AcSUT4 gene of 'Hongyang' kiwi fruit and application of AcSUT4 gene

Abstract

The invention belongs to the field of biology, and particularly relates to a 'Hongyang' kiwi fruit AcSUT4 gene and application thereof. The full-length cDNA of the AcSUT4 gene of the 'Hongyang' kiwi fruit is cloned, a yeast mutant function complementation experiment proves that the AcSUT4 gene has the function of transporting sucrose, the instant silencing AcSUT4 gene has the function of reducing the content of sucrose, fructose, glucose and starch in the fruit, the weight of the fruit can be reduced, the transverse diameter and the longitudinal diameter of the fruit can be reduced, and the yield of the fruit can be increased. A reliable gene target is provided for high-yield molecular improvement of kiwi fruits, and a theoretical basis is provided for molecular breeding for improving the quality of plant fruits.

Description

Technical Field

[0001] The invention belongs to the biological field, and specifically relates to the AcSUT4 gene of 'Hongyang' kiwifruit and its application. Background Art

[0002] Kiwifruit is a fruit with fresh quality, rich nutrition and delicious flavor. The 'Hongyang' kiwifruit is an excellent variety of kiwifruit, with high sugar content, low total acid, high soluble solid content and high vitamin C content. However, there are few studies on the genes that regulate the quality of the 'Hongyang' kiwifruit, so it is difficult to explain why the 'Hongyang' kiwifruit has the above qualities. In-depth research on the genes related to fruit quality regulation in the 'Hongyang' kiwifruit and obtaining the key genes that regulate the excellent quality of the 'Hongyang' kiwifruit will play an important role in the screening and improvement of kiwifruit varieties. Summary of the invention

[0003] The invention provides the 'Hongyang' kiwifruit AcSUT4 gene and its application, and provides a theoretical basis for molecular breeding for improving plant fruit quality.

[0004] The technical solution of the present invention is achieved in this way:

[0005] The first aspect of the present invention is to provide the Hongyang' kiwifruit AcSUT4 gene, 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 the gene of the first aspect of the present invention, whose amino acid sequence is shown in SEQ ID NO:2.

[0007] The 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] Wherein, the original vector of the recombinant vector can adopt the vector commonly used in the field of gene recombination, such as virus, plasmid, etc. The present invention is not limited to this. In a specific embodiment of the present invention, the original vector adopts PDR196 vector, etc., but it should be understood that the present invention can also adopt other plasmids, or viruses, etc.

[0009] The fourth aspect of the present invention is to provide a host bacteria or a transgenic cell line or a recombinant bacteria 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 described in the first aspect of the present invention, or the protein described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention, or the host bacteria or transgenic cell line or recombinant bacteria 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 'Hongyang' kiwifruit.

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

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

[0014] Among them, silencing the gene described in the first aspect of the present invention can reduce the sucrose content of the fruit.

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

[0016] The seventh aspect of the present invention is to provide the use of the gene described in the first aspect of the present invention, or the protein described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention, or the host bacteria or transgenic cell line or recombinant bacteria described in the fourth aspect of the present invention in response to chlorpyrifos, ethylene, abscisic acid and / or gibberellin. Wherein, the gene or protein is down-regulated in ABA treatment; it shows an upward trend in 3-12h in GA3 treatment; the overall expression amount shows a downward trend in ET treatment; and the expression amount shows an upward trend in CPPU treatment.

[0017] The eighth aspect of the present invention is to provide a primer pair, wherein the primer pair is: F (5' end): 5'-ATGGACATGGAGGGAGATGC-3' and R (3' end): 5'-TTAGTGGGAAGTAGTTGAGA-3'; or the primer pair is F: 5'-CGTGGTCGGGTTCTGGA-3' and R: 5'-GAAGGCGTTTGCGTTGC-3'; or the primer pair is 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] The present invention clones the full-length cDNA of the sucrose transporter AcSUT4 gene of "Hongyang" kiwifruit. Gene expression analysis shows that the AcSUT4 gene is expressed in different tissues, with the lowest expression in leaves. The AcSUT4 gene has a higher expression in fruits and is closely related to the development of fruits. At the same time, the expression level is regulated by exogenous hormones. Yeast mutant functional complementation experiments prove that the AcSUT4 gene has the function of transporting sucrose. Sucrose is mainly transported into cells by the sucrose transporter AcSUT4 located on the plasma membrane, and its expression is positively correlated with fruit development. Transient silencing of the AcSUT4 gene has the function of reducing the content of sucrose, fructose, glucose and starch in fruits, and can also reduce the weight of fruits, reduce the horizontal and vertical diameters of fruits, and provide a reliable gene target for molecular improvement of high yield of kiwifruit. Therefore, the gene can be used as an important gene resource and can be applied in the genetic engineering of stress resistance and abiotic stress resistance of other plants or microorganisms other than kiwifruit, providing a theoretical basis for molecular breeding to improve the quality of plant fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 This is tissue-specific expression analysis of the AcSUT4 gene (leaves, buds, bark, fruits, female flowers, and male flowers).

[0022] Figure 2 Expression analysis of AcSUT4 gene at different developmental stages of fruit (18d, 38d, 58d, 88d, 118d, and 138d after flowering).

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

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

[0025] Figure 5 The results of AcSUT4 gene in gene silencing (VIGS). DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The methods described in the following embodiments are all conventional methods unless otherwise specified.

[0027] Example 1. Obtaining genes

[0028] The cDNA from different tissues of "Hongyang" kiwifruit was used as template (obtained by reverse transcription with random primers), F and R were used as primers with a final concentration of 0.5 μmol / L, and PCR amplification was performed in a 25 μL reaction system. The reaction system was: 1 μL cDNA template, 2 μL 10× PCR Buffer, 5 U·μL Taqplus DNA Polymerase -1 )0.2μL, dNTP(2.5mmol·L -1 )1.6μL, upstream and downstream primers (10μmol·L -1 ) 1 μL each, ddH2O 18.2 μL, total 25 μL. Amplification program: 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 about 1500 bp was connected to the pXCS-1301 vector for sequencing. The sequencing showed that the above-obtained fragment was the sucrose transporter gene of the present invention. The fragment had the nucleotide sequence of sequence 1 in the sequence list. The open reading coding frame of sequence 1 in the sequence list was 1557 nucleotides (containing the terminator tga), encoding a protein with a length of 518 amino acids (sequence 2 in the sequence list) and a molecular weight of about 54.7 KDa, which was the sucrose transporter. The sucrose transporter gene was named AcSUT4.

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

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

[0035] Real-time fluorescence quantitative PCR was performed using cDNA randomly reverse transcribed from RNA of male and female flowers, buds, bark, leaves and fruits of "Hongyang" kiwifruit as templates using specific primers for AcSUT4 gene (F: 5'-GATTGCGTGGTTTCCGTTCT-3'; R: 5'-CGCTACCCTGCCCTACTTCC-3'). The reaction system consisted of 1 μL template, 5 μL 2×SYBR Premix, 10 μmol·L -1 The upstream and downstream primers were 0.3 μL and 3.4 μL H2O respectively; the amplification program was 95℃ pre-denaturation for 30 min; 95℃ 5 s, 60℃ 20 s, 40 cycles. The primer amplification efficiency and the corresponding Qr value were calculated using LightCycler4.05 software. It showed that the expression level of this gene in bark was lower than that in the other five tissues ( Figure 1 ). <2> Expression analysis of AcSUT4 gene in kiwifruit "Hongyang" at different developmental stages

[0036] At different developmental stages of the fruit (calculated from 18 days after anthesis, i.e., 18d, 38d, 58d, 88d, 118d, and 138d), real-time fluorescence quantitative PCR was performed using cDNA randomly reverse transcribed from RNA of "Hongyang" kiwifruit fruit as template and specific primers of AcSUT4 gene (F: 5'-GATTGCGTGGTTTCCGTTCT-3'; R: 5'-CGCTACCCTGCCCTACTTCC-3'). The reaction system was 1 μL template, 5 μL 2×SYBR Premix, 10 μmol·L -1 0.3 μL of upstream and downstream primers and 3.4 μL of H2O were added; the amplification program was 95℃ pre-denaturation for 30 min; 95℃ for 5 s, 60℃ for 20 s, 40 cycles. The primer amplification efficiency and the corresponding Qr value were calculated using LightCycler4.05 software. The expression of AcSUT4 gene showed an upward trend ( Figure 2 ), indicating that the AcSUT4 gene plays an important role in fruit development.

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

[0038] Fruits 18 days after flowering were selected and soaked in hormones CPPU (chlorpyrifos, 20 mg / L), ET (ethylene, 50 mg / L), ABA (abscisic acid, 10 mg / L), and GA3 (gibberellic acid, 50 mg / L) for 3 seconds. Three biological replicates were set for each hormone treatment. Fruit samples were collected at 0, 3, 6, 9, 12, and 24 hours after treatment, and were quickly frozen in liquid nitrogen and transported back to the laboratory to extract RNA for subsequent experiments. The response of AcSUT4 gene to ABA, GA3, ET, and CPPU treatment was analyzed, and it was found that AcSUT4 gene in fruit was down-regulated in ABA treatment; it showed an upward trend in GA3 treatment from 3 to 12 hours; the overall expression level showed a downward trend in ET treatment; and the expression level showed an upward trend in CPPU treatment. This shows that the expression of AcSUT4 gene is affected by ABA, GA3, ET, and CPPU. The analysis of their cis-acting elements also identified response elements to abscisic acid, gibberellin, salicylic acid, methyl jasmonate, auxin, light response, growth regulation, drought, low temperature, etc. The results showed that the AcSUT4 gene was indeed regulated by ABA, GA3, ET, and CPPU, which provided a theoretical basis for the subsequent regulation of SUT gene expression by exogenous hormones in 'Hongyang' kiwifruit. Example 3. Functional verification of AcSUT4 gene

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

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

[0041] Design of primers for the coding region of AcSUT4 gene

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

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

[0044] Dip the yeast liquid of the successfully transformed PDR196-AcSUT4 and streak it on SD screening medium with 4% sucrose at 30°C. Observe its growth and take photos. The experiment proves that the protein encoded by AcSUT4 has the function of transporting sucrose. Figure 4 shown.

[0045] Example 4. Functional verification of AcSUT4 gene

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

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

[0048] Design of primers for the coding region of AcSUT4 gene

[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 was as follows: the reaction system was 1 μL of cDNA template, 2 μL of 10×PCR Buffer, and 5 U·μL of Taqplus DNA Polymerase. -1)0.2μL, dNTP(2.5mmol·L -1 )1.6μL, upstream and downstream primers (10μmol·L -1 ) 1 μL each, ddH2O 18.2 μL, a total of 25 μL. The amplification program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, a total of 30 cycles; 72℃ extension for 10 min. The amplified product was connected to the pTRV2 expression vector to obtain a recombinant vector, and the vector primers (pTRV2-F: 5'-ATTCACTGGGAGATGATACGCT-3' and pTRV2-R: 5'-AGTCGGCCAAACGCCGATCTCA-3') were used for colony PCR identification to ensure that the sucrose transporter coding fragment was cloned into the expression vector. The recombinant vector was sequenced and identified, and the sequencing results were consistent with the sequence 1 (700-1010) in the sequence list. The recombinant expression vector was named pTRV2-AcSUT4.

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

[0053] Take out GV3101 Agrobacterium containing the recombinant vector pTRV2-AcSUT4 from the -80℃ refrigerator, use a pipette to draw 100μL of bacterial solution into 10mL liquid LB medium containing 50μg / mL kanamycin in the clean bench, and culture at 28℃, 200rpm shaking for about 12h, until the bacterial solution concentration OD600 value is between 0.8-1.0; centrifuge at 5000rpm for 10min, repeat the operation once, discard the supernatant and add 1mL Agrobacterium. Mix the Agrobacterium infiltration buffer by pipetting, centrifuge again, pour out the supernatant, add the Agrobacterium infiltration buffer again, pipette and mix, and adjust the OD600 value to about 1.0; place the adjusted liquid at 28°C for 3 hours, then mix the bacterial solution containing pTRV1 and pTRV2-AcSUT1 in a 1:1 ratio, and use a needle to inject 300-500μL of the bacterial solution from the four directions of the central axis of the kiwi fruit to obtain kiwi fruit with silenced AcSUT1 gene (AcSUT1 RNAi). Mix the bacterial solution of pTRV1 and pTRV2 in a 1:1 ratio and inject in the same way to obtain the control kiwi fruit (Empty vector). At least 5 kiwi trees were selected for each treatment, with at least 5 fruits per tree. The experiment was repeated at least 3 times for biological replication and 3 times for technical replication. Phenotypic observation and sampling were carried out 30 days after injection. The results are as follows Figure 5 shown.

[0054] Glucose, fructose, sucrose and starch in kiwifruit were determined by referring to the method in the paper "Determination of Glucose, Fructose, Sucrose and Starch in Fruits and Vegetables by Anthrone Spectrophotometry" by Zhang Youjie et al. in 1977; the horizontal and vertical diameters of the fruit were measured with a vernier caliper. The results showed that transient silencing of the AcSUT4 gene has the function of reducing the content of sucrose, fructose, glucose and starch in kiwifruit, and at the same time, the weight, vertical and horizontal diameters of the fruit were reduced to varying degrees.

[0055] 4. Conclusion

[0056] The full-length cDNA of the AcSUT4 gene of the "Hongyang" kiwifruit was cloned for the first time. Gene expression analysis showed that the AcSUT4 gene was expressed in different tissues, with the lowest expression in leaves. The AcSUT4 gene had a higher expression in the fruit and was closely related to the development of the fruit. At the same time, the expression level was regulated by exogenous hormones. The yeast mutant functional complementation experiment proved 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 the molecular improvement of kiwifruit high yield. Therefore, this gene can be used as an important gene resource and can be used in the genetic engineering of stress resistance and abiotic stress resistance of other plants or microorganisms besides kiwifruit.

[0057] The specific embodiments of the present invention are described in detail above, but they are only 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 made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

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

1.

2. The protein encoded by the AcSUT4 gene according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

4.

3. A recombinant vector, host bacteria, transgenic cell line or recombinant bacteria containing the AcSUT4 gene according to claim 1.

4. Use of the AcSUT4 gene as claimed in claim 1, or the protein as claimed in claim 2, or the recombinant vector, host bacteria, transgenic cell line or recombinant bacteria as claimed in claim 3 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.

5. The use according to claim 4, characterized in that The AcSUT4 gene can increase the fruit weight and / or reduce the longitudinal diameter of the plant fruit and / or reduce the transverse diameter of the plant fruit.

6. Use of the AcSUT4 gene according to claim 1, or the protein according to claim 2, or the recombinant vector, host bacteria, transgenic cell line or recombinant bacteria according to claim 3 in regulating the sucrose content of plant fruits.

7. The use according to claim 6, characterized in that Silencing the AcSUT4 gene described in claim 1 can reduce the sucrose content of fruits.

8. The use according to any one of claims 4 to 7, characterized in that: The plant is kiwi fruit.

9. Use of the AcSUT4 gene according to claim 1, or the protein according to claim 2, or the recombinant vector, host bacteria, transgenic cell line or recombinant bacteria according to claim 3 in response to chlorpyrifos, ethylene, abscisic acid and / or gibberellin.

10. 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 site)ATGGAGGGTGGTGGTGGA-3' and R:5'-CCG CTCGAG (XhoI restriction site)TCAATGAAATGCAGCAGC-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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