGGAAAAGTGGGATGAGCTCGTTGGTGAGA

[0028] TGCCTCTGAAGATATGCTATCCTGCTCTCGAGAATCATGAGTGGAGAATTATCACTGGCTGTGACCC

[0029] CAAGAACACCCGGTGGAGTTACCACAATGGAGGATCGTGGCCAGTTCTTCTGTGGCTGCTTGACAGC

[0030] AGCCTGCATCAAGACTGGTAGGCCACAGATGGCAAAACGTGCCATGAGCTCGCTGAGTCGAGGCT

[0031] GCTCAAGGACGGCTGGCCGGAGTACTACGATGGCAAGCTAGGAAGATTCGTTGGTAAGCAGGCCA

[0032] GGAAGTTCCAAACCTGGTCCATTGCAGGTTACCTCGTCGCCCGCATGATGCTGGAGGACCCATCAA

[0033] CACTGATGATGATCTCCATGGAGGAGGACCGGCCTGTGAAGCCGACTATGCGGCGGTCAGCATCATGGAATGCCTGA.

[0034] The present invention also provides a protein encoded by the sugarcane ShN / AINV3.1 gene, the amino acid sequence of which is shown in SEQ ID NO. 2, specifically:

[0035] MKRVSSHVSLASEAEINLDLSRLIIDRPQRFTLERKRSFDEQSWSELSHSHSHRNNDGFDSVLQSPAFPS

[0036] GGFDSPFSIGTHFGGGGPHPLVNEAWEALRKSVVYFREQPVGTVAAVDHASEEVLNYDQVFVRDFVP

[0037] SALAFLMNNETDIVKNFLLKTLHLQSSEKMVDRFKLGAGAMPASFKVDRNKNRNTETLVADFGESAI

[0038] GRVAPVDSGFWWIILLRAYTKYTGDVSLSESPDCQKCMRLILNLCLSEGFDTFPTLLCTDGCSMIDRRM

[0039] GIYGYPIEIQALFYMALRCALQMLKPEGEGKDFIEKIGQRLHALTYHMRNYFWLDFHQLNNIYRYKTE

[0040] EYSHTAVNKFNVIPDSIPDWVDFFMPCRGGYFLGNVSPAMMDFRWFALGNCIAIVSSLATPEQSVAIM

[0041] DLIEEKWDELVGEMPLKICYPALENHEWRIITGCDPKNTRWSYHNGGSWPVLLWLLTAACIKTGRPQ

[0042] MAKRAIELAESRLLKDGWPEYYDGKLGRFVGKQARKFQTWSIAGYLVARMMLEDPSTLMMISMEEEDRPVKPTMRRSASWNA*.

[0043] The ShN / AINV3.1 gene encodes a protein that is localized to the Golgi apparatus, a site for protein processing and involved in plant cell wall formation. This protein can enhance the germination rate of sugarcane stems. The ShN / AINV3.1 gene and its biomaterials can be used to improve sugarcane germplasm resources and increase the germination rate of sugarcane seeds.

[0044] The present invention also provides a biological material containing the sugarcane ShN / AINV3.1 gene, which is a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

[0045] The recombinant expression vector can be constructed using existing plant expression vectors, such as binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment. When constructing a recombinant expression vector using the ShN / AINV3.1 gene, any of a variety of enhancing, constitutive, tissue-specific, or inducible promoters can be added before the transcription initiation nucleotide. These promoters can be used alone or in combination with other plant promoters. Furthermore, when constructing a recombinant expression vector using the ShN / AINV3.1 gene, enhancers, including translational enhancers and transcriptional enhancers, can be used. These enhancer regions can be the ATG start codon or adjacent region start codons, but must be in frame with the coding sequence to ensure correct translation of the entire sequence. The translation control signals and start codons can be derived from a variety of sources, including natural or synthetic sources. The translation initiation region can be derived from the transcriptional initiation region or a structural gene.

[0046] The recombinant expression vector carrying the ShN / AINV3.1 gene can be transformed into plant cells or tissues through conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc.

[0047] The expression cassette particularly refers to an expression cassette containing the sugarcane ShN / AINV3.1 gene.

[0048] The transgenic cell line refers in particular to an artificially constructed transgenic cell line stably expressing the sugarcane ShN / AINV3.1 gene or the protein encoded by the sugarcane ShN / AINV3.1 gene.

[0049] The recombinant bacteria particularly refer to Escherichia coli or Agrobacterium constructed using the ShN / AINV3.1 gene.

[0050] The present invention also provides a primer for amplifying the sugarcane ShN / AINV3.1 gene. The primer sequence is shown in SEQ ID NO.3-4, specifically:

[0051] F:5'-CGA GGATCC ATGAAGCGGGTGTCGTCGCA-3'; (the underline indicates the BamHI restriction site)

[0052] R:5'-CGA AGGCCT GGCATTCCATGATGCTGACCG-3' (the underline indicates the Stul restriction enzyme cleavage site).

[0053] Furthermore, the present invention also protects the use of the sugarcane ShN / AINV3.1 gene, the protein encoded by the sugarcane ShN / AINV3.1 gene, or the biological material containing the gene in regulating sugarcane seed and stalk germination. The biological material refers to a recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacteria containing the sugarcane ShN / AINV3.1 gene.

[0054] Furthermore, the present invention also protects the use of the sugarcane ShN / AINV3.1 gene, the protein encoded by the sugarcane ShN / AINV3.1 gene, or biological materials containing the gene in improving sugarcane germplasm resources. The biological materials include recombinant expression vectors, expression cassettes, transgenic cell lines, or recombinant bacteria containing the sugarcane ShN / AINV3.1 gene.

[0055] Furthermore, the present invention also protects the use of the sugarcane ShN / AINV3.1 gene, the protein encoded by the sugarcane ShN / AINV3.1 gene, or biological materials containing the gene in sugarcane breeding, wherein the breeding objective is to cultivate sugarcane varieties with a high germination rate. The biological materials include recombinant expression vectors, expression cassettes, transgenic cell lines, or recombinant bacteria containing the sugarcane ShN / AINV3.1 gene.

[0056] This study analyzed the function of the sugarcane ShN / AINV3.1 gene through genetic transformation. Using Agrobacterium tumefaciens EHA105, the ShN / AINV3.1 gene, unique to sugarcane, was transferred into sugarcane for functional verification. Results showed that overexpressing the ShN / AINV3.1 gene significantly increased the germination rate of sugarcane, increasing the length of sugarcane sprouts over a given period and accelerating the germination rate. This research has significant implications for sugarcane variety improvement and promoting the development of the sugarcane industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is a clone of the sugarcane ShN / AINV3.1 gene.

[0058] Figure 2 It is the PCR identification of transgenic sugarcane ShN / AINV3.1 gene.

[0059] Figure 3 It is the PCR identification of transgenic sugarcane Bar gene.

[0060] Figure 4 It is the identification of transgenic sugarcane ShN / AINV3.1 protein.

[0061] Figure 5 This is a picture of sugarcane sprouting.

[0062] Figure 6It is the data analysis of sugarcane germination and sprout length. DETAILED DESCRIPTION

[0063] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0064] Unless otherwise specified, the culture media in the examples are all common commercially available products.

[0065] Example 1

[0066] 1. CDS sequence of sugarcane ShN / AINV3.1 gene

[0067] The CDS sequence of the sugarcane ShN / AINV3.1 gene was cloned using the homologous cloning method. According to the rice OsN / AINV3 sequence, the forward primer (5'-CGAGGATCCATGAAGCGGGTGTCGTCGCA-3') and the reverse primer (5'-CGAAGGCCTGGCATTCCATGATGCTGACCG-3') were designed. The target gene ShN / AINV3.1 was amplified using sugarcane cDNA as a template. The reagents used were Max DNA Polymerase (TaKaRa Code No. R045A). PCR amplification reaction system and reaction conditions are shown in Table 1.

[0068] Table 1

[0069]

[0070] Take 5 μL of PCR product and perform 1% agarose gel electrophoresis. Figure 1 The results showed a single amplified product band of approximately 1674 bp in length, consistent with the target product length, indicating successful PCR amplification and the generation of a PCR product of the target length. Sequencing of the PCR product yielded the cDNA sequence of the sugarcane ShN / AINV3.1 gene. The nucleotide sequence of the ShN / AINV3.1 gene described herein is shown in SEQ ID NO. 1.

[0071] 2. Amino acid sequence encoded by the sugarcane ShN / AINV3.1 gene

[0072] The full-length cDNA sequence of the sugarcane ShN / AINV3.1 gene was converted into an amino acid sequence using Snapgene software. It was determined that the sequence encodes 557 amino acids, which is the protein encoded by the ShN / AINV3.1 gene of the present invention. The amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

[0073] 3. Construction of expression vector

[0074] The PCR product was electrophoresed on a 1% agarose gel and excised using a gel recovery kit. The product was then digested with double enzymes and then ligated with the expression vector pCB302 (stored in our laboratory and cited in the literature as “Nannan Zhang, et al., Engineering Artificial MicroRNAs for Multiplex Gene Silencing and Simplified Transgenic Screen, Plant Physiology, Volume 178, Issue 3, November 2018, Pages 989-1001, https: / / doi.org / 10.1104 / pp.18.00828”).

[0075] Enzyme digestion and ligation reaction system (20 μL): Add 2 μL of 10x T4 DNA Ligase Buffer, 100 ng of the digested vector fragment, 100 ng of the target fragment, and 1 μL of T4 DNA Ligase into a centrifuge tube.

[0076] After ligation at 16°C for 30 minutes, 10 μL of the ligation product was transformed into E. coli DH5α competent cells. After transformation, the cells were plated on kanamycin-resistant LB solid medium and cultured at 37°C for 12 hours. PCR identification of the bacterial solution was performed. Ten single colonies were selected for PCR identification. The identification primers are as follows:

[0077] The forward primer was 35S PPDK-F (5'-GTCACGTAGTAAGCAGCTCTCGG-3') and the reverse primer of the target gene was (5'-ATAACCATATATACCCATTCGACGATC-3').

[0078] The PCR amplification reaction system and reaction conditions are shown in Table 2.

[0079] Table 2

[0080]

[0081] 5 μL of the PCR product was run on a 1% agarose gel electrophoresis to detect the target band, which was approximately 810 bp. 100 μL of the bacterial suspension corresponding to 3-5 positive bands was sampled for sequencing. The bacterial suspension containing the correct sequence was saved for plasmid extraction and transfection into Agrobacterium tumefaciens EHA105 for genetic transformation.

[0082] 4. Genetic transformation of sugarcane

[0083] (1) Actively growing ROC22 plants were selected from the field, and callus tissue was induced on MY medium (MS + 2 mg / L 2,4-D + 30 g / L sucrose + 7.0 g / L agar powder) with young heart leaves 5-7 cm above the growth point. The induced callus tissue was subcultured on MY medium every 20 days. After 2-3 subcultures, actively growing callus tissue was selected for Agrobacterium transformation experiments.

[0084] (2) A single clone of Agrobacterium strain EHA105 carrying the pCB302 plasmid was cultured in 25 mL of liquid YEP medium containing 50 mg / L kanamycin and 50 mg / L rifampicin at 28°C with shaking until OD600 = 0.5. The cells were collected by centrifugation at 5000 rpm for 5 min and resuspended in an equal volume of liquid MS medium containing 150 μmol / L acetosyringone (AS). The cells were shaken at 200 rpm for 2 h. The pretreated callus cells were placed in the bacterial solution and co-cultured for 20 min. The bacterial solution was then aspirated, the infected cells were blotted dry on sterile filter paper, and then co-cultured on solid MS medium containing 2 mg / L 2,4-D and 150 μmol / L AS for 3 days. The culture conditions were 28°C and dark.

[0085] After co-cultivation, the embryonic cell clusters were washed with liquid MS medium containing 500 mg / L carbenicillin (Carb) and 2 mg / L 2,4-D, and then selected for culture on MSC medium supplemented with 500 mg / L Carb. Resistant calli were selected for subculture every two weeks. After four weeks of selection, the resulting resistant calli were transferred to MD medium containing 30 mg / L glufosinate and 500 mg / L Carb for differentiation, inducing somatic embryo formation and germination. When the differentiated resistant seedlings reached approximately 2 cm in height, they were transferred to root-promoting medium MR (1 / 2 MS + 50 g / L sucrose + 2.0 mg / L NAA) containing 30 mg / L glufosinate and 500 mg / L Carb for root promotion. When the roots of the seedlings reached 5 m in length, they were moved outdoors for 2-3 days of hardening. The seedlings were then removed, washed, and fixed in a nutrient medium.

[0086] 5. Molecular identification of genetically modified sugarcane

[0087] (1) PCR identification of transgenic sugarcane

[0088] The surviving transgenic sugarcane was marked with a serial number using a pipette tip. The transgenic sugarcane DNA was extracted by cutting sugarcane leaves and PCR identification was performed using specific primers. The identification primers for the target gene ShN / AINV3.1 were: forward primer 35S PPDK-F (5'-GTCACGTAGTAAGCAGCTCTCGG-3') and reverse primer (5'-ATAACCATATATACCCATTCGACGATC-3'). The identification primers for the Bar gene were: Bar-F: 5'-ACAAGCACGGTCAACTTCC-3', Bar-R: 5'-CTTCAGCAGGTGGGTGTAG-3'. The PCR products were detected using 1% agarose gel. The results are shown in Figure 2. Figure 2 、 Figure 3 shown.

[0089] Depend on Figure 2-3 It can be seen that the transgenic sugarcane lines line1, line2, and line3 can all amplify specific target fragments and resistance genes (Bar genes), proving that the target vector has been successfully integrated into the sugarcane genome.

[0090] In addition, among the 22 plants tested, 20 were PCR-positive, accounting for 90.9% of all tested plants. Among them, 4 positive plants could only detect the Basta glufosinate-ammonium resistance exogenous gene, indicating that some gene breakage occurred during the Agrobacterium transformation process. The remaining 16 positive plants contained the target gene ShN / AINV3.1 and the Basta glufosinate-ammonium resistance gene.

[0091] (2) Detection of target gene expression protein in transgenic sugarcane

[0092] Transgenic sugarcane and wild-type WT leaf tissues were used for WB experiments to detect the protein translation of the target gene. Since the target gene was fused with a FLAG tag, we used Anti-FLAG antibody to detect the target gene. The results are as follows Figure 4 As shown, near the 70kDa marker protein, the target protein was detected in transgenic sugarcane lines line1, line2, and line3, while the target protein was not detected in the wild type WT. Therefore, the genes we selected were transcribed and translated normally in sugarcane and were not in a state of gene silencing.

[0093] 6. Analysis of Transgenic Sugarcane Germination Test

[0094] The germination test of sugarcane was carried out in quartz sand, e.g. Figure 5 、 Figure 6As shown in the figure, when the sugarcane germinated for 3 days, the average bud length of the control group WT was 1.16 cm, and the bud lengths of the transgenic sugarcane lines line1, line2, and line3 were 2.50, 2.58, and 2.21 cm, respectively. The data analysis in EXCEL was used to analyze the significance of the differences between the control group WT and line1, line2, and line3. The results showed that the bud lengths of the control group WT were extremely significantly different from those of the transgenic sugarcane, P<0.01, that is, the difference between the two groups of data was extremely significant.

[0095] At 6 days after sugarcane germination, the average bud length of the control group WT was 2.27 cm, while the bud lengths of the transgenic sugarcane lines line1, line2, and line3 were 6.11, 6.84, and 6.32 cm, respectively. The results of variance analysis showed that the P values were all less than 0.01, indicating extremely significant differences.

[0096] At 9 days after sugarcane germination, the average bud length of the control group WT was 5.42 cm, while the bud lengths of the transgenic sugarcane lines line1, line2, and line3 were 10.29, 10.63, and 10.18 cm, respectively. The results of variance analysis showed that the P values were all less than 0.01, indicating extremely significant differences.

[0097] These results indicate that overexpression of the ShN / AINV3.1 gene can significantly increase the germination rate of sugarcane, increase the length of sugarcane sprouts within the same period of time, and accelerate the germination speed of sugarcane.

[0098] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. ShN / AINV3.1 Application of a gene or its encoded protein or a biological material containing the gene in promoting germination of sugarcane stems, ShN / AINV3.1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. ShN / AINV3.1 Application of a gene or its encoded protein or a biological material containing the gene in sugarcane breeding, wherein the breeding purpose is to cultivate sugarcane varieties with high sugarcane stem germination rate, ShN / AINV3.1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that The biological material is a recombinant expression vector, an expression cassette or a recombinant bacterium.

4. A method for promoting sugarcane stem germination, characterized in that: Make sugarcane ShN / AINV3.1 The expression of the gene is increased, thereby promoting the germination of sugarcane stems. ShN / AINV3.1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

Images