AcAAT1 gene and application thereof in regulation and control of formation of fruit ester aroma

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an AcAAT1 gene and application thereof in regulation and control of formation of fruit ester aroma. The invention provides a kiwi fruit AcAAT1 gene related to ester aroma formation and a protein coded by the kiwi fruit AcAAT1 gene, and constructs a corresponding overexpression vector and a transient silence vector which are transferred into kiwi fruits. Results show that the content of ester aroma in kiwi fruits can be increased by overexpressing the AcAAT1 gene, and the content of ester aroma in kiwi fruits can be reduced by silencing the AcAAT1 gene. The actinidia chinensis AcAAT1 gene disclosed by the invention can be used for promoting the formation of ester aroma. The method disclosed by the invention has very important application value in the direction of accurately creating new germplasm of the kiwi fruit with strong fragrance through a genetic improvement mode.

Description

Technical Field

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

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

[0003] The fatty acid metabolism 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 catalyzed by hydroperoxide lyase HPL to generate corresponding aldehydes and oxygen-containing acid compounds. They are then catalyzed by alcohol dehydrogenase ADH to synthesize specific alcohols. The alcohol and acyl-coenzyme A are catalyzed by alcohol acyltransferase AAT to finally form corresponding ester substances.

[0004] Alcohol acyltransferase AAT is the final rate-limiting enzyme involved in the synthesis of ester aroma, and the activity of alcohol acyltransferase is significantly positively correlated with the accumulation of ester substances in the fruit. Alcohol acyltransferase is a protein encoded by members of a multigene family, and 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 an AcAAT1 gene and an application thereof in regulating the formation of fruit ester aroma.

[0006] To achieve the above object, the technical solution adopted by the present invention is: The invention provides a kiwifruit AcAAT1 gene for regulating fruit ester aroma. The nucleotide sequence of the kiwifruit AcAAT1 gene is shown in SEQ ID NO.1.

[0007] The present invention finds that ester aroma substances in Donghong kiwifruit increase as the fruit matures and softens. By sequencing the transcriptomes of Donghong kiwifruit fruits treated with ethylene and 1-MCP and untreated Donghong kiwifruit fruits, the differentially expressed gene AcAAT1 related to ester formation in kiwifruit fruits was screened and cloned. It was found that transient overexpression of the AcAAT1 gene in Donghong kiwifruit fruits can increase the ester aroma content, while transient silencing of the AcAAT1 gene can reduce the ester aroma content.

[0008] The present invention also provides a recombinant overexpression vector, which comprises the above-mentioned kiwifruit AcAAT1 gene.

[0009] The present invention also provides a recombinant cell, which comprises the above-mentioned recombinant overexpression vector.

[0010] The present invention also provides a recombinant transient silencing vector, which comprises a specific fragment for silencing the kiwifruit AcAAT1 gene.

[0011] The present invention also provides application of the kiwifruit AcAAT1 gene in regulating the formation of ester aroma of kiwifruit fruit.

[0012] Furthermore, the kiwifruit ester aroma content is increased by increasing the expression of the kiwifruit AcAAT1 gene.

[0013] Furthermore, the kiwifruit ester aroma content is reduced by reducing the expression of the kiwifruit AcAAT1 gene.

[0014] The present invention also provides the use of the recombinant overexpression vector and the recombinant cell in promoting the formation of ester aroma of kiwi fruit.

[0015] The present invention also provides application of the recombinant transient silencing vector in inhibiting the formation of ester aroma of kiwi fruit.

[0016] The present invention has the following beneficial effects: The present invention provides a kiwifruit AcAAT1 gene related to the formation of ester aroma and a protein encoded by it, and constructs an overexpression vector and a transient silencing vector of the kiwifruit AcAAT1 gene. The overexpression vector and the transient silencing vector containing the kiwifruit AcAAT1 gene are transferred into kiwifruit fruit by Agrobacterium-mediated transformation. The results show that overexpression or silencing of the kiwifruit AcAAT1 gene can increase or decrease the content of ester aroma in kiwifruit fruit. It can be seen that the kiwifruit AcAAT1 gene of the present invention can promote the formation of ester aroma. The present invention uses genetic engineering technology to provide a theoretical basis and technical means for accurately creating new kiwifruit germplasm with strong aroma through genetic improvement, and has very important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the characterization test diagrams of the effects of ethylene and 1-MCP treatment on post-harvest Donghong kiwifruit fruits, where A is the hardness test diagram and B is the ester aroma content test diagram.

[0018] Figure 2 This is the result of PCR amplification of the full-length CDS of the kiwifruit AcAAT1 gene.

[0019] Figure 3This is the expression analysis diagram of kiwifruit AcAAT1 gene during the postharvest ripening and softening process of Donghong kiwifruit fruit.

[0020] Figure 4 These are the results of kiwifruit fruits with transient overexpression of the AcAAT1 gene in kiwifruit, where A is the result of identification of AcAAT1 expression level in kiwifruit fruits with transient overexpression of the AcAAT1 gene in kiwifruit, and B is the analysis of ester aroma content in kiwifruit fruits with transient overexpression of the AcAAT1 gene in kiwifruit.

[0021] Figure 5 The results of transient silencing of the AcAAT1 gene in kiwifruit are shown in Figure 1. A is the result of identifying the expression of AcAAT1 in kiwifruit after transient silencing of the AcAAT1 gene, and B is the analysis of the aroma content of esters in kiwifruit after transient silencing of the AcAAT1 gene. DETAILED DESCRIPTION

[0022] The present invention is described in detail with respect to specific examples below, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0023] Example 1: AcAAT1 protein and its encoding gene sequence of kiwifruit alcohol acyltransferase.

[0024] Donghong kiwifruit fruits with uniform size, no mechanical damage and no pests and diseases were selected and divided into three groups, with about 200 fruits in each group. The ethylene group was treated with 100 μL / L ethylene and fumigated at 20℃ for 24 hours, the 1-MCP group was treated with 0.5 μL / L ethylene signal inhibitor 1-MCP and fumigated at 20℃ for 24 hours, and the control group was not treated. Figure 1 As shown in the data, compared with the control fruit, ethylene treatment can accelerate the ripening and softening process of Donghong kiwifruit fruit and promote the accumulation of ester aroma content in the fruit; while 1-MCP treatment effectively delays the softening of Donghong kiwifruit fruit and hinders the formation of ester aroma in the fruit.

[0025] 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. The obtained cDNA was used as a template to design the full-length amplification primers of AcAAT1, and PCR amplification was performed using a high-fidelity PCR enzyme. The full-length sequence amplification primers of AcAAT1 are shown in Table 1: Table 1: Primers for amplifying the full-length sequence of AcAAT1.

[0026]

[0027] The PCR amplification system was: 2×PCR buffer 25 μL, 2 mM dNTPs 10 μL, AcAAT1-F 1.5 μL, AcAAT1-R 1.5 μL, cDNA 2 μL, KOD FX Neo 1 μL, and ddH2O 9 μL.

[0028] The PCR amplification program was as follows: 94°C, 2 min; 98°C, 10 s; 55°C, 30 s; 68°C, 30 s; return to the second step for 35 cycles; 68°C, 7 min; and store at 4°C.

[0029] The products obtained by PCR amplification were detected by agarose gel electrophoresis. Figure 2 As shown, the specific band obtained is the AcAAT1 gene in kiwifruit. After the PCR product is purified and recovered, it is connected with the TA cloning vector and transferred into the competent E. coli DH5α. The positive clones are screened for sequencing. The sequencing results show that the full length of the kiwifruit AcAAT1 gene is 960bp. The kiwifruit AcAAT1 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0030] SEQ ID NO.1:ATGGTGGAATTGCACGAGAGAAGGGTTTTGTTCATCGAGGCAGATGCAGATCTGAGGTCAACCAGCTTAATGGTGACCACAATTGCATCGGGGTTTCATGACTTAGACGAGCTTTTGCATGACGTGCCTGGTCTGAAGGTGACCCGTTTTAGATGCGGTGGATGGGCGTTTGCCATACGCCTAAACCACACGATGAGTGATGGACCAGGGTTGGTCCAATTTATAACTACAATTGCTGAGTTTGCCCGAGGGGTAGAGGATGCACCTTCTGTGCCACCGGTGTGGCAAAGGGAGTTCCTAGCTGCGAGACAACCTCCCTCCATCACATTCCAGCATCACGAATATGAGCAACTCCACATTCGACGTCTTGCCGCGTGTTTATGGAGATGCCGCACTTGTGCACTTGGGCTAGACCCCGAGAAGATGGTTCGCATCTTGTGCGCAGTCAACGGACGTGGAAAGGCACGACTGCATTTACCGGGGAGGGGGGTCATCTATCTCCTCTGGAATGTCTTTTCTTTTGCCCTTCCGGCTGCAGTTTCTAGAGCTGGAATAATTTCTACTTCCCCATTGCAATACGGGGTGGAGGTGGTGAAGAAGGCGAAGGCAAGAATGACCGGAGAGTATTTGAGGTCAGTGGCAGATCTTATGGTGACCAAGGGACGATCTCCCTTTACGGTGGCGGGGAACTATATTGTATCTGATACGACCCGCATTGGCTTTGACGCGATAGATTTTGGGTGGGGCAAACCGGTATATGGCGGCCTCGCTAGGGCATTACCTTTGATTAGCTTCTATACAAGGTTTAAAGATAATAGAGGGGAAGATGGAACTGTGGTACTAATGTGCTTACCCGAAGAAGCTATGGAAAGGTTTCAAGAGGAGTTGAAGAAAATGACTGAAGGGCATGTGGACGGTCCACTTGATTACAAGCCCACAAAAATTATGTCCATGCTTTAA。

[0031] SEQ ID NO.2: MVELHERRVLFIEADADLRSTSLMVTTIASGFHDLDELLHDVPGLKVTRFRCGGWAFAIRLNHTMSDGPGLVQFITTIAEFARGVEDAPSVPPVWQREFLAARQPPSITFQHHEYEQLHIRRLAACLWRCRTCALGLDPEKMVRILCAVNGRGKARL HLPGRGVIYLLWNVFSFALPAAVSRAGIISTSPLQYGVEVVKKAKARMTGEYLRSVADLMVTKGRSPFTVAGNYIVSDTTRIGFDAIDFGWGKPVYGGLARALPLISFYTRFKDNRGEDGTVVLMCPEEAMERFQEELKKMTEGHVDGPLDYKPTKIMSML.

[0032] Example 2: Analysis of AcAAT1 gene expression.

[0033] RNA extraction and reverse transcription of different treated Donghong kiwifruit pulps were performed as described in Example 1. A fluorescence quantitative PCR instrument was used to detect the relative expression of genes, and 3 replicates were set for each sample. The reaction system was 20 μL, including: 10 μL of SYBRSolution, 2 μL of pulp cDNA, 1 μL of upstream and downstream primers, and 6 μL of ddH2O.

[0034] Reaction conditions: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 58℃ annealing for 10s, 72℃ extension for 30s, 40 cycles in total; 65℃ incubation for 20s, melting temperature from 55℃, each 0.5℃ increase for 1s, stop the reaction at 95℃. Actin was used as the internal reference gene, and the 2-ΔΔCT method was used for relative expression analysis. The qPCR primer sequences are shown in Table 2: Table 2: qPCR primer sequences.

[0035]

[0036] Gene expression results Figure 3 As shown in the data, the relative expression level of kiwifruit AcAAT1 gene gradually increased with the ripening and softening process of Donghong kiwifruit fruit after harvest. Ethylene treatment can significantly induce the expression of AcAAT1, while 1-MCP treatment can obviously inhibit its expression.

[0037] Example 3: Construction of recombinant vector.

[0038] 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 PBI121 empty vector and AcAAT1-PBI121 were respectively transferred into the Agrobacterium competent GV3101 by freeze-thaw method. The overexpression primer sequences are shown in Table 3: Table 3: Overexpression primer sequences.

[0039]

[0040] VIGS vector construction: Design AcAAT1-pTRV2 primers containing restriction sites, obtain a 444bp specific fragment from the SEQ ID NO.1 sequence obtained in Example 1, whose sequence is shown in SEQ ID NO.3, insert it into the pTRV2 vector to construct the AcAAT1-pTRV2 recombinant plasmid, and transfer pTRV1, pTRV2 and AcAAT1-pTRV2 into Agrobacterium competent GV3101 by freeze-thaw method. The silencing vector primer sequences are shown in Table 4: Table 4: Silencing vector primer sequences.

[0041]

[0042] SEQ ID NO.3:TTTGCCATACGCCTAAACCACACGATGAGTGATGGACCAGGGTTGGTCCAATTTATAACTACAATTGCTGAGTTTGCCCGAGGGGTAGAGGATGCACCTTCTGTGCCACCGGTGTGGCAAAGGGAGTTCCTAGCTGCGAGACAACCTCCCTCCATCACATTCCAGCATCACGAATATGAGCAACTCCACATTCGACGTCTTGCCGCGTGTTTATGGAGAT GCCGCACTTGTGCACTTGGGCTAGACCCCGAGAAGATGGTTCGCATCTTGTGCGCAGTCAACGGACGTGGAAAGGCACGACTGCATTTACCGGGGAGGGGGGTCATCTATCTCCTCTGGAATGTCTTTTCTTTTGCCCTTCCGGCTGCAGTTTCTAGAGCTGGAATAATTTCTACTTCCCCATTGCAATACGGGGTGGAGGTGGTGAAGAAGGCGAAGGCAAGA.

[0043] Example 4: Functional study of kiwifruit AcAAT1 gene.

[0044] (1) Transient overexpression of kiwifruit AcAAT1 gene in kiwifruit fruits.

[0045] First, pick up the transformed Agrobacterium and transfer it to 1 mL of LB liquid medium with cannabinoid resistance, and shake it overnight at 28°C. The next day, take 0.2 mL of Agrobacterium and transfer it to 6 mL of LB liquid medium, and shake it at 28°C for 9 hours to make the OD 600 The value reached 0.8. At room temperature, the bacteria were collected by centrifugation at 4000rpm for 10 minutes. After the bacteria were resuspended, the OD value was adjusted to 0.8. The resuspended Agrobacterium was left to stand for about 3 hours. The bacterial solution containing the PBI121 empty vector and the AcAAT1-PBI121 plasmid was injected into the middle column of the Donghong kiwifruit fruit on the day of harvest, and 0.2mL of bacterial solution was injected into each fruit. The expression level of the kiwifruit AcAAT1 gene and the ester aroma content of the injection site were detected 3 days after injection.

[0046] like Figure 4 As shown in A, the relative expression of AcAAT1 gene in the fruit injected with AcAAT1-PBI121 bacterial solution was significantly higher than that in the control fruit, indicating that the transient overexpression of AcAAT1 gene was successful. The ester aroma content in the fruit was tested, and the results were as follows Figure 4 As shown in B, the ester aroma content in the fruit with overexpression of AcAAT1 gene was significantly higher than that in the control fruit.

[0047] (2) Transient silencing of AcAAT1 gene expression.

[0048] First, pick the transformed Agrobacterium to 1mL LB liquid medium and shake it at 28℃ overnight. The next day, take 0.2mL of Agrobacterium to 6mL of LB liquid medium and shake it at 28℃ for 9h to make the OD value reach 0.8. At room temperature, centrifuge at 4000rpm for 10min to collect the bacteria. After resuspending the bacteria, adjust the OD value to 0.8. Let the resuspended Agrobacterium stand for about 3h.

[0049] The bacterial solution containing the plasmid pTRV1:pTRV2 at a mass ratio of 1:1 and the plasmid pTRV1:AcAAT1-pTRV2 at a mass ratio of 1:1 were injected into Donghong kiwifruit fruits on the day of harvest, with 0.2 mL of bacterial solution injected into each fruit. The expression level of the kiwifruit AcAAT1 gene and the ester aroma content at the injection site were detected 3 days after injection.

[0050] like Figure 5As shown in A, the relative expression of the AcAAT1 gene in the fruit injected with the AcAAT1-pTRV2 bacterial solution was significantly lower than that in the control fruit, indicating that the transient silencing expression of the AcAAT1 gene in kiwifruit was successful. The ester aroma content in the fruit was tested, and the results were as follows Figure 5 As shown in B, the ester aroma content in the fruits of kiwifruit in which the AcAAT1 gene was transiently silenced was significantly lower than that in the control fruits.

[0051] This proves that the kiwifruit AcAAT1 gene can promote the accumulation of ester aroma in the fruit, indicating that the kiwifruit AcAAT1 gene plays an important role in regulating the formation of ester aroma in kiwifruit. The present invention can provide a theoretical basis and technical means for accurately creating a new kiwifruit germplasm with a strong aroma through genetic improvement, and has very important application value.

[0052] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

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

1.

2. A recombinant overexpression vector, characterized in that: It comprises the kiwifruit AcAAT1 gene according to claim 1.

3. A recombinant cell, characterized in that It comprises the recombinant overexpression vector according to claim 2.

4. A recombinant transient silencing vector, characterized in that: The recombinant transient silencing vector comprises a specific fragment for silencing the kiwifruit AcAAT1 gene according to claim 1.

5. Use of the kiwifruit AcAAT1 gene according to claim 1 in regulating the formation of ester aroma in kiwifruit.

6. The use according to claim 5, characterized in that: The kiwifruit ester aroma content is increased by increasing the expression of the kiwifruit AcAAT1 gene.

7. The use according to claim 5, characterized in that: The kiwifruit ester aroma content is reduced by reducing the expression of the kiwifruit AcAAT1 gene.

8. Use of the recombinant overexpression vector according to claim 2 and the recombinant cell according to claim 3 in promoting the formation of ester aroma in kiwi fruit.

9. Use of the recombinant transient silencing vector according to claim 4 in inhibiting the formation of ester aroma in kiwi fruit.

Images