AcAAT1 Gene and Its Application in Regulating the Formation of Fruit Ester Aroma

Abstract

The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to the AcAAT1 gene and its application in regulating the formation of fruit ester aroma. The present invention provides a kiwifruit AcAAT1 gene related to the formation of ester aroma and the protein encoded thereby, and constructs corresponding overexpression vectors and transient silencing vectors and transfers them into kiwifruit. The results show that overexpressing the AcAAT1 gene can increase the content of ester aroma in kiwifruit, and silencing the AcAAT1 gene can reduce the content of ester aroma in kiwifruit. The kiwifruit AcAAT1 gene described in the present invention can promote the formation of ester aroma. The present invention has very important application value in the direction of precisely creating new germplasms of kiwifruit with strong aroma through genetic improvement methods.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to the AcAAT1 gene and its application in regulating the formation of fruit ester aroma. Background Art

[0002] Aroma can objectively reflect the flavor characteristics of fruits and is an important indicator for measuring the flavor quality of fruits. The aroma components of kiwifruit are very rich. So far, more than 300 aroma components have been detected, mainly including alcohols, aldehydes, esters, and terpenoids. Among them, esters are not only an important part of the aroma quality of kiwifruit but also the characteristic aroma in ripe fruits.

[0003] The fatty acid metabolic pathway is the most important pathway for the formation of kiwifruit aroma. Linoleic acid and linolenic acid are decomposed into hydroperoxides by lipoxygenase LOX, and then through the catalytic action of hydroperoxide lyase HPL, the corresponding aldehydes and oxygen-containing acid compounds are generated. After that, through the catalysis of alcohol dehydrogenase ADH, specific alcohols are synthesized. The alcohol and acyl-CoA are catalyzed by alcohol acyltransferase AAT, and finally the corresponding ester substances are formed.

[0004] Alcohol acyltransferase AAT is the final rate-limiting enzyme involved in the ester aroma synthesis pathway. The activity of alcohol acyltransferase is significantly positively correlated with the accumulation of fruit ester substances. Alcohol acyltransferase is a protein encoded by members of a multi-gene family. The AcAAT1 gene belongs to the alcohol acyltransferase AAT gene family. Elucidating the role of the AcAAT1 gene in the formation of fruit ester aroma can better improve the flavor of kiwifruit. Summary of the Invention

[0005] The purpose of the present invention is to provide the AcAAT1 gene and its application in regulating the formation of fruit ester aroma.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a kiwifruit AcAAT1 gene for regulating fruit ester aroma, and the nucleotide sequence of the kiwifruit AcAAT1 gene is as shown in SEQ ID NO.1.

[0008] The present invention found that the ester aroma substances in Donghong kiwifruit increase with the process of fruit ripening and softening. By using ethylene and 1-MCP to treat the Donghong kiwifruit fruits and performing transcriptome sequencing on the treated and untreated Donghong kiwifruit fruits, the differential gene AcAAT1 related to fruit ester formation was screened and cloned; it was found that transient overexpression of the AcAAT1 gene in Donghong kiwifruit fruits can increase the content of ester aroma, while transient silencing of the AcAAT1 gene can reduce the content of ester aroma.

[0009] The present invention also provides a recombinant overexpression vector, which contains the kiwifruit AcAAT1 gene described above.

[0010] The present invention also provides a recombinant cell, which contains the recombinant overexpression vector described above.

[0011] The present invention also provides a recombinant transient silencing vector, which contains a specific fragment for silencing the kiwifruit AcAAT1 gene.

[0012] The present invention also provides the application of the kiwifruit AcAAT1 gene in regulating the formation of ester aroma in kiwifruit fruits.

[0013] Furthermore, by increasing the expression of the kiwifruit AcAAT1 gene, the content of ester aroma in kiwifruit is increased.

[0014] Furthermore, by decreasing the expression of the kiwifruit AcAAT1 gene, the content of ester aroma in kiwifruit is decreased.

[0015] The present invention also provides the application of the recombinant overexpression vector and the recombinant cell in promoting the formation of ester aroma in kiwifruit fruits.

[0016] The present invention also provides the application of the recombinant transient silencing vector in inhibiting the formation of ester aroma in kiwifruit fruits.

[0017] The present invention has the following beneficial effects:

[0018] The present invention provides a kiwifruit AcAAT1 gene related to the formation of ester aroma and the protein encoded thereby, and constructs an overexpression vector and a transient silencing vector of the kiwifruit AcAAT1 gene. By using the Agrobacterium-mediated transformation method, the overexpression vector and the transient silencing vector containing the kiwifruit AcAAT1 gene are transferred into kiwifruit fruits. The results show that overexpressing or silencing the kiwifruit AcAAT1 gene can increase or decrease the content of ester aroma in kiwifruit fruits, respectively. It can be seen that the kiwifruit AcAAT1 gene described in the present invention can promote the formation of ester aroma. By using genetic engineering technology, the present invention can provide a theoretical basis and technical means for precisely creating new germplasms of kiwifruit with rich aroma through genetic improvement, and has very important application value. Description of the Drawings

[0019] Figure 1 It is a diagram for the characterization detection of the postharvest Hongdong kiwifruit fruits treated with ethylene and 1-MCP. Among them, A is the diagram for hardness detection, and B is the diagram for the content detection of ester aroma.

[0020] Figure 2 It is a diagram for the PCR amplification result of the full-length CDS of the kiwifruit AcAAT1 gene.

[0021] Figure 3 Expression analysis diagram of kiwifruit AcAAT1 gene during postharvest ripening and softening of 'Donghong' kiwifruit

[0022] Figure 4 Results diagram of transient overexpression of kiwifruit AcAAT1 gene in kiwifruit. Among them, A is the identification result diagram of AcAAT1 expression level in kiwifruit with transient overexpression of kiwifruit AcAAT1 gene, and B is the analysis diagram of ester aroma content in kiwifruit with transient overexpression of kiwifruit AcAAT1 gene

[0023] Figure 5 Results diagram of transient silencing of kiwifruit AcAAT1 gene in kiwifruit. Among them, A is the identification result of AcAAT1 expression level in kiwifruit with transient silencing of kiwifruit AcAAT1 gene, and B is the analysis diagram of ester aroma content in kiwifruit with transient silencing of kiwifruit AcAAT1 gene Specific implementation mode

[0024] The present invention will be described in detail below with specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified

[0025] Example 1: Obtaining of kiwifruit alcohol acyltransferase AcAAT1 protein and its coding gene sequence

[0026] 'Donghong' kiwifruits with consistent size, no mechanical damage and pests and diseases were selected and divided into 3 groups, with about 200 fruits in each group. The ethylene group was treated with 100 μL / L ethylene and fumigated in a sealed container at 20 °C for 24 h. The 1-MCP group was treated with 0.5 μL / L ethylene signal inhibitor 1-MCP and fumigated in a sealed container at 20 °C for 24 h. The control group was not treated. The results are as Figure 1 shown. Compared with the control fruits, ethylene treatment can accelerate the postharvest ripening and softening process of 'Donghong' kiwifruit and promote the accumulation of ester aroma content in the fruits; while 1-MCP treatment effectively delays the softening of 'Donghong' kiwifruit and hinders the formation of ester aroma in the fruits

[0027] The total RNA of kiwifruit pulp was extracted using a polysaccharide and polyphenol plant RNA extraction kit, and the total RNA was reverse transcribed into cDNA using a reverse transcription kit. Using the obtained cDNA as a template, primers for amplifying the full length of AcAAT1 were designed, and PCR amplification was carried out using a high-fidelity PCR enzyme. The primers for amplifying the full length sequence of AcAAT1 are shown in Table 1

[0028] Table 1: Primers for amplifying the full length sequence of AcAAT1

[0029]

[0030] The PCR amplification system is as follows: 2×PCR buffer is 25 μL, 2 mM dNTPs is 10 μL, AcAAT1-F is 1.5 μL, AcAAT1-R is 1.5 μL, cDNA is 2 μL, KOD FX Neo is 1 μL, and ddH2O is 9 μL.

[0031] The PCR amplification program is as follows: 94 °C for 2 min; 98 °C for 10 s; 55 °C for 30 s; 68 °C for 30 s; return to the second step for 35 cycles; 68 °C for 7 min; store at 4 °C.

[0032] The product obtained by PCR amplification was detected by agarose gel electrophoresis, and the results are as Figure 2 shown. The specific band obtained is the AcAAT1 gene in kiwifruit. After purifying and recovering the PCR product, it was ligated to the TA cloning vector and transformed into Escherichia coli competent DH5α. Positive clones were screened for sequencing. The sequencing results showed that the full length of the kiwifruit AcAAT1 gene is 960 bp. The kiwifruit AcAAT1 gene is as shown in SEQ ID NO.1, and the encoded amino acid sequence is as shown in SEQ ID NO.2.

[0033] SEQ ID NO.1: ATGGTGGAATTGCACGAGAGAAGGGTTTTGTTCATCGAGGCAGATGCAGATCTGAGGTCAACCAGCTTAATGGTGACCACAATTGCATCGGGGTTTCATGACTTAGACGAGCTTTTGCATGACGTGCCTGGTCTGAAGGTGACCCGTTTTAGATGCGGTGGATGGGCGTTTGCCATACGCCTAAACCACACGATGAGTGATGGACCAGGGTTGGTCCAATTTATAACTACAATTGCTGAGTTTGCCCGAGGGGTAGAGGATGCACCTTCTGTGCCACCGGTGTGGCAAAGGGAGTTCCTAGCTGCGAGACAACCTCCCTCCATCACATTCCAGCATCACGAATATGAGCAACTCCACATTCGACGTCTTGCCGCGTGTTTATGGAGATGCCGCACTTGTGCACTTGGGCTAGACCCCGAGAAGATGGTTCGCATCTTGTGCGCAGTCAACGGACGTGGAAAGGCACGACTGCATTTACCGGGGAGGGGGGTCATCTATCTCCTCTGGAATGTCTTTTCTTTTGCCCTTCCGGCTGCAGTTTCTAGAGCTGGAATAATTTCTACTTCCCCATTGCAATACGGGGTGGAGGTGGTGAAGAAGGCGAAGGCAAGAATGACCGGAGAGTATTTGAGGTCAGTGGCAGATCTTATGGTGACCAAGGGACGATCTCCCTTTACGGTGGCGGGGAACTATATTGTATCTGATACGACCCGCATTGGCTTTGACGCGATAGATTTTGGGTGGGGCAAACCGGTATATGGCGGCCTCGCTAGGGCATTACCTTTGATTAGCTTCTATACAAGGTTTAAAGATAATAGAGGGGAAGATGGAACTGTGGTACTAATGTGCTTACCCGAAGAAGCTATGGAAAGGTTTCAAGAGGAGTTGAAGAAAATGACTGAAGGGCATGTGGACGGTCCACTTGATTACAAGCCCACAAAAATTATGTCCATGCTTTAA。

[0034] SEQ ID NO.2: MVELHERRVLFIEADADLRSTSLMVTTIASGFHDLDELLHDVPGLKVTRFRCGGWAFAIRLNHTMSDGPGLVQFITTIAEFARGVEDAPSVPPVWQREFLAARQPPSITFQHHEYEQLHIRRLAACLWRCRTCALGLDPEKMVRILCAVNGRGKARLHLPGRGVIYLLWNVFSFALPAAVSRAGIISTSPLQYGVEVVKKAKARMTGEYLRSVADLMVTKGRSPFTVAGNYIVSDTTRIGFDAIDFGWGKPVYGGLARALPLISFYTRFKDNRGEDGTVVLMCLPEEAMERFQEELKKMTEGHVDGPLDYKPTKIMSML。

[0035] Example 2: AcAAT1 gene expression analysis.

[0036] For RNA extraction and reverse transcription of Actinidia chinensis var. rufopulpa fruits under different treatments, refer to Example 1. The relative gene expression levels were detected using a fluorescence quantitative PCR instrument, and three replicates were set for each sample. The reaction system was 20 μL, including: 10 μL of SYBR Solution, 2 μL of pulp cDNA, 1 μL each of the upstream and downstream primers, and 6 μL of ddH2O.

[0037] Reaction conditions: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 58°C for 10 s, extension at 72°C for 30 s, for a total of 40 cycles; incubation at 65°C for 20 s, melting temperature starting from 55°C, holding for 1 s for every 0.5°C increase until 95°C to stop the reaction. Actin was used as the internal reference gene, and finally, the 2-ΔΔCT method was used for relative expression analysis. The qPCR primer sequences are shown in Table 2:

[0038] Table 2: qPCR primer sequences.

[0039]

[0040] The gene expression results are as Figure 3 shown. The relative expression level of the AcAAT1 gene in kiwifruit gradually increased with the post-harvest ripening and softening process of Actinidia chinensis var. rufopulpa fruits. Ethylene treatment could significantly induce the expression of AcAAT1, while 1-MCP treatment could significantly inhibit its expression.

[0041] Example 3: Construction of recombinant vectors.

[0042] Overexpression vector construction: The SEQ ID NO.1 sequence obtained in Example 1 was cloned into the overexpression vector PBI121 using a one-step rapid cloning kit to construct the AcAAT1-PBI121 overexpression recombinant plasmid, and the empty PBI121 and AcAAT1-PBI121 were respectively transferred into the Agrobacterium tumefaciens competent cell GV3101 by the freeze-thaw method. The overexpression primer sequences are shown in Table 3:

[0043] Table 3: Overexpression primer sequences.

[0044]

[0045] VIGS vector construction: AcAAT1-pTRV2 primers containing restriction enzyme sites were designed, and a 444-bp specific fragment was obtained from the SEQ ID NO.1 sequence obtained in Example 1. Its sequence is shown in SEQ ID NO.3 and inserted into the pTRV2 vector to construct the AcAAT1-pTRV2 recombinant plasmid. The pTRV1, pTRV2, and AcAAT1-pTRV2 were respectively transferred into the Agrobacterium tumefaciens competent cell GV3101 by the freeze-thaw method. The silencing vector primer sequences are shown in Table 4:

[0046] Table 4: Silencing vector primer sequences.

[0047]

[0048] SEQ ID NO.3: TTTGCCATACGCCTAAACCACACGATGAGTGATGGACCAGGGTTGGTCCAATTTATAACTACAATTGCTGAGTTTGCCCGAGGGGTAGAGGATGCACCTTCTGTGCCACCGGTGTGGCAAAGGGAGTTCCTAGCTGCGAGACAACCTCCCTCCATCACATTCCAGCATCACGAATATGAGCAACTCCACATTCGACGTCTTGCCGCGTGTTTATGGAGATGCCGCACTTGTGCACTTGGGCTAGACCCCGAGAAGATGGTTCGCATCTTGTGCGCAGTCAACGGACGTGGAAAGGCACGACTGCATTTACCGGGGAGGGGGGTCATCTATCTCCTCTGGAATGTCTTTTCTTTTGCCCTTCCGGCTGCAGTTTCTAGAGCTGGAATAATTTCTACTTCCCCATTGCAATACGGGGTGGAGGTGGTGAAGAAGGCGAAGGCAAGA.

[0049] Example 4: Functional study of Actinidia chinensis AcAAT1 gene.

[0050] (1) Transient overexpression of Actinidia chinensis AcAAT1 gene in Actinidia chinensis fruits.

[0051] First, pick the transformed Agrobacterium tumefaciens into 1 mL of LB liquid medium with kanamycin resistance and shake it overnight at 28 °C. The next day, pipette 0.2 mL of Agrobacterium tumefaciens into 6 mL of LB liquid medium and shake it at 28 °C for 9 h until the OD 600 value reaches 0.8. At room temperature, centrifuge at 4000 rpm for 10 min to collect the bacterial cells. After resuspending the bacterial cells, adjust the OD value to 0.8, and let the resuspended Agrobacterium tumefaciens stand for about 3 h. Inject the bacterial suspensions containing the empty PBI121 vector and the AcAAT1-PBI121 plasmid into the central column of 'Donghong' Actinidia chinensis fruits on the day of harvest, with 0.2 mL of bacterial suspension injected into each fruit. Detect the expression level of Actinidia chinensis AcAAT1 gene 3 days after injection and the content of ester aroma in the injection site.

[0052] As Figure 4 shown in A of, the relative expression level of AcAAT1 gene in the fruits injected with the bacterial suspension containing AcAAT1-PBI121 was significantly higher than that in the control fruits, indicating that the transient overexpression of AcAAT1 gene was successful. Detect the content of ester aroma in the fruits. The results are shown in B of Figure 4 . The content of ester aroma in the fruits with overexpressed AcAAT1 gene was significantly higher than that in the control fruits.

[0053] (2) Transient silencing expression of AcAAT1 gene.

[0054] First, pick the transformed Agrobacterium tumefaciens into 1 mL of LB liquid medium and shake it overnight at 28 °C. The next day, pipette 0.2 mL of Agrobacterium tumefaciens into 6 mL of LB liquid medium and shake it at 28 °C for 9 h until the OD value reaches 0.8. At room temperature, centrifuge at 4000 rpm for 10 min to collect the bacterial cells. After resuspending the bacterial cells, adjust the OD value to 0.8, and let the resuspended Agrobacterium tumefaciens stand for about 3 h.

[0055] Inject the bacterial suspensions containing the plasmids with a mass ratio of 1:1 of pTRV1:pTRV2 and the plasmids with a mass ratio of 1:1 of pTRV1:AcAAT1-pTRV2 into 'Donghong' Actinidia chinensis fruits on the day of harvest, with 0.2 mL of bacterial suspension injected into each fruit. Detect the expression level of Actinidia chinensis AcAAT1 gene 3 days after injection and the content of ester aroma in the injection site.

[0056] As Figure 5As shown in A of , the relative expression level of the AcAAT1 gene in fruits injected with the AcAAT1-pTRV2 bacterial solution was significantly lower than that in the control fruits, indicating that the transient silencing expression of the kiwifruit AcAAT1 gene was successful. The ester aroma content in the fruits was detected, and the results were as Figure 5 shown in B of . The ester aroma content in the fruits with transient silencing expression of the kiwifruit AcAAT1 gene was significantly lower than that in the control fruits.

[0057] Therefore, it can be proved that the kiwifruit AcAAT1 gene can promote the accumulation of ester aroma in fruits, indicating that the kiwifruit AcAAT1 gene plays an important role in regulating the formation of ester aroma in kiwifruit fruits. The present invention can provide a theoretical basis and technical means for precisely creating new germplasms of kiwifruit with strong aroma through genetic improvement, and has very important application value.

[0058] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

Claims

1. Application of the kiwifruit AcAAT1 gene in regulating the formation of ester aroma in kiwifruit, characterized in that: The nucleotide sequence of the kiwifruit AcAAT1 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The kiwifruit ester aroma content is increased by increasing the expression of the kiwifruit AcAAT1 gene.

3. The use according to claim 1, characterized in that: The kiwifruit ester aroma content is reduced by reducing the expression of the kiwifruit AcAAT1 gene.

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