Hongyang kiwifruit acsut4 gene and application thereof

Abstract

The application belongs to the field of biology and particularly relates to 'Hongyang' kiwifruit AcSUT4 gene and application thereof. The full-length cDNA of 'Hongyang' kiwifruit AcSUT4 gene is cloned, the yeast mutant function complementation experiment proves that the AcSUT4 gene has the function of transporting sucrose, the instant silencing of the AcSUT4 gene has the function of reducing the contents of sucrose, fructose, glucose and starch in fruits, and can also reduce the weight, horizontal diameter and vertical diameter of the fruits, which provides a reliable gene target for the high-yield molecular improvement of kiwifruit and a theoretical basis for the molecular breeding of improving the quality of plant fruits.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to the AcSUT4 gene of 'Hongyang' kiwifruit and its applications. Background Technology

[0002] Kiwifruit is a delicious fruit known for its tender texture, rich nutrition, and flavor. The 'Hongyang' kiwifruit is a superior variety, characterized by high sugar content, low total acid, high soluble solids content, and high vitamin C content. However, current research on the genes regulating the quality of 'Hongyang' kiwifruit is limited, making it difficult to elucidate the reasons for its superior qualities. In-depth research on the genes regulating fruit quality in 'Hongyang' kiwifruit, and identifying the key genes controlling its superior quality, will play a crucial role in the screening and improvement of kiwifruit varieties. Summary of the Invention

[0003] This invention provides the AcSUT4 gene of 'Hongyang' kiwifruit and its application, providing a theoretical basis for molecular breeding to improve the quality of plant fruits.

[0004] The technical solution of this invention is implemented as follows:

[0005] The first aspect of the present invention is to provide the AcSUT4 gene of 'Hongyang' kiwifruit, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0006] The second aspect of the present invention is to provide a protein encoded by a gene of the first aspect of the present invention, the amino acid sequence of which is shown in SEQ ID NO:2.

[0007] A third aspect of the present invention is to provide a recombinant vector containing the gene described in the first aspect of the present invention.

[0008] The original vector for the recombinant vector can be a vector commonly used in the field of gene recombination, such as a virus or plasmid. This invention does not limit this. In one specific embodiment of this invention, the original vector is a PDR196 vector, etc., but it should be understood that other plasmids or viruses can also be used.

[0009] A fourth aspect of the present invention is to provide a host bacterium, a transgenic cell line, or a recombinant bacterium containing the gene of the first aspect of the present invention.

[0010] The fifth aspect of the present invention is to provide the use of the gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant vector as described in the third aspect of the present invention, or the host bacterium or transgenic cell line or recombinant bacterium as described in the fourth aspect of the present invention in regulating the weight of plant fruits and / or regulating the longitudinal diameter of plant fruits and / or regulating the transverse diameter of plant fruits.

[0011] Preferably, the plant is the 'Hongyang' kiwifruit.

[0012] Silencing the gene described in the first aspect of the present invention can reduce fruit weight and / or reduce the longitudinal diameter and / or reduce the transverse diameter of the plant fruit.

[0013] The sixth aspect of the present invention is to provide the use of the gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant vector as described in the third aspect of the present invention, or the host bacterium or transgenic cell line or recombinant bacterium as described in the fourth aspect of the present invention in regulating the sucrose content of plant fruits.

[0014] Silencing the gene described in the first aspect of this invention can reduce the sucrose content of the fruit.

[0015] Preferably, the plant is the 'Hongyang' kiwifruit.

[0016] A seventh aspect of the present invention is to provide the use of the gene as described in the first aspect of the present invention, or the protein as described in the second aspect of the present invention, or the recombinant vector as described in the third aspect of the present invention, or the host bacterium or transgenic cell line or recombinant bacterium as described in the fourth aspect of the present invention in response to chlorpyrifos, ethylene, abscisic acid and / or gibberellin. The gene or protein is downregulated in ABA treatment; shows an increasing trend in GA3 treatment over 3-12 hours; shows a decreasing trend in overall expression in ET treatment; and shows an increasing trend in expression in CPPU treatment.

[0017] An eighth aspect of the present invention is to provide a primer pair having the following characteristics: F (5' end): 5′-ATGGACATGGAGGGAGATGC-3′ and R (3' end): 5′-TTAGTGGGAAGTAGTTGAGA-3′; or the primer pair having the following characteristics: F: 5′-CGTGGTCGGGTTCTGGA-3′ and R: 5′-GAAGGCGTTTGCGTTGC-3′; or the primer pair having the following characteristics: F: 5′-G GAATTC (EcoRI restriction site) ATGGACATGGAGGGAGAT-3' and R:5'-CCG CTCGAG (XhoI restriction site) TTAGTGGGAAGTAGTTGAGAT-3'; or the primer pair is: F:5'-G GAATTC (EcoRI restriction site) CGCAAGGCGGCTGAGT-3' and R:5'-C GAGCTC (SacI restriction site)GCTCCCACCACAAATGATGG-3'.

[0018] Beneficial effects:

[0019] This invention cloned the full-length cDNA of the AcSUT4 gene, a sucrose transporter in the 'Hongyang' kiwifruit variety. Gene expression analysis showed that the AcSUT4 gene was expressed in different tissues, with the lowest expression level in leaves. The AcSUT4 gene showed high expression in fruit and was closely related to fruit development, with its expression level regulated by exogenous hormones. Functional complementation experiments with yeast mutants demonstrated that the AcSUT4 gene has the function of transporting sucrose. Sucrose is mainly transported into cells via the sucrose transporter AcSUT4, which is located on the plasma membrane. Its expression is positively correlated with fruit development. Transient silencing of the AcSUT4 gene reduced the content of sucrose, fructose, glucose, and starch in the fruit, and also reduced fruit weight, transverse diameter, and longitudinal diameter, providing a reliable gene target for molecular improvement of high-yield kiwifruit. Therefore, this gene can serve as an important genetic resource and can be applied in genetic engineering for stress and abiotic stress resistance in other plants or microorganisms besides kiwifruit, providing a theoretical basis for molecular breeding to improve fruit quality. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Tissue-specific expression analysis of the AcSUT4 gene (leaf, bud, bark, fruit, female flower, male flower).

[0022] Figure 2 The expression of the AcSUT4 gene was analyzed at different fruit development stages (18d, 38d, 58d, 88d, 118d, and 138d after flowering).

[0023] Figure 3 The expression of the AcSUT4 gene in kiwifruit treated with different hormones.

[0024] Figure 4 To verify the functional complementation of the yeast mutant of the AcSUT4 gene.

[0025] Figure 5 The results for AcSUT4 gene silencing (VIGS) are shown. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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. Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0027] Example 1. Gene Acquisition

[0028] Using cDNA from different tissues of 'Hongyang' kiwifruit as templates (obtained by reverse transcription with random primers), and primers F and R at a final concentration of 0.5 μmol / L, PCR amplification was performed in a 25 μL reaction system. The reaction system consisted of: 1 μL cDNA template, 2 μL 10×PCR Buffer, and 5 U / μL Taqplus DNA Polymerase. -1 0.2 μL, dNTP (2.5 mmol·L) -1 1.6 μL of forward and reverse primers (10 μmol·L⁻¹) -1 1 μL each of 1,000 and 18.2 μL of ddH2O, for a total of 25 μL. The amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s, 32 cycles; 72℃ extension for 10 min.

[0029] The specific primers are as follows:

[0030] F (5′ end): 5′-ATGGAGGGGTGGTGGTGGACT-3′;

[0031] R (3' end): 5'-TCAATGAAATGCAGCAGCGG-3'.

[0032] The obtained nucleotide fragment of approximately 1500 bp was ligated into the pXCS-1301 vector and sequenced. Sequencing showed that the obtained fragment was the sucrose transporter gene of the present invention. The fragment has the nucleotide sequence of Sequence 1 in the sequence listing. The open reading frame of Sequence 1 in the sequence listing is 1557 nucleotides (containing the terminator tga), which encodes a protein with a length of 518 amino acids (Sequence 2 in the sequence listing) and a molecular weight of approximately 54.7 kDa, which is the sucrose transporter. The sucrose transporter gene was named AcSUT4.

[0033] Example 2. Analysis of AcSUT4 gene expression pattern in "Hongyang" kiwifruit

[0034] <1> tissue-specific expression of the AcSUT4 gene in “Hongyang” kiwifruit

[0035] Using cDNA randomly reverse transcribed from RNA of male and female flowers, buds, bark, leaves, and fruits of the 'Hongyang' kiwifruit variety as templates, real-time quantitative PCR was performed using AcSUT4 gene-specific primers (F: 5'-GATTGCGTGGTTTCCGTTCT-3'; R: 5'-CGCTACCCTGCCCTACTTCC-3'). The reaction system consisted of 1 μL template, 5 μL 2×SYBR Premix, and 10 μmol·L⁻¹. -1 0.3 μL of upstream and downstream primers and 3.4 μL of H2O were used respectively; the amplification program was 95℃ pre-denaturation for 30 min; 95℃ for 5 s, 60℃ for 20 s, for 40 cycles. The primer amplification efficiency and corresponding Qr values ​​were calculated using LightCycler 4.05 software. The results showed that the expression level of this gene in the bark was lower than that in the other five tissues (…). Figure 1 ). <2> Expression analysis of the AcSUT4 gene in "Hongyang" kiwifruit at different developmental stages

[0036] At different fruit development stages (calculated from 18 days after flowering, i.e., 18d, 38d, 58d, 88d, 118d, and 138d), cDNA randomly reverse-transcribed from RNA of “Hongyang” kiwifruit fruit was used as a template. Real-time quantitative PCR was performed using AcSUT4 gene-specific primers (F: 5'-GATTGCGTGGTTTCCGTTCT-3'; R: 5'-CGCTACCCTGCCCTACTTCC-3'). The reaction system consisted of 1 μL template, 5 μL 2×SYBR Premix, and 10 μmol·L⁻¹. -1 0.3 μL of upstream and downstream primers and 3.4 μL of H2O were used respectively; the amplification program was 95℃ pre-denaturation for 30 min; 95℃ for 5 s, 60℃ for 20 s, for 40 cycles. The primer amplification efficiency and corresponding Qr values ​​were calculated using LightCycler 4.05 software. The results showed an increasing trend in AcSUT4 gene expression. Figure 2 This indicates that the AcSUT4 gene plays an important role in fruit development.

[0037] <3> Effects of different treatments on AcSUT4 gene expression in kiwifruit

[0038] Fruits 18 days after flowering were treated with CPPU (chlorpyrifos, 20 mg / L), ET (ethylene, 50 mg / L), ABA (abscisic acid, 10 mg / L), and GA3 (gibberellin, 50 mg / L) for 3 seconds, respectively. Three biological replicates were set up for each hormone treatment. Fruit samples were collected at 0, 3, 6, 9, 12, and 24 hours after treatment, flash-frozen in liquid nitrogen, and transported back to the laboratory for RNA extraction for subsequent experiments. The response of the AcSUT4 gene to ABA, GA3, ET, and CPPU treatments was analyzed. It was found that AcSUT4 gene expression was downregulated in fruit under ABA treatment; it showed an upward trend from 3 to 12 hours under GA3 treatment; the overall expression level decreased under ET treatment; and the expression level increased under CPPU treatment. This indicates that AcSUT4 gene expression is responsive to ABA, GA3, ET, and CPPU. Analysis of their cis-acting elements also identified response elements to abscisic acid, gibberellin, salicylic acid, methyl jasmonate, auxin, light response, growth regulation, drought, and low temperature. These results indicate that the AcSUT4 gene is indeed regulated by ABA, GA3, ET, and CPPU, providing a theoretical basis for subsequent regulation of the SUT gene expression in 'Hongyang' kiwifruit using exogenous hormones. Example 3. Functional Verification of the AcSUT4 Gene

[0039] A yeast expression vector for the AcSUT4 gene was constructed using the PDR196 expression vector (provided by Professor Yang Jianghua of the Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences). The specific method is as follows:

[0040] <1> Obtaining a recombinant vector containing the AcSUT4 gene coding region

[0041] Design primers for the AcSUT4 gene coding region

[0042] F:5'-G GAATTC (EcoRI restriction site) ATGGAGGGTGGTGGTGGA-3', R:5'-CCG CTCGAG (XhoI restriction site) TCATGAAATGCAGCAGC-3', using pDR196-AcSUT4 as a template, was used for PCR amplification. The reaction system consisted of 1 μL cDNA template, 2 μL 10×PCR Buffer, and 5 U / μL Taqplus DNA Polymerase. -1 0.2 μL, dNTP (2.5 mmol·L) -1 1.6 μL of forward and reverse primers (10 μmol·L⁻¹) -11 μL of each of the following: 18.2 μL of ddH2O, for a total of 25 μL. The amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, for a total of 30 cycles; 72℃ extension for 10 min. The amplified product was ligated into the PDR196 expression vector to obtain the recombinant vector. Colony PCR was performed using the vector primers (PDR196-F: 5'-CTCTTTTATACACACATTCA-3' and PDR196-R: 5'-CTGGCGAAGAAGTCCAAAGC-3') to ensure that the sucrose transporter coding fragment was positively cloned into the expression vector. The recombinant vector was sequenced, and the sequencing results were completely consistent with the sequence of Sequence 1 in the sequence listing. The recombinant expression vector was named PDR196-AcSUT4.

[0043] <2> PDR196-AcSUT4 recombinant expression vector

[0044] Yeast culture successfully transformed with PDR196-AcSUT4 was streaked onto SD selection medium containing 4% sucrose and incubated at 30°C. Growth was observed and photographed. The experiment demonstrated that the AcSUT4-encoded protein has the function of transporting sucrose. Figure 4 As shown.

[0045] Example 4. Functional verification of the AcSUT4 gene

[0046] A silencing expression vector for the AcSUT4 gene was constructed using the PTRV2 expression vector (provided by Professor Yang Jianghua of the Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences). The specific method is as follows:

[0047] <1> Obtaining a recombinant vector containing the AcSUT4 gene coding region

[0048] Design primers for the AcSUT4 gene coding region

[0049] F:5'-G GAATTC (EcoRI restriction site) CCTCCTGCTTCTCACTC-3',

[0050] R:5'-C GAGCTC (SacI restriction site)GTTTAGCATCAACCCCAACG-3',

[0051] The 700-1010 fragment of AcSUT4 CDS was amplified and inserted into pTRV2 to generate the pTRV2-AcSUT4 vector. The amplification system consisted of 1 μL cDNA template, 2 μL 10×PCR Buffer, and 5 U / μL Taqplus DNA Polymerase. -10.2 μL, dNTP (2.5 mmol·L) -1 1.6 μL of forward and reverse primers (10 μmol·L⁻¹) -1 1 μL of each of the following: 18.2 μL of ddH2O, for a total of 25 μL. The amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, for a total of 30 cycles; 72℃ extension for 10 min. The amplified product was ligated into the pTRV2 expression vector to obtain the recombinant vector. Colony PCR was performed using the vector primers (pTRV2-F: 5'-ATTCACTGGGAGATGATACGCT-3' and pTRV2-R: 5'-AGTCGGCCAAACGCCGATCTCA-3') to ensure that the sucrose transporter coding fragment was cloned into the expression vector. The recombinant vector was sequenced and identified. The sequencing results were consistent with the sequence of sequence 1 (700-1010) in the sequence listing. The recombinant expression vector was named pTRV2-AcSUT4.

[0052] <2> pTRV2-AcSUT4 recombinant expression vector

[0053] Agrobacterium GV3101 containing the recombinant vector pTRV2-AcSUT4 was removed from a -80℃ freezer. In a clean bench, 100 μL of the bacterial culture was pipetted into 10 mL of liquid LB medium containing 50 μg / mL kanamycin. The culture was incubated at 28℃ with shaking at 200 rpm for approximately 12 h, until the OD600 value of the bacterial culture was between 0.8 and 1.0. The culture was then centrifuged at 5000 rpm for 10 min, and this process was repeated once. The supernatant was discarded, and 1 mL of Agrobacterium was added. The Agrobacterium infiltration buffer was mixed by pipetting, centrifuged again, and the supernatant was discarded. Agrobacterium infiltration buffer was added again and mixed by pipetting, adjusting the OD600 value to approximately 1.0. The prepared liquid was incubated at 28°C for 3 hours. Then, a 1:1 mixture of pTRV1 and pTRV2-AcSUT1 bacterial solutions was injected into the kiwifruit fruit from four directions along the central axis using a needle to obtain kiwifruit fruit with the AcSUT1 gene silenced (AcSUT1 RNAi). A 1:1 mixture of pTRV1 and pTRV2 bacterial solutions was injected using the same method to obtain control kiwifruit fruit (Empty vector). At least 5 kiwifruit trees were selected for each treatment, with at least 5 fruits from each tree. The experiment was performed in at least 3 biological replicates and 3 technical replicates. Phenotypic observation and sampling were conducted 30 days after injection. Results are as follows: Figure 5 As shown.

[0054] Following the method described in Zhang Youjie et al.'s 1977 paper, "Determination of Glucose, Fructose, Sucrose, and Starch in Fruits and Vegetables by Anthrone Spectrophotometry," glucose, fructose, sucrose, and starch in kiwifruit were determined. The transverse and longitudinal diameters of the fruit were measured using vernier calipers. The results showed that transient silencing of the AcSUT4 gene reduced the content of sucrose, fructose, glucose, and starch in kiwifruit, while also reducing fruit weight, longitudinal diameter, and transverse diameter to varying degrees.

[0055] 4. Conclusion

[0056] The full-length cDNA of the AcSUT4 gene from the 'Hongyang' kiwifruit variety was cloned for the first time. Gene expression analysis showed that the AcSUT4 gene was expressed in different tissues, with the lowest expression level in leaves. The AcSUT4 gene showed high expression in fruit and was closely related to fruit development, with its expression level regulated by exogenous hormones. Functional complementation experiments with yeast mutants demonstrated that the AcSUT4 gene has the function of transporting sucrose. AcSUT4 can transport sucrose into cells, and its expression is positively correlated with fruit development, providing a reliable gene target for molecular improvement of high-yield kiwifruit. Therefore, this gene can serve as an important genetic resource and can be applied in the genetic engineering of stress and abiotic stress resistance in other plants or microorganisms besides kiwifruit.

[0057] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. 'Hongyang' kiwifruit AcSUT4 Genes, characterized by, The AcSUT4 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. As described in claim 1 AcSUT4 Proteins encoded by genes.

3. A composition comprising the AcSUT4 recombinant vector, host cell, transgenic cell line or recombinant bacteria of claim 1.

4. As described in claim 1 AcSUT4 The application of genes, or the protein of claim 2, or the recombinant vector, host bacteria, transgenic cell line, or recombinant bacteria of claim 3 in regulating plant fruit weight and / or regulating plant fruit longitudinal diameter and / or regulating plant fruit transverse diameter, characterized in that, Silencing the AcSUT4 The gene is capable of reducing kiwifruit weight and / or reducing kiwifruit longitudinal diameter and / or reducing kiwifruit transverse diameter.

5. As described in claim 1 AcSUT4 The application of a gene, or the protein of claim 2, or the recombinant vector, host bacteria, transgenic cell line, or recombinant bacteria of claim 3, in regulating the sucrose content of plant fruits, characterized in that... The silence stated AcSUT4 Genes reduce the sucrose content of kiwifruit.

6. A primer pair, characterized in that, The primer pair is: F (5' end): 5'-ATGGAGGGTGGTGGTGGACT -3' and R (3' end): 5'-TCAATGAAATGCAGCAGCGG-3'; or the primer pair is F: 5'-GATTGCGTGGTTTCCGTTCT -3' and R: 5'-CGCTACCCTGCCCTACTTCC -3'; or the primer pair is F: 5'-G GAATTC (EcoRI restriction sites) ATGGAGGGTGGTGGTGGA-3' and R:5'-CCG CTCGAG (XhoI restriction site) TCATGAAATGCAGCAGC-3'; or the primer pair is: F: 5'-G GAATTC (EcoRI restriction site) CCTCCTGCTTCTCACTC-3' and R:5'-C GAGCTC (SacI restriction site)GTTTAGCATCAACCCCAACG-3'.

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