An AcNAC2 gene that promotes chlorophyll degradation and regulates plant coloration and its applications

Abstract

This invention provides an AcNAC2 gene and its expression product. The nucleotide sequence of the AcNAC2 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. This invention also provides an expression vector containing the above-mentioned AcNAC2 gene and a recombinant bacterial strain. Furthermore, this invention provides the application of the above-mentioned AcNAC2 gene in promoting chlorophyll degradation and regulating plant color phenotype. The AcNAC2 gene of this invention can significantly promote chlorophyll degradation and change the color phenotype of plant leaves; by regulating the expression level of AcNAC2, the purpose of promoting chlorophyll degradation and regulating color phenotype can be achieved.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biotechnology and genetic engineering, and in particular to an AcNAC2 gene that promotes chlorophyll degradation and regulates plant coloration and its applications. Background Technology

[0002] Kiwifruit belongs to the Actinidiaceae family and the Actinididia genus. Kiwifruit is rich in amino acids, vitamins, dietary fiber, minerals and other nutrients, making it highly nutritious and popular among consumers.

[0003] Kiwifruit boasts abundant germplasm resources, with approximately 54 species and 21 varieties worldwide, exhibiting a rich diversity of flesh colors among different species. Flesh color is a crucial indicator of kiwifruit quality. Currently, commercially cultivated kiwifruit can be categorized into green-fleshed and yellow-fleshed varieties (including yellow-fleshed, red-fleshed varieties) based on their flesh color. Compared to common green-fleshed kiwifruit, yellow-fleshed kiwifruit is more competitive in the consumer market. Research indicates that the different colors of kiwifruit are primarily due to variations in the composition and content of pigments such as chlorophyll, carotenoids, and anthocyanins. Chlorophyll metabolism plays a key role in the formation of the yellow / green trait in kiwifruit flesh. During fruit development, the inhibition of chlorophyll degradation in yellow-fleshed kiwifruit directly affects fruit greening, leading to reduced quality. Therefore, identifying key genes regulating chlorophyll degradation is of significant industrial importance for improving the color and quality of kiwifruit.

[0004] Using biotechnology, we isolated NAC transcription factors from kiwifruit and found that AcNAC2 was significantly upregulated during the fruit development of yellow-fleshed and red-fleshed kiwifruit, and could reduce chlorophyll content, thus participating in the regulation of fruit color. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an AcNAC2 gene that promotes chlorophyll degradation and regulates plant coloration and its application. By overexpressing the AcNAC2 gene, chlorophyll degradation in plants can be promoted, thereby achieving the purpose of regulating plant coloration phenotype.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] An AcNAC2 gene that promotes chlorophyll degradation and regulates plant coloration, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the AcNAC2 gene is shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the expression product of the AcNAC2 gene positively regulates chlorophyll degradation and regulates plant coloration.

[0010] An expression product of the AcNAC2 gene described above.

[0011] An expression vector containing the AcNAC2 gene described above.

[0012] A recombinant bacterium containing the AcNAC2 gene described above.

[0013] An application of the AcNAC2 gene in promoting chlorophyll degradation and regulating plant color.

[0014] As one of the preferred embodiments of the present invention, the plants include tobacco and kiwifruit.

[0015] The advantages of this invention compared to the prior art are:

[0016] This invention screened a transcription factor, AcNAC2, that can promote chlorophyll degradation. This gene originates from kiwifruit (Actinidia chinensis) and is a member of the NAC transcription factor family. Analysis revealed that the transcriptional abundance of AcNAC2 was significantly enhanced during fruit development in both yellow-fleshed and red-fleshed kiwifruit varieties, while chlorophyll content was significantly reduced, showing a negative correlation. The open reading frame sequence of AcNAC2 was cloned and loaded into an expression vector, where it was transiently overexpressed in tobacco leaves and stably overexpressed in kiwifruit plants. The results showed that AcNAC2 significantly promoted chlorophyll degradation and altered the color of plant leaves. Therefore, by regulating the expression level of AcNAC2, it is possible to promote chlorophyll degradation and regulate color phenotypes in plants. Attached Figure Description

[0017] Figure 1 This is a diagram showing the changes in chlorophyll content and the gene expression pattern of AcNAC2 during the fruit development of the yellow-fleshed kiwifruit 'Jinshi No. 1' in Example 2 (Figure A shows the changes in chlorophyll content; Figure B shows the gene expression pattern).

[0018] Figure 2 This is a diagram showing the changes in chlorophyll content and the gene expression pattern of AcNAC2 during the fruit development of the red-fleshed kiwifruit 'Hongyang' in Example 3 (Figure A shows the changes in chlorophyll content; Figure B shows the gene expression pattern).

[0019] Figure 3 This is the result of semi-quantitative PCR verification of AcNAC2 overexpression in tobacco leaves in Example 4 (using NtActin-7 gene as internal reference);

[0020] Figure 4The results of the changes in tobacco leaf phenotype and chlorophyll content after transient overexpression of the 35S::AcNAC2 gene in Example 4 are shown in Figure 4 (Figure A shows the tobacco leaf phenotype; Figure B shows the change in chlorophyll content).

[0021] Figure 5 This is the result of the phenotypic and related gene expression changes of kiwifruit plants that stably overexpress the 35S::AcNAC2 gene in Example 5 (Figure A shows the phenotypic comparison between wild-type WT and three kiwifruit plants that stably overexpress AcNAC2; Figures B to D show the expression changes of AcNAC2, AcSGR1, and AcSGR2 genes in different kiwifruit plants; and "#23, #43, #45" represent the three selected positive lines that overexpress AcNAC2, and "***" indicates p<0.001). Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and experimental methods used in the following embodiments are all conventional reagents or methods in the art and will not be described again.

[0023] Example 1

[0024] About the AcNAC2 gene:

[0025] The AcNAC2 gene screened in this invention is derived from kiwifruit (Actinidia chinensis) and is a member of the NAC transcription factor family. The nucleotide sequence of the AcNAC2 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the amino acid residue sequence is a protein composed of 315 amino acid residues, with a molecular weight of approximately 35.43 kD, an isoelectric point of 8.39, and a full gene length of 948 bp.

[0026] Example 2

[0027] Analysis of changes in chlorophyll content in the pulp of yellow-fleshed kiwifruit 'Jinshi No. 1' and expression of the AcNAC2 gene:

[0028] 1. Fruit sampling

[0029] Yellow-fleshed kiwifruit 'Jinshi No. 1' was harvested in 2020 at the experimental base of the Sichuan Academy of Natural Resources Sciences. Sampling points were set at 10, 12, 14, 18, and 22 weeks post-pollination. Fruits of uniform size, free from mechanical damage and pests / diseases were selected at each developmental stage. The outer peel was removed, and the seedless pulp was collected. After flash-freezing in liquid nitrogen, the pulp was stored at -80℃ for long-term preservation.

[0030] 2. Chlorophyll content determination

[0031] Weigh 1g of ground kiwifruit sample and add it to 3mL of 80% acetone. Mix well and incubate on ice in the dark for 30min. Centrifuge at 12000g for 5min at 4℃, collect the supernatant into a new 10mL centrifuge tube, and store on ice in the dark. Add another 3mL of 80% acetone to the precipitate and mix well. Repeat the above steps twice more. Combine the supernatants collected from the three extractions, bring the volume to 10mL, and centrifuge at 10000g for 30min at 4℃. Finally, take 1mL of the supernatant and measure its absorbance at 663.2nm and 646.8nm using a spectrophotometer. Calculate the chlorophyll content per gram of sample, expressed in μg / g. The calculation formula is:

[0032] Chl a(μg / g)=(12.25×A 663.2 -2.79× A646.8 )×V / W;

[0033] Chl b (μg / g) = (21.5 × A 646.8 -5.1×A 663.2 )×V / W;

[0034] Chl(μg / g) = Chl a + Chl b;

[0035] In the formula: Chl a, Chl b, and Chl—the content of chlorophyll a, chlorophyll b, and total chlorophyll, in μg / g; A 663.2 and A 646.8 —Absorbance values ​​of the extract at wavelengths of 663.2 nm and 646.8 nm; V —Total volume of the extract after final dilution, mL; W —Weighing weight of the sample, g.

[0036] 3. AcNAC2 gene expression analysis

[0037] AcNAC2 real-time quantitative PCR primer pairs (SEQ ID NO.3 and SEQ ID NO.4) were designed using the online website Primer3 (v.0.4.0, https: / / bioinfo.ut.ee / primer3-0.4.0 / ), and were double-validated by sequencing of PCR clone products and melting curve analysis of internal reference genes.

[0038] The real-time quantitative PCR reaction system contains a total of 20 μL, including 10 μL of... 480 SYBR Green I Master (Roche, Germany), 2 μL cDNA, 1 μL each of primer pairs SEQ ID NO.3 and SEQ ID NO.4 (10 μM), 6 μL DEPC-H2O. The PCR reaction program was: 95℃, 5 min; 95℃, 10 s; 60℃, 10 s; 75℃, 15 s, 50 cycles. The kiwifruit actin gene (GenBank no. EF063572) was used as an internal control. AcNAC2 expression levels were calculated based on relative quantification. -ΔCt Law.

[0039] The results are as follows Figure 1 As shown, the chlorophyll content of the yellow-fleshed kiwifruit 'Jinshi No. 1' showed an overall decreasing trend during fruit development, and decreased significantly from 14 to 22 weeks after pollination, with a decrease of about 5 times; during this process, AcNAC2 showed a significantly increased expression pattern.

[0040] Example 3

[0041] Analysis of changes in chlorophyll content in the pulp and AcNAC2 gene expression during the development of the red-fleshed kiwifruit 'Hongyang':

[0042] 1. Fruit sampling

[0043] The 'Hongyang' red-fleshed kiwifruit was harvested in 2019 from a commercial orchard in Hangzhou, Zhejiang Province. Sampling was conducted at 6, 8, 10, 14, 18, and 22 weeks post-pollination. Fruits of uniform size, free from mechanical damage and pests / diseases were selected at each developmental stage. The outer peel was removed, and the seedless pulp was collected. After flash-freezing in liquid nitrogen, the pulp was stored at -80℃ for long-term preservation.

[0044] 2. Chlorophyll content determination

[0045] Refer to Example 2.

[0046] 3. AcNAC2 gene expression analysis

[0047] Refer to Example 2.

[0048] The results are as follows Figure 2 As shown, the chlorophyll content of the red-fleshed kiwifruit 'Hongyang' gradually decreased during fruit development, with a decrease of about 2 times; during this process, the relative expression level of AcNAC2 in the fruit development stage of 'Hongyang' increased significantly.

[0049] Example 4

[0050] Phenotypic and chlorophyll content determination of tobacco leaves with transient overexpression of 35S::AcNAC2:

[0051] 1. Transient overexpression in tobacco leaves

[0052] The full-length coding region of AcNAC2 was recombined into the pSAK277 expression vector using primer sequences SEQ ID NO. 5 and SEQ ID NO. 6. The positive recombinant plasmid was transformed into strain EHA105 using a freeze-thaw method, and the glycerol-containing bacteria were preserved. Agrobacterium containing 35S::AcNAC2 and the empty SAK vector was activated twice at 28°C, then resuspended in a permeate buffer (containing 10 mM MES, pH 5.6; 10 mM MgCl2; 150 μM acetylsylcholine) and the OD was adjusted. 600 =1. The prepared negative control (empty SAK vector) and the target gene (35S::AcNAC2 recombinant vector) resuspension were injected into the left and right sides of the main vein of the sixth true leaf of common tobacco, respectively. The injected tobacco was cultured in an artificial climate chamber for 5 days, and then samples were taken for subsequent experiments.

[0053] 2. Semi-quantitative PCR verification of AcNAC2 overexpression in tobacco leaves

[0054] The target fragment of AcNAC2 was amplified using the Phanta Max Super-Fidelity DNA Polymerase kit (Novizan, China) with cDNA synthesized by reverse transcription as a template. The primer sequences used were SEQ ID NO.3 and SEQ ID NO.4. The content of the target fragment in different samples was then analyzed by 1.1% agarose gel electrophoresis. The NtActin-7 gene of common tobacco (GenBank no. XM_016658880) was used as an internal control.

[0055] 3. Chlorophyll content determination:

[0056] The method was optimized and adjusted according to Example 2. 0.05g of ground tobacco leaf sample was weighed and 1mL of 80% acetone was added for chlorophyll extraction, which was repeated three times. Finally, the supernatant from the three extractions was collected and the volume was adjusted to 5mL. After centrifugation, 1mL of the supernatant was used for absorbance measurement.

[0057] Semi-quantitative PCR validation results are as follows Figure 3 As shown, the successful overexpression of 35S::AcNAC2 in tobacco leaves was confirmed.

[0058] The results of transient overexpression of the 35S::AcNAC2 gene in tobacco leaves and changes in chlorophyll content are as follows: Figure 4 As shown, compared with tobacco leaves injected with the empty vector SAK, tobacco leaves transiently overexpressing the 35S::AcNAC2 gene showed obvious yellowing and a significant decrease in chlorophyll content.

[0059] Example 5

[0060] Phenotypic characteristics and expression patterns of related genes in kiwifruit plants with stable overexpression of the 35S::AcNAC2 gene:

[0061] 1. Homologous genetically modified kiwifruit

[0062] The EHA105 strain containing 35S::AcNAC2 (constructed in Example 4) was activated by two rounds of shaking and cultured until the OD600 was about 0.6-0.8. The cells were collected by centrifugation at 10,000 rpm for 10 min at room temperature, and then the cells were resuspended in suspension medium for subsequent infection.

[0063] The plants used for infection were tissue culture seedlings of the Chinese kiwifruit 'Donghong' variety. The leaf disc method was used for transformation. The specific operation was as follows: the tender leaves were cut into rectangular leaf discs and placed in a co-culture medium for dark incubation for 1 day; then the leaf discs were placed in the prepared Agrobacterium infection solution and soaked for 15 minutes, after which the bacterial solution was discarded; then the leaf discs were placed back in the co-culture medium for dark incubation for 2 days; then the leaf discs were subcultured into the selection medium (the selection antibiotic was kanamycin at a concentration of 50 mg / L) for culture, and subcultured every two weeks until callus and adventitious buds grew.

[0064] 2. Identification of positive kiwifruit plants

[0065] Callus materials were collected, RNA was extracted using the CTAB method, and positive lines overexpressing AcNAC2 were identified by real-time PCR (refer to Example 2). The primer sequences used were SEQ ID NO. 3 and SEQ ID NO. 4. The expression of downstream target genes AcSGR1 and AcSGR2 was detected in the positive transgenic lines, using primer sequences SEQ ID NO. 7 and SEQ ID NO. 8, and SEQ ID NO. 9 and SEQ ID NO. 10, respectively.

[0066] The results are as follows Figure 5 As shown, the results are as follows:

[0067] Compared to wild-type plants, kiwifruit materials stably overexpressing AcNAC2 showed significant yellowing and inhibited growth. Figure 5 (A and 5B). Furthermore, overexpression of AcNAC2 significantly induced the expression of downstream key chlorophyll degradation genes AcSGR1 and AcSGR2, indicating that AcNAC2 also plays an important role in kiwifruit chlorophyll degradation (see appendix). Figure 5 C and 5D).

[0068] In summary, this invention provides a transcription factor, AcNAC2, derived from kiwifruit. Using the cloned AcNAC2 gene cDNA sequence, a plant expression vector was constructed, and overexpression was performed in tobacco and kiwifruit leaves. The chlorophyll content in tobacco leaves was significantly reduced, from 1.41 mg / g... -1 FW decreased to 0.53 mg g -1 The reduction was approximately 3-fold. Stable overexpression of AcNAC2 in kiwifruit promotes yellowing of the plant by inducing the expression of key chlorophyll degradation genes AcSGR1 and AcSGR2. Chlorophyll degradation can be promoted by regulating the expression of this NAC transcription factor, thereby reducing chlorophyll content and thus regulating the plant's color phenotype.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method that promotes chlorophyll degradation and regulates plant coloration AcNAC2 Genes, characterized by, The AcNAC2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A device as described in claim 1 AcNAC2 The expression products of genes.

3. A device comprising the features described in claim 1 AcNAC2 Gene expression vectors.

4. A device comprising the features described in claim 1 AcNAC2 Recombinant bacteria.

5. Overexpression of the expression described in claim 1 AcNAC2 The application of genes in promoting chlorophyll degradation and regulating plant color is characterized by, The plants are tobacco and kiwifruit, and the color of the plants being regulated is yellowing.

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