A gene related to yellow-green leaf phenotype of Wucai and its application

Abstract

The present invention provides a gene related to the yellow-green leaf phenotype of Wucai, the gene is a BcAFR gene, and the nucleotide sequence is shown in SEQ ID NO.1. The present invention also provides the use of the above gene in cultivating a new variety of Wucai with a yellow-green phenotype. At the same time, the present invention also provides a BcAFR gene-silenced plant and a method for regulating the yellow-green phenotype of Wucai leaves. The present invention screened the BcAFR gene in Wucai, and verified the role of the BcAFR gene in controlling the yellow-green phenotype of Wucai leaves. At the same time, the Wucai BcAFR gene-silenced plant was obtained through the VIGS experiment, and further verified that BcAFR can regulate the yellow-green phenotype of Wucai leaves, laying a foundation for exploring the molecular mechanism of Wucai leaf color, and providing new gene resources for the creation of breeding materials, and also providing important scientific basis and germplasm materials for the breeding of Wucai varieties with different leaf colors.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding, and in particular to a gene related to the yellow-green leaf phenotype of wucai and an application thereof. Background Art

[0002] Photosynthetic efficiency and the synthesis and accumulation of photosynthetic products have always been the key factors affecting crop yield and quality. Leaves are the main organs of photosynthetic metabolism, and chloroplast pigments are the light receptors of photosynthesis. Leaf color phenotype and pigment content will directly affect photosynthetic efficiency and the synthesis and accumulation of photosynthetic products.

[0003] The synthesis and degradation of chlorophyll is a very complex process, which is related to many factors and involves the participation of a large number of genes. Any gene mutation may affect the synthesis and degradation of chlorophyll, causing plant leaves to show different leaf color changes, thereby affecting plant growth and development.

[0004] Wucai (Brassica campesttris L.ssp.chinesis (L.) Makino var.rosμLaris Tsenet Lee) is a subspecies of Chinese cabbage (B.campestris L.), which is native to China and mainly distributed in the Yangtze and Huaihe River basins. Wucai is a nutritious vegetable rich in minerals and vitamins, and exhibits colorful leaves. Green Wucai is the most common in life. However, for Wucai with different leaf colors, consumers prefer the yellow-green variety, which has beautiful color and more delicious taste.

[0005] Therefore, studying the molecular mechanism of the yellow-green phenotype of Wucai and providing a gene related to the yellow-green phenotype of Wucai leaves is the current research focus. It will provide new genetic resources for the creation of breeding materials and also provide important scientific basis and germplasm materials for the breeding of Wucai varieties with different leaf colors. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a gene related to the yellow-green leaf phenotype of Wucai and an application thereof.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A gene related to the yellow-green leaf phenotype of Wucai, the gene is the BcAFR gene, and the nucleotide sequence is shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the expression level of the BcAFR gene is negatively correlated with the yellow phenotype of Wucai leaves.

[0010] An application of the above-mentioned gene related to the yellow-green leaf phenotype of wucai in breeding new varieties of wucai with yellow-green phenotype.

[0011] A BcAFR gene silenced plant is obtained by silencing the expression of the BcAFR gene in a Wucai plant through a genetic engineering method; wherein the nucleotide sequence of the BcAFR gene is shown in SEQ ID NO.1.

[0012] As one of the preferred methods of the present invention, the specific method is as follows: a 40bp specific sequence is selected from the CDS sequence of the BcAFR gene, and combined with its antisense sequence to form an 80bp fragment, as shown in SEQ ID NO.2; the synthesized SEQ ID NO.2 sequence is inserted into the PTY-S vector to obtain the BcAFR-PTY recombinant vector; then, the constructed BcAFR-PTY recombinant vector is coated with gold powder, and transferred into Wucai seedlings by "gene gun bombardment method" to obtain BcAFR gene silenced plants.

[0013] A method for regulating the yellow-green phenotype of Wucai leaves, wherein the BcAFR gene in Wucai plants is silenced by a genetic engineering method; the nucleotide sequence of the BcAFR gene is shown in SEQ ID NO.1, and is negatively correlated with the yellow phenotype of Wucai leaves.

[0014] The advantages of the present invention over the prior art are as follows: the present invention screened the BcAFR gene in Wucai and verified the role of the BcAFR gene in controlling the yellow-green phenotype of Wucai leaves, and obtained Wucai BcAFR gene silenced plants through VIGS experiments, further verifying that BcAFR can regulate the yellow-green phenotype of Wucai leaves, which can lay a foundation for exploring the molecular mechanism of Wucai leaf color, provide new gene resources for the creation of breeding materials, and provide important scientific basis and germplasm materials for the breeding of Wucai varieties with different leaf colors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a graph showing the expression level of the BcAFR gene in the BcAFR-PTY silenced plants and the PTY silenced plants in Example 2;

[0016] Figure 2 It is a phenotypic diagram of the Wucai BcAFR-PTY silenced plant and the PTY silenced plant in Example 3 (in the figure, Figure A is a leaf diagram of the PTY silenced plant, Figure B is a whole plant diagram of the PTY silenced plant, Figure C is a leaf diagram of the BcAFR-PTY silenced plant, and Figure D is a whole plant diagram of the BcAFR-PTY silenced plant);

[0017] Figure 3It is the phenotypic diagram of the wild-type Arabidopsis WT and the Arabidopsis mutant afr plants in Example 3 (in the figure, Figure A is a diagram of the wild-type Arabidopsis WT plant, Figure B is a diagram of the Arabidopsis mutant afr sample 1 plant, and Figure C is a diagram of the Arabidopsis mutant afr sample 2 plant). DETAILED DESCRIPTION

[0018] The embodiments of the present invention are described in detail below. The embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagents, kits and other materials used in the following embodiments are all commercially available products conventional in the art unless otherwise specified. The experimental methods used in the following embodiments are conventional methods in the art unless otherwise specified, and will not be repeated.

[0019] Example 1

[0020] BcAFR gene:

[0021] The present invention screened out a BcAFR gene related to the yellow-green leaf phenotype of Wucai in Wucai, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0022] Example 2

[0023] Construction of Wucai BcAFR-PTY silenced plants:

[0024] (1) Wucai W7-1 was selected as the material. W7-1 seedlings were grown in a greenhouse at 26±2℃ (daytime) and 20±2℃ (nighttime) with a light intensity of 300μmol m -2 s -1 , relative humidity is 70-80%.

[0025] (2) A 40 bp specific sequence was selected from the CDS sequence of the BcAFR gene and combined with its antisense sequence to form an 80 bp fragment (shown in SEQ ID NO. 2), which was then submitted to GenScript Biotech (Nanjing) for synthesis; the synthesized sequence was inserted into the PTY-S vector to obtain the BcAFR-PTY recombinant vector.

[0026] (3) PTY and BcAFR-PTY plasmids were respectively encapsulated with gold powder and bombarded into Wucai seedlings using a gene gun (1300 psi, PDS-1000 / He, Bio-Rad) to obtain PTY silenced plants and BcAFR-PTY silenced plants, respectively. The specific operation is as follows:

[0027] First, add 8.5 μL gold powder, 4 μg PTY and BcAFR-PTY plasmids, and 20 μL 2.5 M CaC in a 1.5 mL centrifuge tube.2 , 8 μL 0.1 M spermidine. Place on ice for 20 min, vortexing intermittently (once every 5 min).

[0028] Add 80 μL of anhydrous ethanol, vortex, centrifuge at 13,000 x g for 20 s at room temperature, and discard the supernatant.

[0029] Add 20-30 μL of anhydrous ethanol, vortex vigorously for 1 second, add anhydrous ethanol to 200 μL, centrifuge at 13000xg at room temperature for 20 seconds, and discard the supernatant.

[0030] Repeat the previous step and wash twice with anhydrous ethanol.

[0031] Add 10 μL of anhydrous ethanol, suspend and then bombard with a gene gun; bombard each plasmid four times, bombarding 4 to 5 plants each time.

[0032] (3) Two weeks after bombardment, the leaves of the silenced plants gradually showed a disease spot phenotype. Leaves with a susceptible phenotype were selected for sampling, and total RNA was extracted and cDNA was synthesized from the leaves. The specific method was as follows:

[0033] The RNA of samples from three periods was extracted using the TakaRa RNA extraction kit. After quantification, the RNA was reverse transcribed using the TRAN reverse transcription kit. The system is shown in Table 1.

[0034] Table 1 Reverse transcription system

[0035]

[0036] (4) Real-time quantitative PCR was used to identify silent plants. Fluorescent primers (F: SEQ ID NO.3; R: SEQ ID NO.4) were designed based on the gene sequence using Oligo7 software, and Actin from Wucai was used as the internal reference gene. The primers were F: SEQ ID NO.5; R: SEQ ID NO.6. The quantitative PCR reaction system is shown in Table 2.

[0037] Table 2 Quantitative PCR reaction system

[0038]

[0039] The results of qRT-PCR analysis were as follows Figure 1 As shown in Figure 1, compared with the "PTY silenced plants", the expression level of BcAFR gene in "Wucai BcAFR-PTY silenced plants" was significantly downregulated, and the transcription abundance decreased by 64.3%.

[0040] Example 3

[0041] Phenotype, color difference and chlorophyll content determination of "BcAFR gene silenced plants of Wucai" and "Arabidopsis thaliana mutant afr":

[0042] Phenotypic observations and color difference and chlorophyll content determinations were performed on Wucai PTY silenced plants, BcAFR gene silenced plants (constructed in Example 2), wild-type Arabidopsis WT, and Arabidopsis mutant afr plants (Arabidopsis mutants with AFR gene knocked out according to standard genetic engineering procedures).

[0043] (1) Color difference measurement

[0044] Determination of color value: The colorimeter (DS-700D) was used to measure the leaf chromaticity value on the upper surface of the leaf. Ten plants were measured and the measurement was repeated three times at the same leaf position. The CIELAB color coordinate system was used for color data analysis. Among them, the color difference value has three representative color values: L*, a*, and b*. L* represents brightness (black and white), a* represents red and green, and b* represents yellow and blue. In addition, there are two color differences: value H* (Hue angle) and value C* (Chroma).

[0045] (2) Determination of chlorophyll content

[0046] The content of photosynthetic pigments (Chl a, Chl b) was determined by the Arnon method: fresh leaves were ground into powder, and then 2.5 mL of 80% acetone and a small amount of calcium carbonate and quartz sand were added to grind into a homogenate. 3 mL of acetone was added and allowed to stand in the dark for 3 to 5 seconds. A piece of filter paper was placed in a funnel, moistened with ethanol, and the extract was poured into the funnel along a glass rod and filtered into a 25 mL volumetric flask. 95% ethanol was used to wash the mortar with a dropper, and the cleaning solution was also filtered into a volumetric flask, and the pigment was washed around the filter paper with 95% ethanol. After the filter paper and the residue were completely whitened, the volume was finally fixed to the mark with 95% ethanol. The absorbance at wavelengths of 646 and 663 nm was measured using a TU1950 UV-visible spectrophotometer (PER-SEE, Beijing, China). The content of Chla (mg / g) and Chl b (mg / g) was calculated as follows:

[0047] CChl a mg / L=12.21×A663-2.81×A646;

[0048] CChl b mg / L=20.13×A646-5.03×A663;

[0049] Chl a mg / g=CChl a mg / L×V(L) / Mfresh;

[0050] Chl b mg / g=CChl b mg / L×V(L) / Mfresh.

[0051] In this example, the phenotypes of the Wucai BcAFR-PTY silenced plants and the PTY silenced plants are as follows: Figure 2 As shown, the phenotypes of wild-type Arabidopsis WT and Arabidopsis mutant afr plants are as follows Figure 3 The color difference values ​​and total chlorophyll content of each plant are shown in Table 3.

[0052] Table 3 Color difference and total chlorophyll results of BcAFR-PTY and PTY silenced plants

[0053]

[0054] Depend on Figure 2 As shown in Table 3, the leaves of each silenced plant gradually showed a lesion phenotype, and the leaves of the "BcAFR gene silenced plant" also turned yellow; compared with the empty "PTY silenced plant", the L*Value of the "BcAFR gene silenced plant" increased by 38.03%, the a*Value increased by 63.65%, the b*Value increased by 129.27%, and the total Chl content decreased by 97.79%. The above results show that the BcAFR gene plays a key role in the yellow-green phenotype of Wucai.

[0055] Furthermore, this was also demonstrated in the Arabidopsis mutant afr ( Figure 3 and Table 3).

[0056] 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 in the protection scope of the present invention.

Claims

1. A method for regulating the yellow-green phenotype of Wucai leaves, characterized in that: Through genetic engineering, the BcAFR Gene silencing; BcAFR The nucleotide sequence of the gene is shown in SEQ ID NO.1, which is negatively correlated with the yellow phenotype of Wucai leaves; The specific silencing methods are as follows: BcAFR A 40 bp specific sequence was selected from the CDS sequence of the gene and combined with its antisense sequence to form an 80 bp fragment, as shown in SEQ ID NO.2; the synthesized SEQ ID NO.2 sequence was inserted into the PTY-S vector to obtain BcAFR -PTY recombinant vector; then, the constructed BcAFR -PTY recombinant vector was coated with gold powder and transferred into Wucai seedlings by "gene gun bombardment method" to obtain BcAFR Gene-silenced plants.

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