Sugarcane sugar transporter gene SoERDL6-7 and SoTST5 as well as related biological materials and application thereof

Abstract

The invention discloses cane sugar transporter genes SoERDL6-7 and SoTST5 as well as related biological materials and application thereof, and belongs to the technical field of biology. The invention provides two DNA (deoxyribonucleic acid) fragments which are any one of the following DNA fragments: 1) nucleotide sequences of the DNA fragments are SEQ ID NO: 2 and SEQ ID NO: 4, and 2) a nucleotide sequence which has more than 90% of homology with the nucleotide sequence shown in 1) and has the same function. Experiments prove that when the sugarcane SoERDL6-7 or SoTST5 gene is expressed in sorghum, the content of sucrose, glucose and fructose of sorghum stalks can be remarkably increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a sugarcane sugar transporter gene SoERDL6-7 and SoTST5 and related biological materials and applications. BACKGROUND

[0002] Sugarcane is the most important sugar crop in agricultural production, providing raw materials for 80% of global sugar and 40% of bioenergy production. More than 90% of sugar in China comes from sugarcane, and sugar crops have always been a key crop that the country emphasizes to protect. Sugarcane sugar metabolism is regulated by complex genetic control. Identifying genes related to sugarcane sugar synthesis and transport storage can provide a theoretical basis for breeding high-sugar sugarcane varieties.

[0003] In plants, sugars produced by photosynthesis need to be loaded into the phloem by sugar transporters and then transported to the "sink" organs. The unloading of sugars in the "sink" organs also requires the participation of sugar transporters. Identifying and studying key sugar transporters that affect sugar storage can provide molecular targets for breeding high-sugar crops. The vacuole of stem parenchyma cells is the main site for storing sugars. However, the key sugar transporter genes involved in sugar storage in sugarcane have not been identified.

[0004] High-quality genome assembly of sugarcane has laid a good foundation for the identification and study of functional genes in sugarcane. However, sugarcane is a polyploid species with a large and complex genome. As a close relative of sugarcane, sorghum is in the same tribe of the grass family, and its genetic manipulation is relatively simple. Therefore, sorghum is often used as a reference species for the study and verification of functional genes in sugarcane. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a new gene for regulating sugar content. The technical problem to be solved is not limited to the technical subject described, and other technical subjects not mentioned in this article can be clearly understood by those skilled in the art through the following description.

[0006] The present application first identifies key genes involved in stem sugar storage in sorghum ERDL6 and TST Then, using the genome and transcriptome data of sugarcane GT42 (GT42), the homologous genes in sugarcane are cloned SoERDL6-7 and SoTST5 and it is verified that overexpression of SoERDL6-7 or SoTST5 can increase the sugar content of the stem. The present application provides a molecular target and theoretical basis for breeding high-sugar quality sugarcane varieties.

[0007] To solve the above technical problems, the present application provides the following technical solutions: The present application provides a protein, which is A1) or / and A2): The A1) is any one of: A1-1) a protein whose amino acid sequence is SEQ ID NO: 1; A1-2) a protein having 80% or more identity to the protein whose amino acid sequence is shown in A1-1) and having the same function, which is obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in A1-1); A1-3) a fusion protein having the same function, which is obtained by connecting a tag to the N terminus and / or C terminus of the amino acid shown in any one of A1-1) or A1-2); The A2) is any one of: A2-1) a protein whose amino acid sequence is SEQ ID NO: 3; A2-2) a protein having 80% or more identity to the protein whose amino acid sequence is shown in A2-1) and having the same function, which is obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in A2-1); A2-3) a fusion protein having the same function, which is obtained by connecting a tag to the N terminus and / or C terminus of the amino acid shown in any one of A2-1) or A2-2).

[0008] The tag protein includes, but is not limited to, a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0009] Herein, the identity refers to the identity of the amino acid sequence or the nucleotide sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the value of the identity (%) can be obtained by performing a search in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and then calculating the identity of the amino acid sequence.

[0010] Herein, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0011] The present application also provides a biological material related to the protein, which is any one of the following: B1) a nucleic acid molecule encoding the aforementioned protein; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2); B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3); B5) a transgenic plant cell line containing the nucleic acid molecule of B1), or a transgenic plant cell line containing the expression cassette of B2); B6) a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2); B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2).

[0012] The nucleic acid molecule is any one of the following DNA molecules: A1) a DNA molecule with a nucleotide sequence of SEQ ID NO: 2, or a DNA molecule with a nucleotide sequence of positions 2231-6350 of SEQ ID NO: 2; A2) a DNA molecule with a nucleotide sequence of SEQ ID NO: 4, or a DNA molecule with a nucleotide sequence of positions 2001-6792 of SEQ ID NO: 4; A3) a DNA molecule encoding a sequence of SEQ ID NO: 5; A4) a DNA molecule encoding a sequence of SEQ ID NO: 6.

[0013] The present application also provides the use of the aforementioned protein or the aforementioned biological material in any one of the following, M1) increasing the sugar content of a plant; M2) preparing a product for increasing the sugar content of a plant; M3) preparing a plant with increased sugar content; M4) plant breeding.

[0014] The purpose of the plant breeding is to obtain a plant with increased sugar content.

[0015] In the above use, the sugar content includes sucrose content, glucose content or / and fructose content.

[0016] In the above use, the plant is any one of the following: G1) a dicotyledonous plant or a monocotyledonous plant; G2) a plant of the order Poales or a plant of the order Poales; G3) a plant of the family Poaceae or a plant of the family Poaceae; G4) a plant of the genus Saccharum or a plant of the genus Sorghum; G5) sugar cane or sorghum.

[0017] The present application provides a method for increasing sugar content of a plant, comprising increasing or enhancing or up-regulating sugar content of a recipient plant by increasing or enhancing or up-regulating expression of a gene encoding a protein or regulating activity and / or content of the protein in the recipient plant.

[0018] The present application provides a method for increasing sugar content of a plant, comprising increasing or enhancing or up-regulating sugar content of a recipient plant by increasing or enhancing or up-regulating expression of a gene encoding a protein or regulating activity and / or content of the protein in the recipient plant, wherein the amino acid sequence of the protein is SEQ ID NO: 1.

[0019] In the above method, the increasing or enhancing or up-regulating expression of a gene encoding a protein or regulating activity and / or content of the protein in the recipient plant comprises introducing into the recipient plant a substance that increases or enhances or up-regulates expression of a gene encoding a protein or regulates activity and / or content of the protein, wherein the amino acid sequence of the protein is SEQ ID NO: 1.

[0020] The substance is any one of the following: B1) a nucleic acid molecule encoding a protein having an amino acid sequence of SEQ ID NO: 1; B2) an expression cassette comprising the nucleic acid molecule of B1); B3) a recombinant vector comprising the nucleic acid molecule of B1) or an expression cassette of B2); B4) a recombinant microorganism comprising the nucleic acid molecule of B1) or an expression cassette of B2) or a recombinant vector of B3).

[0021] The nucleotide sequence of the nucleic acid molecule of B1) is from the 2231st to the 6350th nucleotide of SEQ ID NO: 2.

[0022] The present application provides a method for increasing sugar content in a plant, the method comprising increasing or enhancing or up-regulating the sugar content in a recipient plant by increasing or enhancing or up-regulating the expression of a gene encoding a protein having an amino acid sequence of SEQ ID NO: 3 or modulating the activity and / or content of the protein.

[0023] In the above method, the increasing or enhancing or up-regulating the expression of a gene encoding a protein or modulating the activity and / or content of the protein in a recipient plant comprises introducing into the recipient plant a substance that increases or enhances or up-regulates the expression of a gene encoding a protein having an amino acid sequence of SEQ ID NO: 3 or modulates the activity and / or content of the protein.

[0024] The substance is any one of the following: B1) a nucleic acid molecule encoding a protein having an amino acid sequence of SEQ ID NO: 3; B2) an expression cassette comprising the nucleic acid molecule of B1); B3) a recombinant vector comprising the nucleic acid molecule of B1) or an expression cassette of B2); B4) a recombinant microorganism comprising the nucleic acid molecule of B1) or an expression cassette of B2) or a recombinant vector of B3).

[0025] The nucleotide sequence of the nucleic acid molecule of B1) is SEQ ID NO: 4 from nucleotide 2001 to 6792.

[0026] The present application also provides a method for preparing a plant with increased sugar content, the method comprising obtaining a target plant by increasing or enhancing or up-regulating the expression of a gene encoding a protein or modulating the activity and / or content of the protein in a starting plant, the target plant having a higher sugar content than the starting plant.

[0027] The present application also provides a method for preparing a plant with increased sugar content, the method comprising obtaining a target plant by increasing or enhancing or up-regulating the expression of a gene encoding a protein or modulating the activity and / or content of the protein in a starting plant, the target plant having a higher sugar content than the starting plant, the protein having an amino acid sequence of SEQ ID NO: 1.

[0028] In the above method, the increasing or enhancing or up-regulating the expression of a gene encoding a protein or modulating the activity and / or content of the protein in a recipient plant comprises introducing into the recipient plant a substance that increases or enhances or up-regulates the expression of a gene encoding a protein having an amino acid sequence of SEQ ID NO: 1 or modulates the activity and / or content of the protein.

[0029] The substance is any one of the following: B1) a nucleic acid molecule encoding a protein having the amino acid sequence of SEQ ID NO: 1; B2) an expression cassette comprising the nucleic acid molecule of B1); B3) a recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2); B4) a recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3).

[0030] The nucleotide sequence of the nucleic acid molecule of B1) is from nucleotide 2231 to 6350 of SEQ ID NO: 2.

[0031] The present application also provides a method for preparing a plant with increased sugar content, comprising obtaining a target plant by increasing or enhancing or up-regulating the expression of a gene encoding the aforementioned protein or regulating the activity and / or content of the protein in a starting plant, the sugar content of the target plant being higher than that of the starting plant, the amino acid sequence of the protein being SEQ ID NO: 3.

[0032] In the above method, the increasing or enhancing or up-regulating the expression of a gene encoding the protein or regulating the activity and / or content of the protein in the recipient plant comprises introducing into the recipient plant a substance that increases or enhances or up-regulates the expression of a gene encoding the protein or regulates the activity and / or content of the protein, the amino acid sequence of the protein being SEQ ID NO: 3.

[0033] The substance is any one of the following: B1) a nucleic acid molecule encoding a protein having the amino acid sequence of SEQ ID NO: 1; B2) an expression cassette comprising the nucleic acid molecule of B1); B3) a recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2); B4) a recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3).

[0034] The nucleotide sequence of the nucleic acid molecule of B1) is from nucleotide 2001 to 6792 of SEQ ID NO: 4.

[0035] In the above method, the plant is any one of the following: G1) a dicotyledonous plant or a monocotyledonous plant; G2) a plant of the order Poales or a plant of the order Poales; G3) a plant of the family Poaceae or a plant of the family Poaceae; G4) a plant of the genus Saccharum or of the genus Sorghum; G5) sugar cane or sorghum.

[0036] The starting plant or the recipient plant comprises the sorghum Wheatland.

[0037] In the above use or method, the expression cassette containing a nucleic acid molecule of B2) refers to DNA capable of expressing the above-mentioned protein in a host cell. The expression cassette can also include single- or double-stranded nucleic acid molecules of all regulatory sequences necessary for expression of any one of the above-mentioned proteins or DNA of the RNA molecule. The regulatory sequences can direct the expression of the coding sequence in a suitable host cell to express any one of the above-mentioned proteins or DNA of the RNA molecule under compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences include a promoter and signals for termination of transcription and translation. To introduce specific restriction enzyme sites for the regulatory sequences to be ligated to the coding region of the nucleic acid sequence encoding the protein or DNA of the RNA molecule, the regulatory sequences can be provided with linkers. The regulatory sequences can be a suitable promoter sequence, i.e., a nucleic acid sequence that is recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the protein or DNA of the RNA molecule. The promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell of choice including mutated, truncated, and hybrid promoters, and can be derived from genes encoding proteins either homologous or heterologous to the host cell. The regulatory sequences can also be a suitable transcription terminator sequence, i.e., a sequence of nucleotides that is recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein or DNA of the RNA molecule. Any terminator that is functional in the host cell of choice can be used in the present application. The regulatory sequences can also be a suitable leader sequence, i.e., an mRNA untranslated region important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein or DNA of the RNA molecule. Any leader sequence that is functional in the host cell of choice can be used in the present application. The regulatory sequences can also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of the protein or DNA of the RNA molecule, which can direct the expressed protein or DNA of the RNA molecule into the cell's secretory pathway. Any signal peptide coding region that is functional in the host cell of choice can be used in the present application. It can also be desirable to add regulatory sequences that allow the regulation of the expression of the protein or DNA of the RNA molecule relative to the growth of the host cell. Examples of regulatory sequences are those that allow expression of the protein or DNA of the RNA molecule in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other examples of regulatory sequences are those that allow the amplification of the gene.

[0038] In the above use or method, the vector can be a plasmid, cosmid, phage, or viral vector.

[0039] In the above-mentioned application or method, the microorganism can be a yeast, a bacterium, an algae or a fungus, such as Agrobacterium.

[0040] In the above-mentioned application or method, the transgenic plant cell lines do not include propagation materials.

[0041] The present application also provides a DNA molecule, the nucleotide sequence of which is any one of the following: A1) the nucleotide sequence of the DNA molecule is SEQ ID NO: 2; A2) the nucleotide sequence of the DNA molecule is SEQ ID NO: 4; A2) a DNA molecule having 90% or more identity with the nucleotide sequence shown in A1) or A2) and having the same function.

[0042] Experiments of the present application demonstrate that the sugarcane SoERDL6-7 or SoTST5 genes can be significantly improved in the stem sugar content of the sorghum recipient material, including sucrose content, glucose content and fructose content, when the genes are expressed heterologously in sorghum. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The stem ERDL6 family genes and TST family genes of sorghum were removed from the sorghum, and the changes in the expression amount of the genes at different time points were detected.

[0044] Figure 2 The expression amount of the transgenic sorghum material expressing the sugarcane SoERDL6-7 and SoTST5 genes was detected. The transgenic lines with the highest expression amount of the SoERDL6-7 and SoTST5 genes are shown respectively relative to the expression fold of the wild type.

[0045] Figure 3 The stem sugar content of the transgenic sorghum material expressing the sugarcane SoERDL6-7 and SoTST5 genes was detected. (a) Sucrose content determination; (b) glucose content determination; (c) fructose content determination. DETAILED DESCRIPTION

[0046] The present application will be further described in detail below in conjunction with the specific embodiments. The examples given are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0047] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0048] In the quantitative experiments in the following examples, three repeated experiments were set up, and the results were averaged.

[0049] Sweet sorghum E048 (variety protection number 20191004941) and wild type Wheatland sorghum are recorded in the non-patent document "Chengxuan Chen 1, Fengyong Ge 1, Huilong Du 2, Yuanchang Sun 3, Yi Sui 4, Sanyuan Tang 5, Zhengwei Shen 3, Xuefeng Li 6, Huili Zhang 5, Cuo Mei 1, Peng Xie 1, Chao Li 5, Sen Yang 1, Huimin Wei 1, Jiayang Shi 1, Dan Zhang 1, Kangxu Zhao 1, Dekai Yang 1, Yi Qiao 1, Zuyong Luo 6, Li Zhang 6, Aimal Khan 1, Baye Wodajo 1, Yaorong Wu 5, Ran Xia 5, Chuanyin Wu 4, Chengzhi Liang 5, Qi Xie 7, Feifei Yu 8. A comprehensive omics resource and genetic tools for functional genomics research and genetic improvement of sorghum. Mol Plant. 2025 Apr 7; 18(4): 703-719. doi: 10.1016 / j.molp.2025.03.005. Epub 2025 Mar 7.", which is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the biological material can only be used for repeating the relevant experiments of the present application and cannot be used for other purposes.

[0050] In the following examples, the data were processed using GraphPad Prism 8 statistical software, and the experimental results were expressed as mean ± standard deviation, and One-way ANOVA test was used, P<0.05 (*) indicating significant difference.

[0051] Example 1, Identification and cloning of sugarcane sugar transporter genes SoERDL6-7 and SoTST5 The vacuoles of stem parenchyma cells are the main sites for sugar storage in sugarcane and sweet sorghum, and the sugar transporters of ERDL6 and TST families located on the vacuole membrane are involved in the storage of vacuole sugars. However, there are many members in these two families, and the key ERDL6 and TST genes involved in the storage of vacuole sugars in sugarcane are still unknown. Sugarcane is a polyploid species with aneuploidy and high complexity of genome. Therefore, the key genes involved in the storage of vacuole sugars in sugarcane were identified by first identifying the key genes involved in the storage of vacuole sugars in the diploid relative of sugarcane, sweet sorghum, and then identifying the homologous genes in sugarcane by phylogenetic analysis combined with transcriptome data.

[0052] In order to identify the key ERDL6 and TST genes involved in the storage of vacuole sugars in sweet sorghum, the panicles of sweet sorghum at the heading stage were removed, and the stems were sampled before the removal of panicles (i.e. 0 h in Figure 1 ) and 1 h, 3 h, 6 h, 9 h after the removal of panicles (i.e. 1 h, 3 h, 6 h, 9 h in Figure 1 ), and the changes in the expression levels of all ERD6L and TST gene family genes in sweet sorghum were analyzed by RT-qPCR. The specific implementation process is as follows: The stem samples of sweet sorghum E048 at the above time points were sampled and quickly frozen in liquid nitrogen.

[0053] ​After grinding the sample into powder with liquid nitrogen in a mortar, about 100 mg of the sample was placed in a 2 mL centrifuge tube, and a plant ultra-pure RNA extraction kit from Beijing Kangwei Century Biotechnology Co., Ltd. was used to extract total RNA: 1 mL TRIzon Reagent was added, the sample was thoroughly mixed by inverting to fully lyse the sample, and the protein-nucleic acid complex was completely separated at room temperature for 5 min; then 200 μL chloroform was added, the centrifuge tube was tightly capped, and then vigorously shaken for 15 s, and left at room temperature for 2 min; centrifuged at 4°C, 12,000 rpm for 10 min, at which time the sample was divided into three layers, and the upper aqueous phase containing RNA was moved to a new RNase-Free centrifuge tube; an equal volume of 70% ethanol (prepared with RNase-free water) was added to the obtained aqueous solution, and mixed by inverting; the above solution was all added to the adsorption column in the collection tube, centrifuged at 12,000 rpm for 20 s, and the waste liquid in the collection tube was discarded, and the adsorption column was placed back into the collection tube; 700 μL buffer RW1 was added to the adsorption column, and centrifuged at 12,000 rpm for 20 s, and the waste liquid in the collection tube was discarded; 500 μL buffer RW2 was added to the adsorption column for washing twice, each time at 12,000 rpm for 20 s; centrifuged at 12,000 rpm for 2 min, and the waste liquid in the centrifuge tube was discarded, and the adsorption column was left to dry at room temperature for a few minutes; the adsorption column was placed in a new RNase-free centrifuge tube, 30 μL RNase-Free water was added to the middle part of the adsorption column, left to stand at room temperature for 1 min, and centrifuged at 12,000 rpm for 1 min to collect the RNA. After measuring the RNA concentration using a NanoDrop 2000, the RNA was stored at -80°C or directly used for the next step of the experiment.

[0054] RNA reverse transcription: RNA was reverse transcribed into cDNA using a Tian Gen Fast Quant RT Kit, and 5 × gDNA buffer, Q-RT Primer Mix, 10 × Fast RT buffer and RNase-Free distilled water were thawed at room temperature, quickly placed on ice, and the gDNA removal reaction system was configured: 5 × gDNA buffer 2 μL, RNA 2 μg, and RNase-Free distilled water was added to 10 μL, and incubated at 42°C for 3 min to remove genomic DNA; then 10 μL of the following reaction system was added: 10 × Fast RT buffer 2 μL, RT Enzyme Mix 1 μL, Q-RT Primer Mix 2 μL, RNase-Free distilled water 5 μL; mixed, then incubated at 42°C for 15 min, and then incubated at 95°C for 3 min to obtain cDNA, which was stored at -20°C or directly used for the next step of the experiment.

[0055] Quantitative Real-time PCR: The cDNA above was diluted 10 times with distilled water as template to quantify the expression of 5 genes of TST family and 8 genes of ERDL6 family in different time points. The following reaction system was configured using TIANGEN Talent qPCR PreMix kit: 2 x Talent qPCR PreMix 10 μL, Primer F 0.6 μL, Primer R 0.6 μL, diluted 10 times cDNA 3 μL, RNase-Free distilled water 5.8 μL. After mixing, centrifugation was performed, and two-step reaction program was used in Biorad CFX96 instrument for analysis: 95°C pre-denaturation for 3 min, 95°C denaturation for 5 s, 60°C annealing / extension for 15 s. After 40 cycles, melting curve analysis was performed. The primer sequences used for each gene are as follows: SbERDL6-1 Forward primer: 5'-TGGTGCTCATTGTGGCAAAC-3'; SbERDL6-1 Reverse primer: 5'-TCCAGAAAACCCGGCAGAAC-3'; SbERDL6-2 Forward primer: 5'-ACATTCTTTGGGGCCCTTCT-3'; SbERDL6-2 Reverse primer: 5'-AGCAGACTCTAGTACTCTCAGAT-3'; SbERDL6-3 Forward primer: 5'-GTTCGGCATCTCGGTTCCA-3'; SbERDL6-3 Reverse primer: 5'-CCAAAGTCCGCCAGGTGATA-3'; SbERDL6-4 Forward primer: 5'-AGTTGGGCTTATGGCCCTTC-3'; SbERDL6-4 Reverse primer: 5'-GTCCGACCATTGGAACCTGT-3'; SbERDL6-5 Forward primer: 5'-ATACCGCACGCCACTACTTC-3'; SbERDL6-5 Reverse primer: 5'-GTGGCCAAGTTGCTGTTTGT-3'; SbERDL6-6 Forward primer: 5'-AGGAATTGGGCTACTTGCGT-3'; SbERDL6-6Forward primer: 5'-GCAACAACCCCGATAGCTCC-3'; SbERDL6-7 Forward primer: 5'-CAGGTTCTTGCCACTGTTGTT-3'; SbERDL6-7 Reverse primer: 5'-AAGGCTTATAGTCATCCCAGCA-3'; SbERDL6-8 Forward primer: 5'-GGCTGGCAAAAATGGGGAAG-3'; SbERDL6-8 Reverse primer: 5'-TCAGCAAACCTTATGGCGGT-3' SbTST1 Forward primer: 5'-GTTGGCCGTCACACTTGTTC-3'; SbTST1 Reverse primer: 5'-GGGCATGAGGGACATTCCAA-3'; SbTST2 Forward primer: 5'-AAATGAGCCCACTGTGGAGG-3'; SbTST2 Reverse primer: 5'-CGGCCGATCCAGTCTGATAC-3'; SbTST3 Forward primer: 5'-CTGCCCACCCTTAGAGACG-3'; SbTST3 Reverse primer: 5'-GGGCATGAGGGACATTCCAA-3'; SbTST4 Forward primer: 5'-CCGACGACCCTGCGTTTTAT-3'; SbTST4 Reverse primer: 5'-CCATCCAAGCATTGCATACCC-3'; SbTST5 Forward primer: 5'-TGGTGCTCATTGTGGCAAAC-3'; SbTST5 Reverse primer: 5'-AACCCCATGACGAAGCAACA-3'.

[0056] The results of the fluorescent quantitative PCR showed that, ERDL6-2 and TST3 The expression of the two genes in the stem node was very low, so the two genes were deleted (data not shown). The expression changes of the remaining 7 ERDL6 genes and 4 TST genes were analyzed, and the results showed thatSbERDL6-7 and SbTST5 The gene expression amount changed most significantly at the 6th and 9th hours after the removal of the panicle, so the two genes were taken as candidate genes Figure 1 ).

[0057] The SbERDL6-7 ( Sobic.009G235900 ) and SbTST5 ( Sobic.010G276100 ) gene sequences were taken as query sequences to conduct a Blast search in the GT42 genome, and 7 SbERDL6-7 and 10 SbTST5 orthologous genes of orthologous genes were searched. In order to further determine the ERDL6-7 and TST5 genes expressed in the sugarcane stalk, the transcriptome data of different tissues of GT42 were combined, and it was found that h1tg00041 and h1tg000788 had a specific high expression in the stem node in the development of sugarcane, so the two genes were taken as candidate genes and were named SoERDL6-7 and SoTST5 .

[0058] The SoERDL6-7 gene was cloned by using the forward primer 1 and the reverse primer 1 and the SoTST5 gene was cloned by using the forward primer 2 and the reverse primer 2, with the genomic DNA of the sugarcane GT42 as a template. The primer sequences are as follows: Forward primer 1: 5'-agagtgtcgtgctccaccatgTGTATTGATAATGCTCGAGCGC-3' Reverse primer 1: 5'-caggtcgactctagaggatccCTGCACCATAGTAGGAGAATAA-3' Forward primer 2: 5'-gagagtgtcgtgctccaccatgAAAGAGAAGATGCACATCATGC-3' Reverse primer 2: 5'-gcaggtcgactctagaggatccACGAAAATCTATAGTTCAAATTC-3' PCR reaction system: 2X KOD one Mix 25 μL, forward primer 2 μL, reverse primer 2 μL, DNA template 2 μL, and ddH2O was supplemented to 50 μL.

[0059] PCR amplification procedure: 95℃ pre-denaturation for 3 minutes; 98℃ denaturation for 10 seconds, 65℃ annealing for 30 seconds, 68℃ extension for 2 minutes and 30 seconds, a total of 33 cycles; 68℃ terminal extension for 5 minutes.

[0060] The PCR products were subjected to 1% agarose gel electrophoresis, and the target band was excised and recovered.

[0061] Sequencing revealed that SEQ ID NO:2 is sugarcane. SoERDL6-7 The gene sequence, where nucleotides 1 to 2230 are the promoter, nucleotides 2231 to 2530 are the first exon, nucleotides 2640 to 2729 are the second exon, nucleotides 3142 to 3201 are the third exon, nucleotides 3295 to 3360 are the fourth exon, nucleotides 3608 to 3673 are the fifth exon, nucleotides 3780 to 3855 are the sixth exon, nucleotides 4059 to 4123 are the seventh exon, nucleotides 4213 to 4305 are the eighth exon, and nucleotides 4467 to 454 are the fifth exon. Nucleotides at position 7 are exon 9, nucleotides 4637 to 4721 are exon 10, nucleotides 5128 to 5175 are exon 11, nucleotides 5259 to 5320 are exon 12, nucleotides 5402 to 5461 are exon 13, nucleotides 5640 to 5705 are exon 14, nucleotides 5793 to 5852 are exon 15, nucleotides 6018 to 6132 are exon 16, and nucleotides 6238 to 6350 are exon 17, encoding the protein with the amino acid sequence SEQ ID NO:1 (coding sequence SEQ ID NO:5).

[0062] SEQ ID NO:4 is sugarcane SoTST5 The sequence of the gene, wherein nucleotides 1 to 2000 are the promoter, nucleotides 2001 to 2079 are the first exon, nucleotides 3640 to 4206 are the second exon, nucleotides 4853 to 5817 are the third exon, nucleotides 6067 to 6254 are the fourth exon, and nucleotides 6354 to 6792 are the fifth exon, encoding the protein whose amino acid sequence is SEQ ID NO:3 (encoding sequence is SEQ ID NO:6).

[0063] Example 2: Sugarcane transporter gene SoERDL6-7S and oTST5 Application I. SoERDL6-7 and SoTST5 Construction of gene hyperexpression vectors SoERDL6-7 and SoTST5 The gene encodes a sugar transporter located on the vacuolar membrane, used in the study of sugarcane. SoERDL6-7 and SoTST5The relationship between the genes and sugar content, and the two genes are expressed in a heterologous manner in a sorghum relative, sorghum.

[0064] The pCAMBIA2300-Myc vector is described in the non-patent literature "Natural variation in Glume Coverage 1 causes naked grains in sorghum. Nature Communications 25 Feb 2022 Vol 13, Issue 1. DOI: 10.1038 / s41467-022-28680-3", which is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the biological material is only used for repeating the related experiments of the present application and cannot be used for other purposes.

[0065] The pCAMBIA2300-Myc vector is digested using BstXI and BamHI, and the enzyme digestion system is as follows: 10X Cutsmart buffer 5 μL, plasmid vector 1 μg, BstXI 1 μL, BamHI 1 μL, ddH2O to 50 μL. Enzyme digestion at 37°C for 1 hour. After enzyme digestion products are electrophoresed using a 1% agarose gel, the target band is recovered, and a linearized pCAMBIA2300-Myc carrier is obtained.

[0066] The linearized pCAMBIA2300-Myc carrier recovered from the gel above is ligated with the SoERDL6-7 gene and SoTST5 gel recovery product fragment obtained in Example 1 using a method of homologous recombination, and the reaction system is as follows: 5X CE buffer 4 μL, linearized pCAMBIA2300-Myc carrier 5 μL, gel recovery SoERDL6-7 and SoTST5 gene fragment 2 μL 2 μL, EnaseII 2 μL, ddH2O 7 μL. Reaction at 37°C for 30 minutes to obtain the ligation product.

[0067] Transforming E. coli with the ligation product: Take E. coli XL1-blue competent cells, add the ligation product after thawing on ice, mix gently, ice bath for 30 minutes; 42°C heat shock for 90 seconds, immediately place on ice for 3 minutes, then add 500 μL of antibiotic-free LB liquid medium, incubate at 37°C, 220 rpm for 1 hour; centrifuge at 5,000 rpm for 1 minute, collect the bacterial liquid precipitate; discard the excess liquid LB medium and resuspend the precipitate with the remaining liquid LB, spread it on a selection plate containing kanamycin, and incubate at 37°C for 16 hours; pick a single colony from the plate, and use forward primer 3 and reverse primer 3 to amplify the target gene. SoERDL6-7PCR verification was performed on the gene, and 1586 bp was obtained as a positive clone; forward primer 4 and reverse primer 4 were used to amplify the gene SoTST5 PCR verification was performed on the gene, and 1517 bp was obtained as a positive clone.

[0068] Forward primer 3: 5'-CAGCAAACTTGCTGATCAGC-3'; Reverse primer 3: 5'-gctgcaaggcgattaagttg-3'.

[0069] Forward primer 4: 5'-ACAGTCTTCCTGTGATGCTG-3'; Reverse primer 4: 5'-gctgcaaggcgattaagttg-3'; PCR reaction system: 2X SuperTaq Mix 10 μL, forward primer 1 μL, reverse primer 1 μL, template 2 μL, ddH2O to 20 μL.

[0070] PCR amplification program: 95°C pre-denaturation for 3 minutes; 98°C denaturation for 10 seconds, 56°C annealing for 30 seconds, 72°C extension for 45 seconds, a total of 33 cycles; 72 final extension for 5 minutes.

[0071] The above PCR product was subjected to agarose gel electrophoresis, and the positive clone with the desired band was sent to the company for sequencing for further verification, and the high-expression vector plasmid was extracted and saved according to the following method: The verified positive monoclonal was inoculated in 5 mL of LB liquid medium containing the corresponding antibiotic, and cultured at 37°C and 220 rpm for 14 hours; the bacterial solution was collected by centrifugation at 12,000 rpm for 30 seconds, and the supernatant was discarded; 250 μL of P1 buffer was added, vortexed to precipitate; 250 μL of P2 buffer was added, and the bacterial solution was mixed slowly for 4 to 6 times to make it transparent and clear, and this step should not exceed 5 minutes; 350 μL of N3 was added, and the bacterial solution was mixed slowly immediately; centrifugation at 12,000 rpm for 5 minutes, the supernatant was removed to the absorption column, centrifugation at 12,000 rpm for 30 seconds, the filtrate was discarded; 150 μL of PB was added, centrifuged for 30 seconds, and the filtrate was discarded; 700 μL of washing buffer was added, centrifuged for 1 minute, and the filtrate was discarded; 12,000 rpm empty centrifugation for 1 minute, the absorption column was moved to a new 1.5 mL centrifuge tube, and placed at room temperature for 2 minutes; after the washing buffer was completely dried, 30 μL of EB buffer was added, and the mixture was placed at room temperature for about 2 minutes; centrifugation at 12,000 rpm for 2 minutes, and the plasmid was eluted with EB buffer to obtain the high-expression vector.

[0072] The concentration was measured using NanoDrop 2000, and stored at -20°C.

[0073] The high expression vector SoERDL6-7::pCAMBIA2300 is a vector obtained by replacing the fragment between the BstXI and BamHI enzyme cutting sites of the pCAMBIA2300-Myc vector with SEQ ID NO: 2 while keeping other nucleotides of the pCAMBIA2300 plasmid unchanged.

[0074] The high expression vector SoTST5::pCAMBIA2300 is a vector obtained by replacing the fragment between the BstXI and BamHI enzyme cutting sites of the pCAMBIA2300-Myc vector with SEQ ID NO: 4 while keeping other nucleotides of the pCAMBIA2300 plasmid unchanged.

[0075] II. Transformation of sorghum with high expression vectors The recombinant vectors SoERDL6-7::pCAMBIA2300 and SoTST5::pCAMBIA2300 were respectively transformed into the sorghum recipient material Wheatland using Agrobacterium-mediated genetic transformation.

[0076] 1. The above "Construction of high expression vectors of genes" I. SoERDL6-7 and SoTST5 The recombinant vector plasmids SoERDL6-7::pCAMBIA2300 and SoTST5::pCAMBIA2300 constructed in the "Construction of high expression vectors of genes" were respectively transformed into Agrobacterium EHA105 by electroporation to obtain recombinant bacteria EHA105 / SoERDL6-7::pCAMBIA2300 and EHA105 / SoTST5::pCAMBIA2300.

[0077] The Agrobacterium EHA105 competent cells were thawed on ice and then the plasmid to be transformed was added; the mixture of competent cells and plasmid was added to an electroporation cup, 1.8 kV, and electroporated for 5.6 ms; the competent cells in the electroporation cup were sucked into a 1.5 mL centrifuge tube and 500 μL of antibiotic-free LB liquid medium was added, and the mixture was cultured at 28°C and 200 rpm for 3 hours; after the culture was completed, 30 μL of bacterial liquid was spread on LB solid medium containing kanamycin and rifampicin antibiotics, and cultured at 28°C in an incubator for 2 days. After the colonies grew, a single colony was used for PCR detection with forward primer 3 and reverse primer 3, and the PCR reaction system and amplification program were as described in Example 2 "Construction of high expression vectors of genes". I. SoERDL6-7 and SoTST5Construction of gene overexpression vectors. The 1586 bp fragment was amplified from positive colony EHA105 / SoERD16-7::pCAMBIA2300; the single colony was used for PCR detection with forward primer 4 and reverse primer 4, and the 1517 bp fragment was amplified from positive colony EHA105 / SoTST5::pCAMBIA2300.

[0078] Generation of transgenic sorghum plants using Agrobacterium-mediated genetic transformation system: Immature embryos were isolated from 12-14 day old developing seeds of sorghum Wheatland. The immature seeds were surface sterilized with 50% bleach containing 0.1% Tween-20 for 30 minutes, and then rinsed 3 times with distilled water. The immature embryos were dissected out using sterile forceps and dissecting tools, and rinsed 2-3 times with IM solution before transferring the immature embryos into fresh IM in a 43°C water bath for 3 minutes, and then immediately transferred to a 25°C water bath for at least 2 minutes.

[0079] The immature embryos were mixed with Agrobacterium cells (1 mL, OD600 = 0.35-0.4) resuspended in IM for 10 minutes at room temperature. After removing the liquid, the infected immature embryos were transferred to sterile filter paper to drain the remaining liquid, and then plated on solid infection medium (co-M). The plates were incubated in the dark at 25°C for 3 days.

[0080] The immature embryos were transferred to resting medium (R) and incubated in the dark at 25°C for 5 days, and then subcultured for 10 days.

[0081] The immature embryos were transferred from the resting medium to callus induction medium (CIM) using forceps. 18 to 20 immature embryos were placed on each plate. The plates were incubated in the dark for 10 days for callus induction and selection at 26°C.

[0082] The primary shoots were removed using forceps and a surgical blade, and the callus was transferred to shoot induction medium (SM) plates. The plates were placed in a growth chamber for shoot induction under 80 μιηοΙ m"2 sec"1 light and 24°C, 18 hours / light, 6 hours / dark. The embryogenic callus was subcultured every 2 weeks with fresh SM for 6-10 weeks.

[0083] The small shoots (with 3 to 4 healthy leaves) were transferred to sterile boxes containing 30 mL of rooting medium (RM) and incubated for 2-3 weeks under the conditions of the previous step. After 1-2 weeks of root formation, the plants with healthy shoots and roots were moved to pots and grown to maturity in a greenhouse to obtain T0 generation transgenic sorghum lines SoERD6-7-OE and SoTST5-OE.

[0084] The medium formulations used in the above process are as follows: IM solution (per liter): 4.3 g MS salts, 68.5 g sucrose, 0.5 g 2-(N-morpholino)ethanesulfonic acid (MES), 1.5 ml 2,4-dichlorophenoxyacetic acid (2,4-D), 10 ml B5 vitamins. Adjust pH to 5.2. Filter sterilize and add acetylpyrazole to a final concentration of 1X before use.

[0085] Co-M solution (per liter): 4.3 g MS salts, 20 g sucrose, 10 g glucose, 0.7 g L-proline, 0.5 g 2-(N-morpholino)ethanesulfonic acid (MES), 1.5 ml 2,4-dichlorophenoxyacetic acid (2,4-D). Adjust pH to 5.8. Then add 8 g agar, 10 mg ascorbic acid, 10 g polyvinylpyrrolidone (PVPP). After autoclaving, cool to 50°C to 55°C, then add filter-sterilized 10 ml B5 vitamins, 1 ml 1000X acetylpyrazole, pour into sterile Petri dishes.

[0086] R solution (per liter): 4.3 g MS salts, 30 g sucrose, 1 g L-proline, 0.5 g 2-(N-morpholino)ethanesulfonic acid (MES), 1.5 ml 2,4-dichlorophenoxyacetic acid (2,4-D). Adjust pH to 5.8. Then add 8 g agar, 10 g polyvinylpyrrolidone (PVPP). After autoclaving, cool to 50°C to 55°C, then add filter-sterilized 10 ml B5 vitamins, 1 g / 1 asparagine, 1 g / 1 potassium phosphate monobasic (KH2PO4). Pour into sterile Petri dishes.

[0087] CIM (per liter): Same as R medium recipe above, plus 300 mg / 1 cefotaxime, 0.1 ml / 1 copper sulfate (CuSO4), 0.125 ml / 1 carbenicillin. Pour into sterile Petri dishes.

[0088] SM (per liter): 4.3 g MS salts, 30 g sucrose, 0.5 g 2-(N-morpholino)ethanesulfonic acid (MES), adjust pH to 5.8. Then add 8 g agar, 10 g polyvinylpyrrolidone (PVPP). After autoclaving, cool to 50°C to 55°C, then add filter-sterilized 10 ml B5 vitamins, 1 ml 6-benzylaminopurine (BAP), 1 ml indole-3-acetic acid (IAA), 300 mg / 1 cefotaxime, 0.1 ml / 1 copper sulfate (CuSO4), 0.5 ml / 1 carbenicillin. Pour into sterile Petri dishes.

[0089] RM (per liter): Same as SM medium recipe, but do not add 6-benzylaminopurine (BAP) and carbenicillin.

[0090] 2. Identification of transgenic plants Genomic DNA was extracted from the leaves of T0 generation transgenic sorghum plants using the CTAB method. Take approximately 100 mg of sorghum sample and place it in a 2 mL centrifuge tube along with a 5 mm steel ball. After quick-freezing in liquid nitrogen, grind the sample into powder using a grinder at 900 rpm for 2 min. Add 650 μL of DNA extraction buffer and mix well. Incubate at 65°C in a water bath or constant temperature oven for 40 min, then add 650 μL of chloroform / isoamyl alcohol (24:1). Vigorously vortex and centrifuge at 12,000 rpm for 10 min at 4°C. Take 500 μL of the supernatant and add an equal volume of isopropanol. Incubate at -20°C for at least 30 min to precipitate the DNA. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. Wash the precipitate with 1 mL of 70% ethanol and centrifuge at 12,000 rpm for 2 min. Repeat this process once. After centrifugation, dry the precipitate at room temperature, add 100 μL of sterile water, and store at -20°C after the DNA has completely dissolved.

[0091] Using the extracted DNA as a template, PCR amplification was performed using forward primer 3 and reverse primer 3. The genomic DNA amplification product of wild-type Wheatland sorghum was used as a negative control. Plants amplified with a 1586 bp fragment were identified as positive transgenic plants of SoERDL6-7::pCAMBIA2300. PCR amplification was performed using forward primer 4 and reverse primer 4, again using the genomic DNA amplification product of wild-type Wheatland sorghum as a negative control. Plants amplified with a 1517 bp fragment were identified as positive transgenic plants of SoTST5::pCAMBIA2300. The PCR reaction system and amplification procedure are as described in Example 2. I. SoERDL6-7 and SoTST5 Construction of gene high expression vectors.

[0092] Gene expression levels were verified in the positive plants identified above. Total RNA was extracted from the DNA-identified positive transgenic plants according to the method described in Example 1, and reverse transcribed into cDNA. Using the cDNA as a template, [the following was performed / conducted / etc.]. SoERDL6-7 and SoTST5 Gene expression levels were detected using quantitative real-time PCR. The system and procedures for quantitative real-time PCR are as described in Example 1. SoERDL6-7 The primers used for quantitative detection of the gene are forward primer 5 and reverse primer 5. SoTST5 The primers used for quantitative detection of the gene were forward primer 6 and reverse primer 6. According to 2^ （-ΔΔCt） Method Calculation SoERDL6-7 Genes and SoTST5 The relative expression level of genes.

[0093] Forward primer 5: 5'-AAAGAAATACCGAACGCCCC-3'; Reverse primer 5: 5'-CAACAGTGGCAAGAACCTGAA-3'; Forward primer 6: 5'-TGGTCCCCGAATGTCTATGT-3'; Reverse primer 6: 5'-CCCCTGACCCACTGAACTG-3'; Internal control EIF forward primer: 5'-ATGTTGCGTAGGCAGTCTCT-3'; Internal control EIF reverse primer: 5'-GCATGGTAGCTGAGAACACAC-3'; The results are shown in the following table: Figure 2 The expression level of the transgenic line with the highest expression level is shown in the following table. The expression level of the wild type plant was normalized to 1, SoERD6-7 The expression level of the positive transgenic line was 42.10 times that of the wild type, SoTST5 The expression level of the positive transgenic line was 427.46 times that of the wild type. SoERD6-7 SoERD6-7 III. Determination of sugar content in stems of positive transgenic sorghum lines SoTST5 The sugar content of the positive transgenic sorghum plants was determined. The sugar content in stems was determined 15 days after flowering, using wild type sorghum Wheatland plants as controls. The sugar content determination kit from Megazyme (Cat. No. K-SUFRG) was used to determine the sucrose, glucose and fructose content. The determination method was as follows: SoTST5 Sample preparation: about 0.1 g of stem tissue was weighed into a 1.5 mL centrifuge tube, 500 μL of ultrapure water was added, and the stem tissue was ground into a homogenate using a grinding rod; centrifuged at 12,000 rpm for 2 min, the supernatant was diluted 4 times to obtain the sample to be tested; 1 blank control and 3 experimental replicates were set during determination.

[0094] III. Determination of sugar content in stems of positive transgenic sorghum lines The sugar content of the positive transgenic sorghum plants was determined. The sugar content in stems was determined 15 days after flowering, using wild type sorghum Wheatland plants as controls. The sugar content determination kit from Megazyme (Cat. No. K-SUFRG) was used to determine the sucrose, glucose and fructose content. The determination method was as follows: SoERDL6-7 Sample preparation: about 0.1 g of stem tissue was weighed into a 1.5 mL centrifuge tube, 500 μL of ultrapure water was added, and the stem tissue was ground into a homogenate using a grinding rod; centrifuged at 12,000 rpm for 2 min, the supernatant was diluted 4 times to obtain the sample to be tested; 1 blank control and 3 experimental replicates were set during determination. SoTST5 III. Determination of sugar content in stems of positive transgenic sorghum lines

[0095] ​​Determination of sucrose content: Prepare 4 disposable cuvettes (1 blank control, 3 experimental groups), according to the kit instructions, configure solution 2, add 0.2 mL solution 2 to each cuvette; except for the blank control, add 0.1 mL sample to each of the 3 experimental groups, mix well and stand for 5 min; according to the kit instructions, configure solution 1, add 2 mL ultrapure water, 0.1 mL bottle 1 buffer, and 0.1 mL solution 1 to the blank control group, add 1.9 mL ultrapure water, 0.1 mL bottle 1 buffer, and 0.1 mL solution 1 to the experimental groups, mix well and stand for 3 min, use a spectrophotometer to measure the absorbance at 340 nm, record as A1 value (record the A1 value of the blank and control groups respectively); add 0.02 mL bottle 3 buffer to the blank and experimental groups respectively, mix well and react for 5 min, then use a spectrophotometer to measure the absorbance at 340 nm, record as A2 value (note: if the reaction does not stop at 5 min, it should be continuously measured at 2 min intervals until the A2 value no longer changes); according to the calculation table provided on the Megazyme company website, input the sample weight volume ratio, dilution multiple, A1 and A2 values to calculate the stem sucrose content, unit g / 100g.

[0096] Determination of total glucose and fructose content: Prepare 4 disposable cuvettes (1 blank control, 3 experimental groups), except for the blank control, add 0.1 mL sample to each of the 3 experimental groups; according to the kit instructions, configure solution 1, add 2.2 mL ultrapure water, 0.1 mL bottle 1 buffer, and 0.1 mL solution 1 to the blank control group, add 2.1 mL ultrapure water, 0.1 mL bottle 1 buffer, and 0.1 mL solution 1 to the experimental groups, mix well and stand for 3 min, use a spectrophotometer to measure the absorbance at 340 nm, record as A1 value (record the A1 value of the blank and control groups respectively); add 0.02 mL bottle 3 buffer to the blank and experimental groups, mix well and react for 5 min, then use a spectrophotometer to measure the absorbance at 340 nm, record as A2 value (note: if the reaction does not stop at 5 min, it should be continuously measured at 2 min intervals until the A2 value no longer changes); add 0.02 mL bottle 4 buffer to the blank and experimental groups respectively, mix well and react for 10 min, then use a spectrophotometer to measure the absorbance at 340 nm, record as A3 value.

[0097] Calculate the sucrose, glucose, and fructose content in the stems using the following formula or the calculation table provided on the Megazyme website (enter the sample weight-to-volume ratio and dilution factor). The unit is g / 100g.

[0098] ΔA (Total Glucose) = Sample (A2 - A1) - Control (A2 - A1) [Note: Measured value when measuring sucrose] ΔA (glucose) = Sample (A2 - A1) - Control (A2 - A1) [Note: Measured values ​​for glucose and fructose] ΔA (sucrose) = ΔA (total glucose) - ΔA (glucose) ΔA (fructose) = Sample (A3 - A2) - Control (A3 - A2) [Note: Measured values ​​for glucose and fructose] Sucrose concentration (g / L) = 1.315 × ΔA (sucrose) Glucose concentration (g / L) = 0.6920 × ΔA (glucose) Fructose concentration (g / L) = 0.6978 × ΔA (fructose) Sucrose content (g / 100g) = [Sucrose concentration (g / L) / Sample weight to solution volume ratio (g / L)] × 100 Glucose content (g / 100g) = [Glucose concentration (g / L) / Sample weight to solution volume ratio (g / L)] × 100 Fructose content (g / 100g) = [Fructose concentration (g / L) / Sample weight to solution volume ratio (g / L)] × 100 The results are as follows Figure 3 As shown in Figure a, it can be seen that the sucrose content of transgenic plants expressing ERDL6-7 or TST5 was significantly increased compared with the wild-type control. The average sucrose content of the wild-type control was 0.097 g / 100g, while the sucrose content of plants expressing ERDL6-7 or TST5 was 3.64 g / 100g and 0.23 g / 100g, respectively, representing increases of 37.6 and 2.4 times. Figure 3 Figure b shows that, compared with the wild-type control, the glucose content of the transgenic plants expressing TST5 was significantly increased. The average glucose content of the wild-type control was 0.016 g / 100g, while the glucose content of the TST5-expressing plants was 0.338 g / 100g, which is 20.7 times higher. Figure 3The results above demonstrate that expression of sugarcane

[0099] sucrose transporter gene in sorghum can significantly increase the sucrose, glucose and fructose content in the stem. SoTST5 and SoERDL6-7 sucrose, glucose and fructose content in the stem.

[0100] The above detailed the present application. For those skilled in the art, without departing from the spirit and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

Claims

1. A protein, which is A1) or / and A2): said A1) is any one of: A1-1) a protein whose amino acid sequence is SEQ ID NO: 1; A1-2) a protein having 80% or more identity to the protein whose amino acid sequence is shown in A1-1) and having the same function, obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in A1-1); A1-3) a fusion protein having the same function, obtained by linking a tag to the N terminus and / or C terminus of the amino acid shown in any one of A1-1) or A1-2); said A2) is any one of: A2-1) a protein whose amino acid sequence is SEQ ID NO: 3; A2-2) a protein having 80% or more identity to the protein whose amino acid sequence is shown in A2-1) and having the same function, obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in A2-1); A2-3) a fusion protein having the same function, obtained by linking a tag to the N terminus and / or C terminus of the amino acid shown in any one of A2-1) or A2-2).

2. A biological material related to the protein described in claim 1, which is any one of: B1), a nucleic acid molecule encoding the protein described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6), a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2); B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

3. The biomaterial of claim 2, wherein, said nucleic acid molecule is any one of a DNA molecule: A1) a DNA molecule whose nucleotide sequence is SEQ ID NO: 2 or a DNA molecule whose nucleotide sequence is positions 2231-6350 of SEQ ID NO: 2; A2) a DNA molecule whose nucleotide sequence is SEQ ID NO: 4 or a DNA molecule whose nucleotide sequence is positions 2001-6792 of SEQ ID NO: 4; A3) a DNA molecule whose coding sequence is SEQ ID NO: 5; A4) a DNA molecule whose coding sequence is SEQ ID NO:

6.

4. Use, characterized in that, the use of the protein described in claim 1 or the biological material described in claim 2 in any one of: M1) increasing the sugar content of a plant; M2) producing a product for increasing the sugar content of a plant; M3) producing a plant with increased sugar content; M4) plant breeding.

5. Use according to claim 4, characterized in that, said sugar content includes sucrose content, glucose content, or / and fructose content.

6. Use according to claim 4 or 5, characterized in that, said plant is any one of: G1) a dicotyledonous plant or a monocotyledonous plant; G2) a plant of the order Poales or a plant of the order Poales; G3) a plant of the family Poaceae or a plant of the family Poaceae; G4) a plant of the genus Saccharum or the genus Sorghum; G5) a plant of the species Saccharum officinarum or the species Sorghum bicolor.

7. A method of increasing the sugar content of a plant, the method comprising increasing or enhancing or up-regulating the sugar content of a recipient plant by increasing or enhancing or up-regulating the expression of a gene encoding the protein of claim 1 or modulating the activity and / or amount of the protein in the recipient plant.

8. A method of producing a plant having an increased sugar content, the method comprising obtaining a plant of interest by increasing or enhancing or up-regulating the expression of a gene encoding the protein of claim 1 or modulating the activity and / or amount of the protein in a starting plant, the plant of interest having a higher sugar content than the starting plant.

9. The method of claim 7 or 8, wherein the nucleotide sequence of the encoding gene is nucleotides 2231 to 6350 of SEQ ID NO: 2 or nucleotides 2001 to 6792 of SEQ ID NO:

4.

10. The method according to any one of claims 6-8, characterized in that, the plant is any one of: G1) a dicotyledonous plant or a monocotyledonous plant; G2) a plant of the order Poales or a plant of the order Poales; G3) a plant of the family Poaceae or a plant of the family Poaceae; G4) a plant of the genus Saccharum or the genus Sorghum; G5) a plant of the species Saccharum officinarum or the species Sorghum bicolor.

Images