JP7292753B2 - Applications of genes that negatively regulate tomato leaf photosynthesis - Google Patents

Description

The present invention belongs to the field of crop molecular genetics, and relates to a gene for improving the photosynthetic efficiency of tomato leaves and its use.

Photosynthesis is the process in which green plants use light energy to synthesize carbon dioxide and water into organic substances and release oxygen. is. About 220 billion tons of organic matter is synthesized annually through photosynthesis on Earth, making it the most important chemical reaction on Earth (Zhang Lixin et al., 2016). Without the use of light energy by plants, the survival and sustainable development of human society would be impossible. With the development of modern molecular biology techniques, several new genes controlling the efficiency of photosynthesis have been rapidly unearthed. For example, proteins encoded by two plant genes, PGR5 and PGRL1, instantaneously form a super-protein complex with other chromoprotein complexes, and directly transmit energy within the super-complex to effectively utilize light energy. significantly enhanced (Munekage et al. 2004; DalCorso et al. 2008). A research team led by Li Zhensheng of the Chinese Academy of Sciences has selected and bred new varieties of wheat with high photosynthetic efficiency, such as Xiaowian 81, by using strong photooxidation-resistant genes. Li et al. (2011) cloned a new gene PHD1 that encodes a chloroplast locator protein in rice. This gene not only improves photosynthetic capacity, but can also promote tillering and 1000-grain weight in rice. Yuan et al. (2018) reported that SlARF10 plays an important role in chlorophyll, and overexpression of this gene can improve photosynthesis in tomato leaves and fruits, as well as increase the accumulation of starch, fructose and sucrose in fruits. discovered. Zhang et al. (2019) found that transgenic tomato photosynthesis and fruit size could be significantly improved by converting genes encoding betaine aldehyde dehydrogenase and choline oxidase into tomatoes. From the above, it can be seen that sufficiently discovering new genes with high photosynthetic efficiency in plants and promoting efficient use of light energy in crops is an effective means for increasing the production of crops.

Tomato is a nutritious, multi-eared berry with strong adaptability, high yield and good texture. It is the second largest vegetable crop in the world and is loved by consumers. Leaves are the main organ of photosynthesis in tomato, and by increasing the photosynthetic efficiency of tomato leaves, it promotes better plant growth and increases fruit yield and quality, thus leading to higher economic and social benefits. can bring. Therefore, discovering new functional genes and using gene editing technology to select and breed new tomato cultivars with high photosynthetic efficiency is a relatively quick method.

An object of the present invention is to effectively improve the photosynthetic efficiency of tomato leaves and promote the synthesis of organic matter.

To solve the above problems, the present invention provides the use of genes that negatively regulate tomato leaf photosynthesis. Photosynthetic efficiency of tomato leaves can be improved (effectively improved) by knocking out the Solyc05g005230 gene in tomato.
The nucleotide sequence of the gene Solyc05g005230 is shown in SEQ ID NO:1.
In improving the use of genes that negatively regulate photosynthesis in tomato leaves of the present invention,
A target sgRNA sequence to be edited with CRISPR/Cas9 in the Solyc05g005230 gene is designed: 5′-GATGATCGAGAGGTGCTTAG-3′, primers are artificially synthesized based on this sgRNA sequence and constructed in the CRISPR/Cas9 carrier.
In a further improvement of the use of genes that negatively regulate tomato leaf photosynthesis of the present invention,
The synthesized primers are
Upstream: 5′-TGATTGATGATCGAGAGGTGCTTAG-3′
Downstream: 5'-AAACCTAAGCACCTCTCGATCATCA-3'
is.
In a further improvement of the use of genes that negatively regulate tomato leaf photosynthesis of the present invention,
A plant 5230-KO in which the gene Solyc05g005230 is knocked out is obtained.
The contents of photosynthetic pigments such as chlorophyll a, chlorophyll b and carotenoids in the leaves of the Solyc05g005230 gene knockout strain 5230-KO are all increased (remarkably increased), and the net photosynthetic rate of the leaves of the Solyc05g005230 gene knockout strain 5230-KO, All of the major indicators of photosynthesis, such as stomatal conductance and transpiration, are enhanced (significantly enhanced).
The nucleotide sequence of the Solyc05g005230 gene in this 5230-KO plant is shown in SEQ ID NO:2.
In the present invention, the gene Solyc05g005230 that negatively regulates tomato leaf photosynthesis is shown in SEQ ID NO: 1 as the nucleotide sequence encoding the protein.

The present invention further provides a method of knocking out the Solyc05g005230 gene in tomato. This method includes the following steps.
Step 1): Design target sgRNA sequence for gene editing by CRISPR/Cas9 technology: 5′-GATGATCGAGAGGTGCTTAG-3′.
Step 2): Two primers are synthesized based on the sequence obtained in step 1), artificially annealed, and then constructed on the CRISPR/Cas9 carrier.
Step 3): Transform the carrier obtained in step 2) into the wild-type tomato cultivar MicroTom to obtain the corresponding genetically modified plant. A plant in which the Solyc05g005230 gene has been knocked out from the genetically modified tomato plant is identified.

Specific technical means of the present invention are as follows.
synthesizing an sgRNA sequence specifically targeting the Solyc05g005230 gene based on the protein-encoding nucleotide sequence (SEQ ID NO: 1) in the Solyc05g005230 gene by CRISPR/Cas9 gene editing technology and constructing it into the corresponding CRISPR/Cas9 carrier; By transforming wild-type tomato cultivar MicroTom, the Solyc05g005230 gene in the genome is specifically edited to obtain a transgenic plant. The Solyc05g005230 gene in the transgenic plant is PCR amplified and sequenced to obtain the Solyc05g005230 gene knockout plant 5230-KO (Fig. 1). The mutated sequence of the Solyc05g005230 gene in the 5230-KO plant is SEQ ID NO:2. The Solyc05g005230 gene knockout plant has a darker green color than the control cultivar MicroTom (Fig. 3), its leaf photosynthetic pigment content is significantly higher than that of the control cultivar (Fig. 4), and the leaf photosynthetic efficiency is also higher than that of the control cultivar. (Fig. 5). In addition, in order to verify that the Solyc05g005230 gene is involved in the regulation of photosynthesis, the present inventor constructed an overexpression carrier for this gene, obtained a transgenic plant, and identified the overexpressed Solyc05g005230 gene strain 5230. - OE (Fig. 2) is obtained, the plant turns yellow, the content of photosynthetic pigments in the leaves is reduced, and the photosynthetic efficiency of the leaves is reduced (Figs. 3-5) compared to the control cultivar MicroTom. These results amply demonstrate that the Solyc05g005230 gene negatively regulates tomato leaf photosynthesis.

We discovered a new functional gene that negatively regulates photosynthesis. Gene-editing technology yields a plant in which only the Solyc05g005230 gene is knocked out, and significantly improves the photosynthesis of the tomato plant.

The '**' in Figures 2, 4 and 5 below indicates that there is a highly significant difference (P<0.01) in the t-test compared to the wild-type control MicroTom.

Sequencing analysis of CRISPR/Cas9 targets of tomato Solyc05g005230 gene knockout mutants. Here, WT is the wild-type control variety MicroTom, 5230-KO is the Solyc05g005230 gene knockout strain of MicroTom, and so on. Fig. 2 shows an analysis of the expression level of the tomato Solyc05g005230 gene. Here, WT is the wild-type control cultivar MicroTom, and 5230-OE is the Solyc05g005230 gene overexpressing strain of MicroTom. A comparison of leaf color of tomato Solyc05g005230 gene knockout strain, overexpression strain and its wild-type control plants is shown. A comparison of the content of photosynthetic pigments in leaves of the tomato Solyc05g005230 gene knockout strain, the overexpression strain and its wild-type control is shown. Here, A is the content of chlorophyll a, B is the content of chlorophyll b, and C is the content of carotenoids. A comparison of leaf photosynthesis of the tomato Solyc05g005230 gene knockout strain, the overexpression strain and its wild-type control is shown. where A is the net photosynthetic rate, B is the intercellular _CO2 concentration, C is the stomatal conductance and D is the transpiration rate.

Specific embodiments of the present invention will be described in more detail below with reference to the drawings.

<Step 1 Construction of Solyc05g005230 gene knockout mutant>
Based on the coding sequence of the Solyc05g005230 gene (SEQ ID NO: 1), improved by CRISPR/Cas9 target analysis software (http://crispr.mit.edu/) and CRISPR/Cas9 edited into the Solyc05g005230 gene sgRNA sequence: 5 '-GATGATCGAGAGGTGCTTAG-3' (the first base was originally a T, but was replaced with a G to improve the efficiency of gene knockout), and the corresponding primers below were synthesized according to this sequence. .
Upstream: 5′-TGATTGATGATCGAGAGGTGCTTAG-3′
Downstream: 5'-AAACCTAAGCACCTCTCGATCATCA-3'
Corresponding CRISPR/Cas9 carriers were constructed by CRISPR/Cas9 kit (Biogle, China). The construction was operated according to the product manual.

<Step 2 Transfer transformation of Solyc05g005230 gene knockout carrier into tomato>
The CRISPR/Cas9 carrier constructed in step 1 was transformed into tomato cultivar MicroTom to specifically edit the Solyc05g005230 gene in the genome. The genetic recombination method was the method proposed by Kimura et al. (Kimura S et al, CHS Protoc, 2008) to obtain the corresponding genetically engineered tomato plants.

<Step 3 Extraction of genomic DNA from genetically modified tomato leaves>
After taking 0.1 g of tomato leaves and polishing them with liquid nitrogen, add ultrapure water to 600 μl of extract (15.76 g of Tris-cl, 29.22 g of NaCl, 15.0 g of SDS powder until the volume becomes 1 L). , adjusted to pH=8.0), incubated at 65° C. for 60 min, added 200 μl of KAC (5 mol/L), mixed uniformly, cooled in an ice bath for 10 minutes, and added 500 μl of chloroform. , Mix evenly, centrifuge at 10000 rpm for 5 min, remove the supernatant, add 500 μl isopropanol, mix evenly, centrifuge at 12000 rpm for 3 minutes, discard the supernatant, wash the precipitate with 75% ethanol, After centrifugation at 12,000 rpm for 3 minutes, the supernatant was discarded, and the DNA was dried by turning it upside down for 15 minutes, 30 μl of pure water was added to dissolve the DNA.

<Step 4 PCR amplification and sequencing of Solyc05g005230 gene knockout target>
Primers (upstream: 5'-GAAAGCAGAATCGAACCC-3', downstream: 5'-CATAATCCAACCATAAAACAA-3') were synthesized by PCR-amplifying the Solyc05g005230 gene by PCR. The Solyc05g005230 gene was PCR amplified using the Fidelity Taq enzyme PrimeSTAR® HS DNA Polymerase (TaKaRa, Japan).

The PCR amplification system was PrimeSTAR HS (Premix) 25 μl; upper and lower primers (10 μM): 1 μl each; template DNA: 2 μl (<200 ng); sterile water: 21 μl. The PCR amplification program was pre-denaturation: 95°C, 5 min; denaturation: 98°C, 10 sec; annealing: 55°C, 15 sec; extension: 72°C, 60 sec; .
Sequencing analysis of the PCR product yielded a successful Solyc05g005230 gene knockout strain 5230-KO. Thus, a 14-base deletion in the coding region of the Solyc05g005230 gene caused a frameshift mutation in this gene. The nucleotide sequence of the Solyc05g005230 gene in the 5230-KO plant is shown in SEQ ID NO:2.

<Step 5 Tomato leaf total RNA extraction and cDNA reverse transcription>
Total RNA was extracted from leaves of tomato wild-type cultivar MicroTom according to the product manual using RNeasy Plant Mini Kit (QIAGEN, Germany) and reverse transcribed into cDNA using PrimeScript ^™ 1st Strand cDNA Synthesis Kit (TaKaRa, Japan).

<Step 6 PCR cloning of Solyc05g005230 gene>
The Solyc05g005230 gene was PCR amplified with PrimeSTAR® HS DNA Polymerase (TaKaRa, Japan). The formulation of the reaction system is operated according to the product manual. cDNA obtained in step 5, the program for PCR amplification is: pre-denaturation: 95°C, 5 min; denaturation: 98°C, 10 sec; annealing: 55°C, 15 sec; extension: 72°C, 30 sec; : 72°C for 5 minutes.
PCR primers:
Upstream primer: 5'- cggggtacc ATGTATCAACAAAACCAGGAGTAC-3'
Downstream primer: 5'- aactgcag TTACTGCCAATAATATAGTTTCCT-3'
Note: Underlined letters are restriction enzyme recognition sequences.

<Step 7 Construction of carrier for overexpression of Solyc05g005230 gene>
The pCAMBIA1300-2×35S carrier was double digested with restriction enzymes Kpn I and Pst I (TaKaRa, Japan). Reactions contained Kpn I and Pst I: 1 μl each; 4 μl Buffer (supplied with the product) _; Treated for 4 h. The enzymatic cleavage products were purified using the AxyPrep PCR cleaning kit (Axygen, USA) according to the manufacturer's manual. The amplification product of the Solyc05g005230 gene was purified by the same method. Enzymatically cleaved and purified carriers and PCR products were ligated by T4 ligase kit from Promega (USA). The ligation system contained 1 μl of enzyme-cleaved carrier plasmid, 2 μl of gene enzyme cleavage fragment, 0.5 μl of T4 ligase, 1 μl of Buffer, supplemented to 10 μl with ddH ₂ O and incubated overnight (12 hours) at 4°C. placed. After converting the reaction products into JM109 competent cells by thermal excitation and obtaining positive clones, sequencing analysis was performed by a biotechnology company to confirm the sequence of the fragment inserted into the carrier (SEQ ID NO: 1). , Solyc05g005230 gene expression carriers were obtained.

<Step 8 Transformation of Solyc05g005230 gene overexpression carrier into tomato>
The expression carrier for the Solyc05g005230 gene obtained in step 7 was transformed into tomato wild-type cultivar MicroTom. For the genetic recombination method, the method of Kimura et al. (Kimura S et al, CHS Protoc, 2008) was adopted. The corresponding transgenic tomato plant, namely Solyc05g005230 gene overexpressing plant 5230-OE, was obtained.

<Step 9 RT-PCR identification of Solyc05g005230 gene overexpressing plant>
Total RNA of the transgenic tomato (5230-OE) leaf tissue obtained in step 8 was extracted by the method described in step 5 and reverse transcribed into cDNA, TB cDNA.
Synthesis of PCR primers Upstream primer: 5'-GTGCTTAGTGGTTGGGATGG-3'
Downstream primer: 5'-CCTTTCCCCAAATCTTGGTTC-3'

Real time PCR amplification: TB Green ^™ Premix Ex Taq ^™ kit (TaKaRa) was used as follows. To the mixed system were added 2 μL of TB cDNA, 10 μL of Green Premix Ex Taq, 1 μL each of upstream and downstream primers (concentration of both is 10 μM), 5.6 μL of ddH ₂ O, and 0.4 μL of ROX Reference Dye. PCR was performed by the StepOne Plus ^™ Real-Time PCR System (Applied Biosystems). The running program was predenaturation: 95° C., 30 s; 95° C., 5 s; 60° C., 30 s; 40× cycles. The obtained data were analyzed for significant difference by t-test.
The results are shown in FIG. The expression level of the Solyc05g005230 gene in the overexpression line 5230-OE plant was remarkably increased (up-regulation) compared to the wild-type cultivar MicroTom.

<Step 10 Measurement of photosynthetic pigments in tomato leaves>
The Solyc05g005230 gene knockout strain 5230-KO, the overexpressing strain 5230-OE, and their wild-type control cultivar MicroTom obtained above were cultivated in a greenhouse at 25° C. under the conditions of 16 hours of illumination and 8 hours of darkness. After about 45 days of growth, randomly select 6 plants from each cultivar, select 3 leaves from each plant, remove the main vein, cut into small pieces, weigh 0.1 g and add 3 mL of 80% (v/w) acetone. and extracted at 28° C. in the dark for 48 hours. The extracted solution was taken, and numerical values at wavelengths of 664 nm, 647 nm and 470 nm were measured with a UV spectrophotometer (NanoDrop 2000C). The contents of chlorophyll a, chlorophyll b and carotenoids were then calculated by the method of Amon (1949). The formula is as follows.
Chlorophyll a = (13.71 x OD ₆₆₄ -2.85 x OD ₆₄₇ ) x V/W
Chlorophyll b = (22.39 x OD ₆₄₇ -5.42 x OD ₆₆₄ ) x V/W
carotenoid = (1000 x _OD470 - 3.27 x chlorophyll a - 104 x chlorophyll b)/229
where V is the volume of the extract (3 mL), W is the leaf mass of 0.1 g, OD ₆₆₄ , OD ₆₄₇ and OD ₄₇₀ were measured by UV spectrophotometer at wavelengths 664 nm, 647 nm and 470 nm, respectively. It is a numerical value (unit: mg/g). Measured results were analyzed for significant differences between mutants and wild-type controls by t-test.
The results are shown in FIG. Compared to the control variety MicroTom, chlorophyll a, chlorophyll b and carotenoids in the leaves of the Solyc05g005230 gene knockout strain 5230-KO are all significantly increased, and chlorophyll a, chlorophyll b and carotenoids in the leaves of the overexpression strain 5230-OE are all decreased significantly.

<Step 11 Measurement of photosynthesis of tomato leaves>
Cultivated in the same manner as in step 10, after about 45 days of growth, 6 strains were randomly selected from each cultivar, and their leaf net photosynthetic rate, intercellular CO _Two concentrations, stomatal conductance and transpiration were measured. After the photosynthesis meter automatically preheated for 15 min, set the environment options, _H2O and _CO2 parameters, mixing fan, temperature, and external light source options in order, create a new recording file, and set the leaf area S and porosity K. After set-up, tomato leaf clamping, and data stabilization, net photosynthetic rate, intercellular _CO2 concentration, stomatal conductance and transpiration rate were read out. Measured results were analyzed for significant differences between mutants and wild-type controls by t-test.
The results are shown in FIG. Compared to the control cultivar MicroTom, the net photosynthetic rate, stomatal conductance and transpiration of the leaves of the Solyc05g005230 gene knockout strain 5230-KO were all significantly improved, and the net photosynthetic rate, stomatal conductance and transpiration of the leaves of the overexpressing strain 5230-OE were significantly improved. Both effects were reduced.

The above descriptions are only some specific examples of the present invention. The present invention is not limited to the above examples, and includes various modifications. A person skilled in the art can directly derive or conjecture all variations from the content disclosed in the present invention, which should fall within the protection scope of the present invention.

Claims (6)

Use of the Solyc05g005230 gene to negatively regulate tomato leaf photosynthesis, comprising:
Photosynthetic efficiency of tomato leaves can be improved by knocking out the Solyc05g005230 gene in tomato,
Use, characterized in that the nucleotide sequence of said gene Solyc05g005230 is shown in SEQ ID NO:1. In the Solyc05g005230 gene, designing sgRNA against CRISPR/Cas9 edited target sequence : 5′-GATGATCGAGAGGTGCTTAG-3′,
Use according to claim 1, characterized in that, based on this sgRNA sequence, primers are artificially synthesized and built into the CRISPR/Cas9 carrier. The synthesized primer is
Upstream: 5′-TGATTGATGATCGAGAGGTGCTTAG-3′
Downstream: 5'-AAACCTAAGCACCTCTCGATCATCA-3'
3. Use according to claim 2, characterized in that Use according to claim 2 or 3, characterized in that the Solyc05g005230 gene knockout plant 5230-KO is obtained. The contents of chlorophyll a, chlorophyll b and carotenoids in the leaves of the Solyc05g005230 gene knockout plant 5230-KO are all increased, and the net photosynthetic rate, stomatal conductance and transpiration of the leaves of the Solyc05g005230 gene knockout plant 5230-KO are all increased. 5. The use according to claim 4, characterized in that the is also improved. Use according to claim 4 or 5, characterized in that the nucleotide sequence of the Solyc05g005230 gene in the 5230-KO plant is shown in SEQ ID NO:2.

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