CN112280782B - Application of negative regulation tomato leaf photosynthesis gene - Google Patents
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Abstract
The invention discloses an application of a negative regulation tomato leaf photosynthesis gene, and the Solyc05g005230 gene knocked out in a tomato can increase the photosynthesis efficiency of the tomato leaf; the nucleotide sequence of the gene Solyc05g005230 is shown as SEQ ID NO:1 is shown. The contents of photosynthetic pigments such as chlorophyll a, chlorophyll b, carotenoid and the like in the leaves of 5230-KO of the Solyc05g005230 gene knockout strain are obviously increased; the main indexes of photosynthesis such as net photosynthetic rate, stomatal conductance, transpiration and the like of 5230-KO leaves of the Solyc05g005230 gene knockout strain are obviously improved.
Description
Technical Field
The invention relates to a gene for increasing photosynthesis efficiency of tomato leaves and application thereof, belonging to the field of crop molecular genetics.
Background
Photosynthesis is a process that green plants utilize light energy to synthesize organic matters from carbon dioxide and water and release oxygen, more than 90% of plant dry matters are from photosynthesis and are the basis of crop yield formation. The organic matter synthesized annually on earth by photosynthesis is about 2200 million tons, the most important chemical reaction on earth (Zhang Xin et al, 2016). Without the use of light energy by plants, the survival and sustained development of human society is not possible. With the development of modern molecular biology technology, some new genes for regulating and controlling photosynthesis efficiency are rapidly discovered, for example, proteins coded by two genes of plant PGR5 and PGRL1 can instantly form a super protein complex with other pigment protein complexes, so that energy can be directly transferred in the super complex, and the effective utilization of light energy is greatly improved (Munekage et al 2004; DalCorso et al 2008); a Chinese academy Lizhen academy team breeds a new wheat variety with high photosynthetic efficiency such as 'Elytrigia 81' by using a strong photooxidation resistant gene; li and the like (2011) are cloned to a novel gene PHD1 for encoding chloroplast localization protein in rice, and the gene can not only improve the photosynthetic capacity of crops under low light intensity, but also promote tillering and thousand-grain weight of the rice; yuan et al (2018) find that SlARF10 plays an important role in chlorophyll, and overexpression of the gene can increase photosynthesis of tomato leaves and fruits and increase accumulation of starch, fructose and sucrose in the fruits; zhang et al (2019) found that the photosynthesis and fruit size of transgenic tomatoes can be significantly improved by transferring genes encoding betaine aldehyde dehydrogenase and choline oxidase into tomatoes. Therefore, the method fully explores new genes with high photosynthetic efficiency of plants, promotes the efficient utilization of light energy of crops, and is an effective means for increasing crop yield.
The tomato is a multi-cluster berry rich in nutrition, has strong adaptability, high yield and good taste, is the second vegetable crop in the world, and is deeply loved by consumers. The leaves are the main organs of the tomato for photosynthesis, the photosynthesis efficiency of the tomato leaves is enhanced, the plants can be promoted to grow better, and the fruit yield and quality are improved, so that higher economic and social benefits are brought. Therefore, the method is a relatively rapid method for breeding a new tomato variety with high photosynthetic efficiency by digging new functional genes and utilizing a gene editing technology.
Disclosure of Invention
The invention aims to solve the technical problem of how to effectively improve the photosynthesis efficiency of tomato leaves and promote the synthesis of organic matters.
In order to solve the technical problems, the invention provides the application of the negative regulation tomato leaf photosynthesis gene: knocking out the Solyc05g005230 gene in tomato can increase (effectively increase) the photosynthesis efficiency of tomato leaves;
the nucleotide sequence of the gene Solyc05g005230 is shown as SEQ ID NO:1 is shown.
As an improvement of the use of the present genes for down-regulating photosynthesis in tomato leaves:
designing a sgRNA sequence of a CRISPR/Cas9 editing target in a Solyc05g005230 gene: 5'-GATGATCGAGAGGTGCTTAG-3', respectively; primers were artificially synthesized from the sgRNA sequence and constructed into the CRISPR/Cas9 vector.
As a further improvement of the application of the negative regulation tomato leaf photosynthesis gene, the synthesized primers are as follows:
upstream of the flow path 5'-TGATTGATGATCGAGAGGTGCTTAG-3', it is preferred that,
downstream 5'-AAACCTAAGCACCTCTCGATCATCA-3'.
As a further improvement of the use of the present genes for down-regulating photosynthesis in tomato leaves: obtaining a knock-out plant 5230-KO of the gene Solyc05g 005230.
The contents of photosynthetic pigments such as chlorophyll a, chlorophyll b and carotenoid in the leaves of the Solyc05g005230 gene knockout strain 5230-KO are increased (obviously increased); the main indexes of photosynthesis such as net photosynthetic rate, stomatal conductance and transpiration of the leaves of the Solyc05g005230 gene knockout strain 5230-KO are all increased (remarkably increased).
The nucleotide sequence of the Solyc05g005230 gene in the 5230-KO plant is shown in SEQ ID NO: 2.
In the invention, a gene Solyc05g005230 for negatively regulating photosynthesis of tomato leaves has a nucleotide sequence of a coding protein shown in SEQ ID NO:1 is shown.
The invention also provides a method for knocking out the Solyc05g005230 gene in tomato, which comprises the following steps:
1) designing a target sgRNA sequence for gene editing by using a CRISPR/Cas9 technology: 5'-GATGATCGAGAGGTGCTTAG-3', respectively;
2) synthesizing two primers by using the sequence obtained in the step 1), and constructing the two primers into a CRISPR/Cas9 vector after artificial annealing;
3) genetically transforming the carrier obtained in the step 2) into a wild tomato variety MicroTom so as to obtain a corresponding transgenic plant; identifying a plant with Solyc05g005230 gene knocked out from the transgenic tomato plant.
The technical scheme of the invention is as follows:
a CRISPR/Cas9 gene editing technology is adopted, a sgRNA sequence of a specific target Solyc05g005230 gene encoding protein is synthesized according to a nucleotide sequence (SEQ ID NO:1) of a Solyc05g005230 gene, a corresponding CRISPR/Cas9 vector is constructed and is genetically transformed into a wild tomato variety MicroTom, the Solyc05g005230 gene in a genome is directionally edited to obtain a transgenic plant, and PCR amplification and sequencing are carried out on the Solyc05g005230 gene in the transgenic plant to obtain a Solyc05g005230 gene knockout plant 5230-KO (figure 1). 5230 the mutant sequence of the Solyc05g005230 gene in the KO plant is SEQ ID NO. 2. The Solyc05g005230 gene knockout plant is darker green than a control variety MicroTom (figure 3), the content of photosynthetic pigments in the leaf is higher than that of the control variety (figure 4), and the photosynthesis efficiency of the leaf is also higher than that of the control variety (figure 5). Meanwhile, in order to verify the function of the Solyc05g005230 gene participating in photosynthesis regulation, the inventor constructs an overexpression vector of the gene to obtain a transgenic plant, identifies and obtains a Solyc05g005230 gene overexpression strain 5230-OE (figure 2), and compared with a control variety MicroTom, the plant yellowing, the content of chromochrome in leaf light and the photosynthesis efficiency of leaves are reduced (figures 3-5). These results fully demonstrate that the Solyc05g005230 gene is negatively regulating photosynthesis in tomato leaves.
The invention has the following technical advantages: a new functional gene for negatively regulating photosynthesis is found, and a plant with the Solyc05g005230 gene knocked out can be obtained by a gene editing technology, and the photosynthesis of a tomato plant is obviously improved.
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The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows CRISPR/Cas9 target sequencing analysis of tomato Solyc05g005230 gene knockout mutant;
WT: wild type control variety MicroTom; 5230-KO: solyc05g005230 gene knockout strain from MicroTom, as follows.
FIG. 2 is the expression analysis of tomato Solyc05g005230 gene;
WT: wild type control variety MicroTom; 5230-OE: solyc05g005230 gene overexpression strain from MicroTom.
FIG. 3 is a comparison of leaf color of tomato Solyc05g005230 knock-out line, over-expression line and wild type control.
FIG. 4 is a comparison of photosynthetic pigment content in leaf blades of a tomato Solyc05g005230 gene knock-out strain, an overexpression strain and wild type controls thereof;
a: chlorophyll a content, B: chlorophyll b content, C: carotenoid content.
FIG. 5 is a comparison of leaf photosynthesis of tomato Solyc05g005230 knock-out strain, over-expression strain and wild type control.
A is net photosynthetic rate and B is intercellular CO2Concentration, C is the stomatal conductance, D is the transpiration rate.
The "×" in fig. 2, 4 and 5 indicate that there was a very significant (P <0.01) difference in the t-test compared to the wild type control MicroTom.
Detailed Description
According to the coding sequence (SEQ ID NO:1) of the Solyc05g005230 gene, an sgRNA sequence edited by CRISPR/Cas9 is designed in the Solyc05g005230 gene through improvement on the basis of CRISPR/Cas9 target analysis software (http:// CRISPR. mit. edu.): 5'-GATGATCGAGAGGTGCTTAG-3' (the first base was originally T, and is forcibly changed to G in order to improve the efficiency of gene knock-out); and synthesizing corresponding primers according to the sequence:
upstream of the flow path 5'-TGATTGATGATCGAGAGGTGCTTAG-3', it is preferred that,
downstream 5'-AAACCTAAGCACCTCTCGATCATCA-3'.
The CRISPR/Cas9 kit (Biogle, China) is adopted to construct a corresponding CRISPR/Cas9 vector, and the construction method is operated according to the product instruction.
And (2) genetically transforming the CRISPR/Cas9 vector constructed in the step (1) into a tomato variety MicroTom to directionally edit the Solyc05g005230 gene in a genome, wherein the corresponding transgenic tomato plant is obtained by a transgenic method according to a Kimura method and the like (Kimura S et al, CHS Protoc, 2008).
Grinding 0.1g of tomato leaf with liquid nitrogen, adding 600 μ L of extractive solution (15.76g Tris-cl, 29.22g NaCl, 15.0g SDS powder, adding ultrapure water to constant volume to 1L, adjusting pH to 8.0), and incubating at 65 deg.C for 60 min; adding 200 μ L KAC (5mol/L), mixing, and ice-cooling for 10 min; adding 500 μ l chloroform, mixing, centrifuging at 10000rpm for 5 min; taking the supernatant, adding 500 mul of isopropanol, mixing uniformly, centrifuging at 12000rpm for 3min, and discarding the supernatant; washing the precipitate with 75% ethanol, centrifuging at 12000rpm for 3min, and discarding the supernatant; after drying the DNA by inversion for 15min, 30. mu.l of pure water was added to dissolve the DNA.
Synthesizing a primer for PCR amplification of Solyc05g005230 gene: upstream 5'-GAAAGCAGAATCGAACCC-3' and downstream 5'-CATAATCCAACCATAAACAA-3', using transgenic tomato plant and its control variety MicroTom genome DNA as template, adopting high fidelity Taq enzyme
HS DNA Polymerase (TaKaRa, Japan) PCR-amplified the Solyc05g005230 gene.
The PCR amplification system is as follows: PrimeSTAR HS (Premix) 25. mu.l, upstream and downstream primers (10. mu.M) 1. mu.l each, template DNA 2. mu.l (<200ng), sterile water 21. mu.l; the PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10sec, annealing at 55 ℃ for 15sec, extension at 72 ℃ for 60sec, 30 cycles; extension at 72 ℃ for 5 min.
After sequencing analysis of PCR products, 1 knock-out strain 5230-KO of the Solyc05g005230 gene is successfully obtained, namely 14 bases are deleted in the coding region of the Solyc05g005230 gene, so that the gene is subjected to frame shift mutation. 5230 the nucleotide sequence of Solyc05g005230 gene in KO plant is shown in SEQ ID NO: 2.
Step 5, extracting total RNA of tomato leaves and carrying out reverse transcription on cDNA
Extracting total RNA of leaves of a tomato wild type variety MicroTom by using an RNeasy Plant Mini Kit (QIAGEN, Germany) according to product specifications; and using PrimeScriptTM1st Strand cDNA Synthesis Kit (TaKaRa, Japan) was reverse-transcribed into cDNA according to the product instructions.
By using
The Solyc05g005230 gene is PCR-amplified by HS DNA Polymerase (TaKaRa, Japan), the configuration of a reaction system is operated according to the product instruction, the cDNA obtained in the step 5 is utilized, and the PCR amplification program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10sec, annealing at 55 ℃ for 15sec, extension at 72 ℃ for 30sec, 30 cycles; extension at 72 ℃ for 5 min. The PCR primers were as follows:
an upstream primer: 5' -cggggtaccATGTATCAACAAAACCAGGAGTAC-3’
A downstream primer: 5' -aactgcagTTACTGCCAATAATATAGTTTCCT-3’
Note: underlined letters are restriction enzyme recognition sequences.
Step 7, construction of Solyc05g005230 gene overexpression vector
The pCAMBIA 1300-2X 35S vector was double digested with restriction enzymes Kpn I and Pst I (TaKaRa, Japan) in the following reaction scheme: 1. mu.l each of Kpn I and Pst I, 4. mu.l of Buffer (product from product), 15. mu.l of pCAMBIA 1300-2X 35S vector plasmid, ddH2The amount of O was increased to 40. mu.l, and the mixture was digested at 37 ℃ for 4 hours. The cleavage products were purified using AxyPrep PCR clean-up kit (Axygen, USA) according to the product instructions. The amplification product of Solyc05g005230 gene was purified in the same manner as described above. The vector thus digested and purified was ligated to the PCR product using T4 ligase kit from Promega, USAThe connecting body is: mu.l of vector plasmid after enzyme digestion, 2. mu.l of target gene enzyme digestion fragment, 0.5. mu. l T4 ligase, 1. mu.l Buffer, ddH2Make up to 10. mu.l of O, overnight (12 hours) at 4 ℃. The reaction product is transformed into JM109 competent cells by a heat shock method, after positive cloning is obtained, the JM109 competent cells are sent to a biotechnology company for sequencing, the sequence of an insert fragment in a vector is verified (SEQ ID NO:1), and the expression vector of the Solyc05g005230 gene is obtained.
The result of step 7 is: the expression vector of the Solyc05g005230 gene is genetically transformed into a tomato wild type variety MicroTom, and a corresponding transgenic tomato plant, namely a Solyc05g005230 gene over-expressed plant- - -5230-OE, is obtained by a transgenic method according to a Kimura method and the like (Kimura S et al, CHS Protoc, 2008).
Step 9, RT-PCR identification of Solyc05g005230 gene overexpression plant
The total RNA of the leaf tissue of the transgenic tomato (5230-OE) obtained in step 8 is extracted according to the method described in step 5 and is reversely transcribed into cDNA, namely TB cDNA.
Synthesizing a PCR primer:
an upstream primer: 5'-GTGCTTAGTGGTTGGGATGG-3'
A downstream primer: 5'-CCTTCTCCCAAATCTTGGTTC-3'
Real time PCR amplification: use of TB GreenTM Premix Ex TaqTMThe kit (TaKaRa) is specifically operated as follows: 2. mu.L of TB cDNA, 10. mu.L of Green Premix Ex Taq, and 1. mu. L, ddH each of upstream and downstream primers (10. mu.M each) were added to the mixture2O5.6. mu.L, ROX Reference Dye 0.4. mu.L. In StepOne PlusTMReal-Time PCR System (Applied Biosystems) runs PCR with the following program: pre-denaturation at 95 ℃ for 30 s; and (3) analyzing the significant difference of the obtained data by adopting a t test method at 95 ℃ for 5s, 60 ℃ for 30s and 40 cycles.
The results are shown in FIG. 2, and compared with the wild type variety MicroTom, the expression level of Solyc05g005230 gene in the over-expression strain 5230-OE plant is significantly up-regulated.
The Solyc05g005230 gene knockout strain 5230-KO, the overexpression strain 5230-OE and the wild type control variety MicroTom obtained in the above are planted in a greenhouse at 25 ℃ under 16-hour light and 8-hour dark. After about 45 days of growth, 6 plants are randomly selected from each variety, 3 leaves are taken from each plant, the main veins are removed, the plant is cut into small pieces, 0.1g is weighed and soaked in 3mL of 80% (v/w) acetone, and dark extraction is carried out for 48 hours at the temperature of 28 ℃. The extracted solution was measured by an ultraviolet spectrophotometer (NanoDrop 2000C) at 664nm, 647nm, and 470 nm. Then, the contents of chlorophyll a, chlorophyll b and carotenoid were calculated according to the method of Amon (1949), and the calculation formula is as follows:
chlorophyll a ═ (13.71 XOD)664-2.85×OD647)×V/W
Chlorophyll b ═ 22.39 XOD647-5.42×OD664)×V/W
Carotenoid ═ 1000 XOD470-3.27 Xchlorophyll a-104 Xchlorophyll b)/229
Wherein: v is the volume of the extract (3mL), W is the leaf mass 0.1g, OD664、OD647And OD470The values are read by an ultraviolet spectrophotometer under three wavelengths of 664nm, 647nm and 470nm, and the unit is mg/g. Assay results significant differences between the mutant and wild-type controls were analyzed using the t-test.
As shown in FIG. 4, compared with the control variety MicroTom, the leaves of the Solyc05g005230 gene knockout strain 5230-KO have significantly increased chlorophyll a, chlorophyll b and carotenoid, while the leaves of the over-expression strain 5230-OE have significantly decreased chlorophyll a, chlorophyll b and carotenoid.
Step 11, determination of photosynthesis of tomato leaves
Planting according to the method of step 10, after about 45 days of growth, randomly selecting 6 plants from each variety, and measuring net photosynthetic rate and intercellular CO of folium xiphocae with Li-6400(Li-COR, USA) photosynthetic tester2Concentration, stomatal conductance and transpiration. After the photosynthetic apparatus is automatically preheated for 15min, sequentially setting environment options H2O and CO2Parameter, mixingSelecting fan, temperature and external light source, creating a record file, setting leaf area S and air hole proportion K, clamping tomato leaves, waiting for data stabilization, checking net photosynthetic rate and intercellular CO2And (3) numerical values of concentration, stomatal conductance and transpiration rate, and analyzing the significant difference between the mutant and the wild type control by using a t test according to the determination result.
As shown in FIG. 5, compared with the control variety MicroTom, the net photosynthetic rate, stomatal conductance and transpiration of the leaves of the Solyc05g005230 gene knock-out strain 5230-KO are all increased remarkably, while the net photosynthetic rate, stomatal conductance and transpiration of the leaves of the over-expression strain 5230-OE are all decreased remarkably.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
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<120> application of negative regulation tomato leaf photosynthesis gene
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cttaggcttt caaagagaac caagatttgg gagaaggaaa ctatattatt ggcagtaa 298
Claims (4)
1. The application of the gene for negatively regulating the photosynthesis of tomato leaves is characterized in that: the gene has a nucleotide sequence shown as SEQ ID NO:1, knocking out the gene Solyc05g005230 in tomato can increase the photosynthesis efficiency of tomato leaves.
2. Use of a gene for down-regulating photosynthesis in tomato leaves according to claim 1, wherein:
designing a sgRNA sequence of a CRISPR/Cas9 editing target in a Solyc05g005230 gene: 5'-GATGATCGAGAGGTGCTTAG-3', respectively; primers were artificially synthesized from the sgRNA sequence and constructed into the CRISPR/Cas9 vector.
3. Use of a gene for down-regulating photosynthesis in tomato leaves according to claim 2, wherein the synthetic primers are:
upstream of the flow path 5'-TGATTGATGATCGAGAGGTGCTTAG-3', it is preferred that,
downstream 5'-AAACCTAAGCACCTCTCGATCATCA-3'.
4. Use of a gene for down-regulating photosynthesis in tomato leaves according to claim 3, wherein: compared with a wild tomato variety MicroTom, the contents of chlorophyll a, chlorophyll b and carotenoid of plant leaves with a knockout gene Solyc05g005230 are increased; the net photosynthetic rate, stomatal conductance and transpiration of the plant leaves with the gene Solyc05g005230 knocked out are all increased.
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| CN112280782A (en) | 2021-01-29 |
| JP7292753B2 (en) | 2023-06-19 |
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