A method for creating kiwifruit germplasm resources with low citric acid

Abstract

The present invention provides a method for creating kiwifruit germplasm resources of the low-citric-acid type, which targets the key gene AcNAC1 that regulates citric acid synthesis, and combines CRISPR-Cas9 gene editing and kiwifruit transgenic technology to create loss-of-function kiwifruit germplasm resources with mutations in the AcNAC1 gene sequence. The low-citric-acid type kiwifruit germplasm materials have no special requirements for cultivation management measures such as fertilizer and water. The citric acid content in the fruits produced remains at a low level throughout the entire development process from fruit setting to maturity, which is 15-20% of the fruits of Actinidia chinensis 'Donghong'. The method for creating germplasm resources provided by the present invention is reasonably designed and simple to operate, and can effectively break through the bottleneck problem of the randomness of hybridization phenotypes encountered in conventional cross-breeding, providing an effective strategy for the targeted and precise creation of innovative kiwifruit varieties.

Description

Technical Field

[0001] The present invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a method for creating germplasm resources of low-citric acid kiwifruit. The fruits produced by this kiwifruit germplasm resource have extremely low citric acid content, and are suitable for the mechanism research of organic acid metabolism in kiwifruit fruits and the breeding research for improving fruit flavor. Background Art

[0002] Kiwifruit (Actinidia spp.) is an economic fruit worldwide and is known as the "king of fruits" because of its rich bioactive substances and unique flavor. Organic acids are a common type of primary plant metabolites and metabolic intermediates, and are one of the important indicators affecting fruit flavor. They not only determine the sensory quality of fruits, but also affect secondary metabolic pathways and post-harvest shelf life. The types of organic acid components in fruits of different species are diverse, and there are also significant differences in content. According to the main organic acids accumulated in mature fruits, they can be generally divided into malic acid-dominant type, citric acid-dominant type, and tartaric acid-dominant type. The main organic acid components in mature kiwifruit fruits are citric acid, quinic acid, and malic acid, among which citric acid accounts for 40-60% of the total acid, quinic acid is 40-60%, and malic acid is 10%. This ratio also varies due to different varieties, which is an important reason for the different flavors of kiwifruit fruits of different varieties.

[0003] Changing the types or contents of organic acid components is one of the core tasks in the breeding research for improving fruit flavor. However, fruit organic acids have obvious quantitative trait genetic characteristics, including that the phenotypes of hybrid offspring are significantly affected by the environment, the variation is continuous, it is controlled by multiple genes, and the genetic mode is complex, etc. This causes great difficulties in using cross-breeding means to change specific organic acid components. Summary of the Invention

[0004] The present invention provides a technical approach to solve the bottleneck problems encountered in cross-breeding, and provides a method for creating germplasm resources of low-citric acid kiwifruit. The obtained germplasm resource materials are not only suitable for the mechanism research of organic acid metabolism in kiwifruit fruits, but also suitable for the breeding research for improving fruit flavor.

[0005] A method for creating germplasm resources of low-citric acid kiwifruit, using the key gene AcNAC1 (nucleotide sequence is SEQ ID NO.2, and the encoded amino acid sequence is SEQ ID NO.3) that regulates citric acid synthesis as a target, and at least including the following steps:

[0006] First, two target gRNA sequences (SEQ ID NO.4 and SEQ ID NO.5) were designed and synthesized based on the AcNAC1 genomic sequence (SEQ ID NO.1), and then a recombinant CRISPR-Cas9 expression vector was constructed;

[0007] Second, the recombinant CRISPR-Cas9 expression vector was transformed into the leaf discs of 'Donghong' kiwifruit by Agrobacterium-mediated transgenic technology, and transgenic kiwifruit was obtained by combining plant tissue culture technology;

[0008] Third, gene sequence sequencing technology was used to analyze the changes in the AcNAC1 genomic sequence in the obtained transgenic kiwifruit, and transgenic kiwifruit germplasm materials with changes in the amino acid sequence encoded due to mutations in the AcNAC1 genomic sequence, resulting in the loss of AcNAC1 gene function, were screened out. The AcNAC1 sequence mutations leading to gene function loss included frameshift mutations and premature termination of the coding sequence;

[0009] Fourth, it was detected that the citric acid content in the fruits with AcNAC1 gene function loss changed little during the entire development process from fruit setting to maturity and remained at a low level, only 15-20% of that of 'Donghong'. It was determined that the obtained AcNAC1 gene function loss type kiwifruit germplasm material was a low-citric-acid type kiwifruit germplasm resource.

[0010] The plants of the low-citric-acid type kiwifruit germplasm resource created by the present invention have no significant particularities in growth and have no special requirements for cultivation management measures such as fertilizer and water. The creation of the low-citric-acid type kiwifruit germplasm resource provides an efficient reference method for cultivating specific organic acid type kiwifruit germplasm resources and excellent kiwifruit varieties. The application of other functional genes with a sequence similarity higher than 90% to the AcNAC1 gene sequence in this regard is also within the protection scope of this invention patent.

[0011] Based on the identification of the key gene AcNAC1 controlling citric acid synthesis in kiwifruit, combined with CRISPR-Cas9 gene editing and kiwifruit tissue culture technology, the present invention precisely created kiwifruit germplasm materials with extremely low fruit citric acid content. This creation method is an effective strategy to solve the bottleneck problems encountered in cross-breeding, that is: identifying the major genes controlling quantitative traits and combining CRISPR-Cas9 technology to create innovative fruit tree varieties. The germplasm resource creation method provided by the present invention is reasonably designed and easy to operate, and can effectively break through the bottleneck problem of the randomness of hybrid phenotypes encountered in conventional cross-breeding, providing an effective strategy for precisely creating innovative kiwifruit varieties. Brief Description of the Drawings

[0012] Appendix Figure 1They are target 1 and target 2 for gene editing of AcNAC1.

[0013] Appendix Figure 2 It is the sequencing result of the acnac1 mutation target.

[0014] Appendix Figure 3 They are fruit pictures of 'Donghong' and low-citric-acid kiwifruit at different development stages.

[0015] Appendix Figure 4 It is a comparison graph of the growth trends of 'Donghong' plants and low-citric-acid kiwifruit plants.

[0016] Appendix Figure 5 It is a comparison of the citric acid content in the fruit development stages of 'Donghong' low-citric-acid kiwifruit. Specific implementation method

[0017] Taking the combination of the attached drawings and the Agrobacterium-mediated kiwifruit transgenic method as an example, the present invention elaborates in detail the creation process of low-citric-acid germplasm materials.

[0018] Example 1. Creation of kiwifruit germplasm resources with AcNAC1 gene editing

[0019] I. Research methods:

[0020] 1. Construction of CRISPR-Cas9 gene editing vector and Agrobacterium transformation

[0021] Using the CRISPR-P website, 2 target sequences were designed on the exon of the AcNAC1 gene, and the nucleotide sequences of the 2 target sequences are shown in SEQ ID NO.4 and SEQ ID NO.5 (as shown in the appendix Figure 1 shown); then, after gene synthesis of the AtU6-26 promoter together with the sgRNA sequence AtU6-26-tRNA-gRNA1-tRNA-gRNA2 containing the 2 target sequences, it was carried on the gene editing vector pDE-KRS to obtain the AcNAC1 gene editing vector; the correctly sequenced AcNAC1 gene editing vector was transformed into the host cell Agrobacterium tumefaciens EHA105 by methods such as electroporation or liquid nitrogen freeze-thaw, and the Agrobacterium monoclonal colonies were picked out and the bacterial liquid was stored in an ultra-low temperature refrigerator at -80 °C for standby.

[0022] 2. Kiwifruit genetic transformation and detection

[0023] The cotyledon explants of 'Donghong' kiwifruit were infected with the host cell Agrobacterium prepared above, and tissue culture seedlings were obtained through callus induction, resistance induction and differentiation, and rooting culture. The genomic DNA of the plants was extracted and verified by PCR and sequencing techniques to screen out AcNAC1 gene mutant kiwifruit plants. The primer sequence of AcNAC1-F1 for amplifying the gene fragment containing the above target sequence 1 is shown in SEQ ID NO.6; the primer sequence of AcNAC1-R1 for amplifying the gene fragment containing the above target sequence 1 is shown in SEQ ID NO.7; the primer sequence of AcNAC1-F2 for amplifying the gene fragment containing the above target sequence 2 is shown in SEQ ID NO.8; the primer sequence of AcNAC1-R2 for amplifying the gene fragment containing the above target sequence 2 is shown in SEQ ID NO.9.

[0024] II. Research results:

[0025] Sequencing revealed that compared with the AcNAC1 nucleotide sequence SEQ ID NO.2 in 'Donghong' kiwifruit, in the mutant kiwifruit plants, acnac1-1 had 55.38% no mutation and 43.09% insertion of 1 base at target site 1, and 55.02% deletion of 5 bases and 44.98% deletion of 4 bases at target site 2; acnac1-2 had 54.81% deletion of 3 bases and 42.92% no mutation at target site 1, and 50.59% deletion of 7 bases and 48.10% insertion of 1 base at target site 2; while acnac1-3 had no base mutation at target site 1 and deletion of 10 bases at target site 2 (as shown in the appendix Figure 2 ). Compared with the AcNAC1 amino acid sequence SEQ ID NO.3 in 'Donghong' kiwifruit, the amino acid sequences encoded by acnac1-1 and acnac1-3 in the mutant kiwifruit plants were prematurely terminated, and the sequence encoded by acnac1-2 had a frameshift mutation, all resulting in the loss of AcNAC1 function, that is, all the obtained kiwifruit germplasm resources were AcNAC1 function-deficient materials.

[0026] Example 2. Determination of citric acid content at different fruit development stages of low-citric-acid kiwifruit

[0027] I. Research methods:

[0028] 1. Collection of fruits at different development stages of low-citric-acid kiwifruit

[0029] The AcNAC1 function-deficient kiwifruit germplasm materials created in Example 1 and 'Donghong' kiwifruit plants were planted in the greenhouse, and the culture conditions were light intensity 300 μmol m -2 s -1, temperature 25°C, photoperiod 16 h / 8 h (light / dark). During the flowering period, artificial pollination was carried out on the blooming flowers, and the fruits at the 6th, 9th, 13th, and 17th weeks after pollination were picked (as shown in Figure 3 ). The pulp part of the fruit samples was collected, the peel, the seeded part, and the central column were removed, and after being frozen in liquid nitrogen, it was stored at -80°C for subsequent analysis.

[0030] 2. Determination of citric acid content at different fruit development stages

[0031] Weigh 0.1000 g of the ground and frozen kiwifruit samples into a 1.5 ml screw centrifuge tube, add 1.4 ml of chromatographic methanol (stored at -20°C), vortex thoroughly 3 times, incubate at 70°C for 15 minutes, and centrifuge at 11000 g for 10 minutes at 4°C. Transfer all the supernatant to a 10 ml centrifuge tube, add 750 μl of chloroform (stored at -20°C) and 1.5 ml of mili-Q double-distilled water (stored at 4°C), vortex thoroughly 3 times, then centrifuge at 2200 g for 10 minutes, and extract 1 ml of the supernatant for derivatization for subsequent citric acid content analysis.

[0032] The derivatization steps are as follows: Take 100 μl of the supernatant into a 1.5 ml centrifuge tube, add 20 μl of 0.2 mg / ml ribitol as an internal standard, evaporate to dryness under vacuum, dissolve the precipitate thoroughly in 60 μl of 20 mg / ml methoxyamine hydrochloride (dissolved in pyridine), seal the tube and incubate at 37°C for 1.5 h, then add 40 μl of Bis(trimethylsilyl)trifluoroacetamide (1% trimethylchlorosilane) for treatment, seal the tube, and incubate at 37°C for 0.5 h. After brief centrifugation, add it into the sample bottle for gas chromatography (GC) to detect the citric acid content, and each sample is replicated biologically three times.

[0033] The chromatographic conditions for citric acid content determination are as follows: Gas chromatography (GC) (7890N - 5975C, Agilent Technologies, USA), the chromatographic column is HP-5MS (30 m × 0.25 mm × 0.25 μm, J&W Scientific Folsom, CA). The inlet temperature is set at 250°C. Nitrogen is used as the carrier gas, and the column flow rate is 1 ml / min. The injection volume is 1 μl, and the split ratio is set at 10:1. The temperature programming is as follows: maintain at 100°C for 1 min, increase to 185°C at 3°C / min, increase to 230°C at 15°C / min, and maintain for 1 min.

[0034] II. Research results:

[0035] The growth of the AcNAC1 loss-of-function kiwifruit germplasm material created in Example 1 and the 'Donghong' kiwifruit plants is not significantly different, and there are no special requirements for cultivation management measures such as fertilizer and water (as shown in the attachment). Figure 4 The citric acid content in the fruits of the AcNAC1 loss-of-function kiwifruit was 0.37 mg / g at the 6th week, and slowly increased to 2.39 mg / g (at the 17th week) as the fruits developed. However, the citric acid content in the fruits of the 'Donghong' kiwifruit had reached 2.61 mg / g at the 6th week, and continued to accumulate during the development process, reaching 12.50 mg / g at the 17th week. Compared with the fruits at the development stage of the 'Donghong' kiwifruit, the citric acid content in the fruits of the AcNAC1 loss-of-function kiwifruit always remained at a low level, and it was determined as a low-citric-acid type of kiwifruit germplasm resource (as shown in the attachment). Figure 5 Shown).

Claims

1. A method for creating kiwifruit germplasm resources of low citric acid type, characterized in that: Using the key gene that regulates citric acid synthesis AcNAC1 as a target, comprising at least the following steps: Step 1, based on AcNAC1 the genomic sequence, design and synthesize two target gRNA sequences, and then construct a recombinant CRISPR-Cas9 expression vector; In the second step, the recombinant CRISPR-Cas9 expression vector was transformed into the leaf discs of 'Donghong' kiwifruit by means of Agrobacterium-mediated transgenic technology, and transgenic kiwifruit was obtained by combining plant tissue culture technology; Step 3. Analyze the changes in the genomic sequences of the obtained transgenic kiwifruits, and screen out the transgenic kiwifruit germplasm materials in which the genomic sequence mutations lead to changes in the encoded amino acid sequences, thereby resulting in AcNAC1 gene function loss; AcNAC1 due to genomic sequence mutations, the encoded amino acid sequences change, and further AcNAC1 result in gene function loss; Step 4, detect AcNAC1 The citric acid content in the fruits with gene function deletion is only 15-20% of that in 'Donghong', and it is determined that the obtained AcNAC1 Kiwi germplasm materials with gene function deletion are low-citric-acid kiwi germplasm resources; The AcNAC1 genomic sequence is as shown in SEQ ID NO.1, the nucleotide sequence is as shown in SEQ ID NO.2, the encoded amino acid sequence is as shown in SEQ ID NO.3, and the two designed target gRNA sequences are as shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

2. The creation method of a low-citric-acid kiwifruit germplasm resource according to claim 1, characterized in that: Resulting in gene function loss AcNAC1 Sequence mutation situations leading to gene function loss include frameshift mutations and premature termination of the coding sequence.

3. The method for creating a low-citric-acid kiwifruit germplasm resource according to claim 1, wherein: The plants of the created low-citric acid kiwifruit germplasm resources have no significant particularities in growth and have no special requirements for fertilizer and water cultivation management measures.

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