AcSUT1 gene of 'Hongyang' kiwi fruit and application of AcSUT1 gene

Abstract

The invention belongs to the field of biology, and particularly relates to a 'Hongyang' kiwi fruit AcSUT1 gene and application thereof. According to the invention, the full-length cDNA of the AcSUT1 gene of the 'Hongyang' kiwi fruit is cloned, and gene expression analysis shows that the expression quantity of the AcSUT1 gene in leaves is the lowest, and the expression quantity in fruits is relatively high. Yeast mutant function complementation experiments prove that the AcSUT1 gene has the function of transporting sucrose, the instant silent AcSUT1 gene has the function of reducing the content of sucrose, fructose, glucose and starch in fruits, the weight of the fruits can be reduced, the transverse diameter and the longitudinal diameter of the fruits can be reduced, and a reliable gene target is provided for improvement of high-yield molecules of kiwi fruits. The gene can be used as an important gene resource, can be applied to stress resistance and abiotic stress resistance gene engineering of other plants except kiwi fruits or microorganisms, and provides a theoretical basis 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 AcSUT1 gene of 'Hongyang' kiwifruit and its application. Background Art

[0002] The fruit of 'Hongyang' kiwifruit is medium to large, neat, very juicy, moderately sweet and sour, and refreshing. The total sugar content in the fruit can reach 13.45%, which is nearly 5 percentage points higher than the world's popular variety Hayworth, while the total acid content is only 0.49%, and the soluble solids content is 16.5%. It is rich in calcium, iron, potassium and other minerals and 17 kinds of amino acids, making it an excellent variety of kiwifruit. However, there are not many studies on the regulatory mechanism of its fruit quality, especially on the genes related to the regulation of the quality of 'Hongyang' kiwifruit. Therefore, in-depth research on the genes related to the regulation of fruit quality in 'Hongyang' kiwifruit and their functions can not only clarify the principles of physiological and biochemical regulation of 'Hongyang' kiwifruit and realize the scientific cultivation and management of 'Hongyang' kiwifruit, but also play a positive role in comprehensively improving the yield and quality of kiwifruit, and improving and breeding excellent kiwifruit varieties. Summary of the invention

[0003] The present invention provides the 'Hongyang' kiwifruit AcSUT1 gene and application thereof, 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 AcSUT1 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 the fruit weight, and / or reduce the longitudinal diameter of the plant fruit, 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 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 sugar content of plant fruits.

[0014] Among them, silencing the gene described in the first aspect of the present invention can reduce the content of sucrose, and / or fructose, and / or glucose, and / or starch in fruits.

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

[0016] The 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 bacteria or transgenic cell line or recombinant bacteria as described in the fourth aspect of the present invention in response to chlorpyrifos, ethylene, abscisic acid and / or gibberellin. Wherein, the expression level of the gene, when treated with ABA, first decreases and then increases with the extension of the treatment time; when treated with GA3 for 0-12h, the expression level has no obvious change, and the expression begins to be down-regulated in the later period (24h); when treated with ET for 0-24h, the overall expression level has no obvious change; when treated with CPPU for 0-6h, the expression level shows a significant upward trend, and decreases after 6h, but is still higher than that at 0-3h.

[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 GAATTCATGGACATGGAGGGAGAT-3' and R:5'-CCG CTCGAG TTAGTGGGAAGTAGTTGAGAT-3'; or the primer pair is: F: 5'-G GAATTC CGCAAGGCGGCTGAGT-3' and R:5'-C GAGCTC GCTCCCACCACAAATGATGG-3'.

[0018] Beneficial effects:

[0019] The present invention cloned the full-length cDNA of the AcSUT1 gene of the 'Hongyang' kiwifruit. Gene expression analysis showed that the AcSUT1 gene was expressed in different tissues, with the lowest expression in leaves. The AcSUT1 gene had a higher expression in fruits and was closely related to the development of fruits. At the same time, the expression level was regulated by exogenous hormones. Yeast mutant functional complementation experiments proved that the AcSUT1 gene had the function of transporting sucrose. Transient silencing of the AcSUT1 gene had the function of reducing the content of sucrose, fructose, glucose and starch in fruits, and could also reduce the weight of fruits, and reduce the horizontal and vertical diameters of fruits, providing 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 AcSUT1 gene (leaves, buds, bark, fruits, female flowers, and male flowers).

[0022] Figure 2 Expression analysis of AcSUT1 gene at different developmental stages of fruit (18d, 38d, 58d, 88d, 118d, and 138d after flowering).

[0023] Figure 3 The expression of AcSUT1 gene in kiwifruit treated with different hormones.

[0024] Figure 4 Functional complementation verification of yeast mutants of the AcSUT1 gene.

[0025] Figure 5 The results of AcSUT1 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 specific primers, and their final concentration was 0.5 μmol / L. 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, ddH2 18.2 μL, a total of 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. The primer amplification efficiency and the corresponding Qr value were calculated using LightCycler 4.05 software. The specific primer sequences are as follows:

[0029] F (5′ end): 5′-ATGGACATGGAGGGAGATGC-3′;

[0030] R (3' end): 5'-TTAGTGGGAAGTAGTTGAGA-3'.

[0031] 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, and the fragment had the nucleotide sequence shown in SEQ ID NO: 1, and its open reading coding frame was 1542 nucleotides (including the terminator TAA), encoding a protein with a length of 513 amino acids (SEQ ID NO: 2) and a molecular weight of about 54.6 KDa, named: AcSUT1.

[0032] Example 2. Analysis of the expression pattern of AcSUT1 gene in 'Hongyang' kiwifruit

[0033] <1> Tissue-specific expression of AcSUT1 gene in 'Hongyang' kiwifruit

[0034] 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 AcSUT1 gene (F: 5'-CGTGGTCGGGTTCTGGA-3'; R: 5'-GAAGGCGTTTGCGTTGC-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 LightCycler 4.05 software. It showed that the expression level of this gene in leaves was lower than that in the other five tissues ( Figure 1 ).

[0035] <2> Expression analysis of AcSUT1 gene in different developmental stages of ‘Hongyang’ kiwifruit fruit At different developmental stages of ‘Hongyang’ kiwifruit fruit (calculated from 18 days after flowering, 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 for AcSUT1 gene (F: 5'-CGTGGTCGGGTTCTGGA-3'; R: 5'-GAAGGCGTTTGCGTTGC-3'). The results showed that the relative expression level of AcSUT1 gene reached the highest level on the 38th day, and then gradually decreased ( Figure 2 ), indicating that the AcSUT1 gene plays an important role in fruit development.

[0036] <3> Effects of different treatments on the expression of AcSUT1 gene in kiwifruit

[0037] 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 AcSUT1 gene to abscisic acid (ABA), gibberellin (GA3), ethylene (ET), and N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) treatment was analyzed. The results are shown in Figure 3 As shown. It was found that the expression of AcSUT1 gene in the fruit of 'Hongyang' kiwifruit decreased first and then increased with the extension of treatment time when treated with ABA; there was no significant change in the expression level during GA3 treatment for 0-12h, and the expression began to be downregulated in the later period (24h); there was no significant change in the overall expression level during ET treatment for 0-24h; there was a significant upward trend during CPPU treatment for 0-6h, and it decreased after 6h, but it was still higher than that during 0-3h. This indicates that the expression of AcSUT1 gene is responsive to ABA, GA3, ET, and CPPU. Analysis of their cis-acting elements also identified response elements such as abscisic acid, gibberellin, salicylic acid, methyl jasmonate, auxin, light response, growth regulation, drought, and low temperature. The results show that the AcSUT1 gene is indeed regulated by ABA, GA3, ET, and CPPU, which provides a theoretical basis for the subsequent regulation of SUT gene expression by exogenous hormones in 'Hongyang' kiwifruit.

[0038] Example 3. Functional verification of AcSUT1 gene

[0039] The yeast expression vector of AcSUT1 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 AcSUT1 gene

[0041] Design of primers for the coding region of AcSUT1 gene

[0042] F:5'-G GAATTC (EcoRI restriction site)ATGGACATGGAGGGAGAT-3', R:5'-CCG CTCGAG(XhoI restriction site) TTAGTGGGAAGTAGTTGAGAT-3', pDR196-AcSUT1 was used as a template for PCR amplification. The reaction system was 1 μL of cDNA template, 2 μL of 10×PCR Buffer, and 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 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 PDR196 expression vector to obtain a 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 positively cloned into the expression vector. The recombinant vector was sequenced and identified, and the sequencing results were completely consistent with the sequence of SEQID NO: 1. The recombinant expression vector was named PDR196-AcSUT1.

[0043] <2> PDR196-AcSUT1 recombinant expression vector

[0044] The constructed recombinant expression vector PDR196-AcSUT1 was transferred into yeast, and the yeast liquid of the successfully transformed PDR196-AcSUT1 was streaked and cultured on SD screening medium containing 4% sucrose by mass fraction at 30°C. Its growth was observed and photographed. The experiment proved that the protein encoded by AcSUT1 has the function of transporting sucrose. Figure 4 shown.

[0045] Example 4. Functional verification of AcSUT1 gene

[0046] The AcSUT1 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 AcSUT1 gene

[0048] Design of primers for the coding region of AcSUT1 gene

[0049] F:5'-G GAATTC (EcoRI restriction site)CGCAAGGCGGCTGAGT-3', R:5'-C GAGCTC (SacI restriction site)GCTCCCACCACAAATGATGG-3',

[0050] The 1139-1438 fragment of AcSUT1 CDS was amplified and inserted into pTRV2 to generate the pTRV2-AcSUT1 vector. The amplification system was: cDNA template 1 μL, 10×PCRBuffer 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 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 (1139-1438) in the sequence list. The recombinant expression vector was named pTRV2-AcSUT1.

[0051] <2> pTRV2-AcSUT1 recombinant expression vector

[0052] Take out GV3101 Agrobacterium containing the recombinant vector pTRV2-AcSUT1 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.

[0053] Glucose, fructose, sucrose and starch in kiwifruit were determined by referring to the method in the 1977 paper "Determination of glucose, fructose, sucrose and starch in fruits and vegetables by anthrone spectrophotometry" by Zhang Youjie et al.; the horizontal and vertical diameters of the fruit were measured using a vernier caliper. The results showed that the relative expression of the AcSUT1 gene was significantly reduced after transient silencing of the AcSUT1 gene. After silencing the AcSUT1 gene, the content of sucrose, fructose, glucose and starch in kiwifruit can be reduced, and the weight, vertical and horizontal diameters of the fruit are reduced to varying degrees.

[0054] 4. Conclusion

[0055] The full-length cDNA of the AcSUT1 gene of the 'Hongyang' kiwifruit was cloned for the first time. Gene expression analysis showed that the AcSUT1 gene was expressed in different tissues, with the lowest expression in leaves. The AcSUT1 gene had a higher expression in fruits and was closely related to the development of fruits. At the same time, the expression level was regulated by exogenous hormones. Functional complementation experiments of yeast mutants proved that the AcSUT1 gene had the function of transporting sucrose. AcSUT1 can transport sucrose into cells, and its expression is positively correlated with fruit development, providing a reliable gene target for molecular improvement of kiwifruit high yield. Therefore, this gene can be used as an important gene resource and can be used in genetic engineering of stress resistance and abiotic stress resistance of other plants or microorganisms besides kiwifruit.

[0056] 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 AcSUT1 gene of 'Hongyang' kiwifruit is characterized by: The nucleotide sequence of the AcSUT1 gene is shown in SEQ ID NO:

1.

2. The protein encoded by the AcSUT1 gene according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. A recombinant vector, host bacteria, transgenic cell line or recombinant bacteria containing the AcSUT1 gene according to claim 1.

4. Use of the AcSUT1 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 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 Silencing the AcSUT1 gene can reduce fruit weight, and / or reduce the longitudinal diameter of plant fruits, and / or reduce the transverse diameter of plant fruits.

6. Use of the AcSUT1 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 sugar content of plant fruits.

7. The use according to claim 6, characterized in that Silencing the AcSUT1 gene according to claim 1 can reduce the content of sucrose, and / or fructose, and / or glucose, and / or starch in 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 AcSUT1 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'-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'-GGAATTCATGGACATGGAGGGAGAT-3' and R: 5'-CCGCTCGAGTTAGTGGGAAGTAGTTGAGAT-3'; or the primer pair is F: 5'-GGAATTCCGCAAGGCGGCTGAGT-3' and R: 5'-CGAGCTCGCTCCCACCACAAATGATGG-3'.

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