Application of LTT protein in improving photosynthetic rate of plants and / or improving salt tolerance of plants

Abstract

The invention relates to the technical field of gene engineering, in particular to application of an LTT gene in improving the photosynthetic rate of plants and / or improving the salt tolerance of the plants. The invention provides application of LTT protein in improving the photosynthetic rate of plants and / or improving the salt tolerance of the plants. The amino acid sequence of the LTT protein is as shown in SEQ ID NO.1. The invention also provides application of the LTT protein in improving the photosynthetic rate of the plants and / or improving the salt tolerance of the plants. According to the invention, an LTT gene of arabidopsis thaliana is obtained through plant genome cloning, an LTT overexpression vector is constructed, and an LTT overexpression rice transgenic line is obtained by using a method of infecting rice callus with agrobacterium. A culture medium salt stress simulation experiment shows that compared with wild rice, LTT overexpressed rice is better in salt tolerance, and gene resources are provided for salt-tolerant molecular breeding of crops. At the same time, a photosynthetic instrument is used for determination, it is found that overexpression of LTT in rice can improve the photosynthetic efficiency of rice, and technical support is provided for molecular breeding and genetic modification of plants.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to the application of LTT protein in improving plant photosynthesis rate and / or improving plant salt tolerance. Background Art

[0002] Soil salinization is one of the environmental problems faced by the whole world, which seriously affects agricultural and forestry production. Soil salinization, also known as soil salinization, is caused by the accumulation of salt under the ground on the soil surface through capillary action. When the water evaporates, the salt accumulates excessively in the soil. Salt stress can cause water exudation in plants, reject the absorption of other nutrients, and affect normal metabolism, thus seriously affecting the growth and development of plants. Many soils in my country are in a salinized state, which leads to a large reduction in crop yields. Therefore, obtaining salt-alkali tolerant crops is crucial for agricultural production and alleviating global food pressure. As we all know, photosynthesis is crucial to plant growth, development and yield, so increasing food production by improving photosynthesis efficiency is also a reliable way.

[0003] LTT protein is a protein with 6 transmembrane domains located in the chloroplast envelope. LTT protein has no known conserved domains. According to literature reports, LTT protein is a component of chloroplast membrane Ycf2-FtsHi. The Ycf2-FtsHi complex has ATP hydrolase activity and may serve as a motor component for chloroplast protein input. However, the function of LTT protein in chloroplast potassium ion transport has not been clearly studied. At the same time, LTT protein has been lost in Poaceae for some unknown reasons, and its transgenic in rice and its functional research on rice photosynthesis have not been reported. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of LTT protein in improving plant photosynthetic rate and / or improving plant salt tolerance. The present invention finds that LTT protein can improve rice photosynthetic rate and salt tolerance, providing technical support for plant molecular breeding and genetic modification.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the use of LTT protein or a biological material for increasing the expression amount of LTT protein in increasing the photosynthetic rate of plants and / or increasing the salt tolerance of plants. The amino acid sequence of the LTT protein is shown in SEQ ID NO.1.

[0007] Preferably, the plant comprises Arabidopsis thaliana and / or rice.

[0008] Preferably, the rice is Nipponbare rice.

[0009] Preferably, the salt includes potassium chloride; and the tolerance concentration range of the salt resistance is 5mM-30mM.

[0010] Preferably, the biological material for increasing the expression amount of LTT protein comprises an expression vector containing an LTT gene and / or an engineered bacterium containing an LTT gene, and the LTT gene is a gene encoding the LTT protein.

[0011] The present invention provides a biological material for improving plant photosynthetic rate and / or improving plant salt tolerance, including an expression vector containing an LTT gene and / or an engineered bacterium containing an LTT gene, wherein the amino acid sequence of the protein encoded by the LTT gene is shown in SEQ ID NO.1.

[0012] Preferably, the engineered bacteria is Agrobacterium into which a recombinant expression vector is introduced; the recombinant expression vector is an expression vector containing the LTT gene; and the Agrobacterium includes EHA105.

[0013] Preferably, the recombinant expression vector containing the LTT gene includes a basic vector and the LTT gene inserted into the basic vector; the promoter of the LTT gene on the recombinant expression vector is the ubi promoter; the nucleotide sequence of the ubi promoter is shown in SEQ ID NO.5; and the basic vector includes the pCAMBIA1303 vector.

[0014] The present invention provides a method for improving plant photosynthetic rate and / or plant salt tolerance, comprising the following steps: introducing the biological material described in the above technical solution into a target plant to obtain a transgenic plant with improved photosynthetic rate and / or salt tolerance.

[0015] Preferably, the target plant includes Arabidopsis thaliana and / or rice.

[0016] Beneficial effects:

[0017] The present invention provides an application of LTT protein or a biological material for increasing the expression amount of LTT protein in increasing the photosynthetic rate of plants and / or increasing the salt tolerance of plants, wherein the amino acid sequence of the LTT protein is shown in SEQ ID NO.1. The present invention obtains the LTT gene of Arabidopsis thaliana by cloning the plant genome, constructs an LTT overexpression vector, and obtains an LTT overexpression rice transgenic line by infecting rice callus with Agrobacterium. The present invention finds through a culture medium salt stress simulation experiment that LTT overexpression rice has better salt tolerance than wild-type rice, providing gene resources for molecular breeding of crop salt tolerance. At the same time, the present invention uses a photosynthetic instrument to measure and finds that overexpression of the LTT gene in rice can improve the photosynthesis efficiency of rice, providing technical support for molecular breeding and genetic modification of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 The figure is a verification result of rice LTT-1303FLAG overexpression; A is the Western blotting test result, 1-21 is the number of the plants to be tested; B is the GUS staining result of LTT-FLAG seedlings, the upper side is the GUS staining result of LTT-FLAG, the lower side is the CK staining result, and the scale is 1 cm; C is a figure containing protein markers, and I is the number of the plants to be tested;

[0020] Figure 2 This is the result diagram of rice LTT-FLAG photosynthetic rate;

[0021] Figure 3 The results of the investigation on agronomic traits of LTT overexpressing plants; A is the phenotype of LTT-FLAG and CK rice (scale is 10 cm); B is the grain length of LTT-FLAG and CK; C is the grain length of LTT-FLAG and CK; D-J are the ear length, number of grains per ear, plant height, 100-grain weight, grain width, number of effective tillers and ear length of CK and LTT-FLAG, respectively; n>=10, * indicates P<0.05, ** indicates P<0.01, ns indicates no significant difference;

[0022] Figure 4 The results of different salt concentration treatments on LTT overexpressing plants; A is the rice phenotype after 14 days of culture in rice nutrient solution after soaking (not treated with KCl); B is the rice phenotype of the plants in A after 20 days of treatment with different concentrations of KCl; C is a schematic diagram of different rice groupings in B. DETAILED DESCRIPTION

[0023] The present invention provides an application of LTT protein or a biological material for increasing the expression amount of LTT protein in increasing the photosynthetic rate of plants and / or increasing the salt tolerance of plants. The amino acid sequence of the LTT protein is shown in SEQ ID NO.1, which is as follows:

[0024] MTLLALSSSLSPSFFCRNSRIRFSPVSRISSLNGDNKLTNSPPQFLNAGASLRRFVAPGKFPSVRLRVLTRCEKENAPKGGIEDDAERFARRESTMPDRFRYLTKEAPDSPIIWPWFVALGFLVYAWRAVLFELSNWRKAAFAILAFVGDLSKFALALLVFHFIGDPITSLISL VETAMYSVRAFYSGIVAYTPVRELTTVILLASSVLAIGEAVAPESISKQPYVVTIAGLVGYAAVQSYISEPFFWTVLLGLYGYSRLIKKRDDVTSALPSAAVLAGVGEPWVRVVAITGYLALAMYHNSTKTSSEEESQILRRAPPMPLLAAALAIGVRLAAKWAGYRHLTWMIV.

[0025] The present invention found that: under normal growth conditions, the photosynthetic rate of rice plants in the LTT gene overexpression strain is higher than that of the wild type; under salt stress, the tolerance of plants in the LTT gene overexpression strain to potassium salt is higher than that of the wild type. According to its function, salt-tolerant and high-photosynthetic-efficiency plants can be obtained by transgenic means. Specifically, a transgenic plant can be obtained by introducing an LTT recombinant expression vector into a target plant, and the salt tolerance and photosynthetic rate of the plant are higher than those of the target plant.

[0026] As an embodiment, the LTT recombinant expression vector can be transformed into plant cells or tissues using common biological methods such as Ti plasmid, microinjection, electroporation, etc., and the cells or tissues can be cultured into plants.

[0027] As one embodiment, the plant includes Arabidopsis thaliana or rice.

[0028] As an embodiment, the rice is Nipponbare rice.

[0029] As an embodiment, the salt includes potassium chloride; the tolerance concentration range of the salt resistance is 5mM-30mM.

[0030] As an embodiment, the biological material for increasing the expression of LTT protein includes an expression vector containing an LTT gene and / or an engineered bacterium containing an LTT gene, wherein the LTT gene is a gene encoding the LTT protein; the nucleotide sequence of the LTT gene is shown in SEQ ID NO.2, and is specifically as follows:

[0031]

[0032] Based on the above advantages, the present invention provides a biological material for improving plant photosynthetic rate and / or improving plant salt tolerance, including an expression vector containing an LTT gene and / or an engineered bacterium containing an LTT gene, wherein the amino acid sequence of the protein encoded by the LTT gene is shown in SEQ ID NO.1.

[0033] As an embodiment, the engineered bacteria is Agrobacterium into which an expression vector is introduced; the expression vector is an expression vector containing the LTT gene; and the Agrobacterium includes EHA105.

[0034] As an embodiment, the expression vector containing the LTT gene includes a basic vector and the LTT gene inserted into the basic vector; the promoter of the LTT gene on the expression vector is the ubi promoter; the nucleotide sequence of the ubi promoter is shown in SEQ ID NO.5; the basic vector includes the pCAMBIA1303 vector.

[0035] Based on the above advantages, the present invention provides a method for improving plant photosynthetic rate and / or improving plant salt tolerance, comprising the following steps: introducing the biological material described in the above technical solution into the target plant to obtain a transgenic plant with improved photosynthetic rate and / or salt tolerance.

[0036] As an embodiment, the target plant includes Arabidopsis thaliana or rice.

[0037] As an embodiment, the rice is Nipponbare rice.

[0038] To further illustrate the present invention, the application of the LTT protein provided by the present invention in improving plant photosynthetic rate and / or improving plant salt tolerance is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] 1. Gene source and isolation:

[0041] Based on the Arabidopsis genome sequencing published on the TAIR official website (http: / / www.arabidopsis.org / ), the nucleotide sequence information of Arabidopsis was obtained. The nucleotide sequence of the LTT gene (AT4G28210) is 1301 bp long, composed of 348 amino acids (SEQ ID NO.1), and has a molecular weight of approximately 38.34 kD. Specific primers were designed based on the nucleotide sequence of the LTT gene, as follows:

[0042] F: 5'-ATGACGCTTTTAGCTCTCTCTTC-3' (SEQ ID NO. 3);

[0043] R: 5'-TCAAACAATCATCCAAGTCAAGT-3' (SEQ ID NO. 4).

[0044] The cDNA of Arabidopsis thaliana seedlings of Col-0 ecotype was used as a template and PCR amplification was performed using PhantaMax Super-Fidelity DNA Polymerase (Vazyme) with an annealing temperature of 57°C.

[0045] 2. Functional identification of LTT gene

[0046] 2.1 Construction of overexpression materials

[0047] 1) The conventional commercially available pCAMBIA1303 vector was selected for overexpression strain construction. First, it was modified to add the ubi promoter shown in nucleotide sequence SEQ ID NO.5, as follows:

[0048]

[0049] The amplification template of the ubi promoter is derived from corn genomic DNA, and the amplification primers are as follows:

[0050] Upstream primer (SEQ ID NO. 6): 5′-AAACGACGGCCAGTGCCAAGCTTGCATGCCTGCAGTGCAGCGTGACC-3′;

[0051] Downstream primer (SEQ ID NO. 7): 5′-GGGACTGACCTACCCGGGGATCCTCTAGAGTCGACCTGCAGAAGTAACACCAAACAAC-3′;

[0052] PhantaMax Super-Fidelity DNA Polymerase high-fidelity enzyme amplification (Vazyme) was used, and the amplification system was: 2×PhantaMax Buffer 25μl, dNTPMix (10mM each) 1μl, upstream primer (10μM) 2μl, downstream primer (10μM) 2μl, Phanta Max Super-Fidelity DNA Polymerase 1μl, template DNA 1μl, ddH2O was added to 50μl.

[0053] The amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec; annealing at 56°C for 15 sec; extension at 72°C for 2.5 min, 40 cycles; and complete extension at 72°C for 5 min.

[0054] The amplified product was inserted between HindIII and BamHI of the pCAMBIA1303 vector to obtain the modified pCAMBIA1303 vector.

[0055] 2) The modified pCAMBIA1303 vector was double-digested with BamHI and SacⅠ to obtain a double-digested vector.

[0056] 3) Using Arabidopsis cDNA as a template, PhantaMax Super-Fidelity DNA Polymerase high-fidelity enzyme amplification (Vazyme) was used. The amplification system and amplification procedure were the same as above to obtain the LTT gene containing the restriction site. The primer sequences are as follows:

[0057] Upstream primer (SEQ ID NO. 8): 5′-CTGCAGGTCGACTCTAGAATGACGCTTTTAGCTCTCTCTTCTTCTC-3′;

[0058] Downstream primer (SEQ ID NO. 9): 5′-GAACGATCGGGGAAATTCTTACTTATCGTCGTCATCCTTGTAATCCTTATCGTCGTCAT-3′.

[0059] 4) The LTT gene containing the restriction site is connected to the double restriction enzyme digestion vector to obtain an LTT gene overexpression vector (denoted as LTT1-pCAMBIA1303 vector).

[0060] 5) The LTT1-pCAMBIA1303 vector was transformed into Escherichia coli. Then, the bacterial solution containing the LTT1-pCAMBIA1303 vector was subjected to PCR identification, and the primer sequences were shown in SEQ ID NO.3 and SEQ ID NO.4. The correct bacterial solution was selected to extract the plasmid and send it for sequencing.

[0061] 2.2 Screening of LTT-overexpressing transgenic positive strains

[0062] The LTT1-pCAMBIA1303 vector constructed in step 2.1 and sequenced correctly was used to infect Nipponbare rice. The Agrobacterium strain used was EHA105. After receiving the T1 seeds, T2 breeding, sowing and identification were carried out. The expression of the target protein in the transgenic plants with the tag was detected by Western blotting. The results showed that 8 plants with expression were screened out from the overexpression plants with the Flag tag ( Figure 1 A and C in the figure), and then these 8 plants were stained with GUS to observe whether they were homozygous or heterozygous. The transgenic plants that showed expression in the Western blotting results were stained with GUS. By detecting whether there was a strong GUS blue signal in the entire rice seedling, it was identified whether it was homozygous or heterozygous. When the plant was completely blue, it was homozygous, if part of it was blue and the other part was not blue, it was heterozygous, and if none of it was blue, it was wild type. Based on this, according to the staining results, a total of 5 homozygous transgenic rice plants ( Figure 1 (B).

[0063] 2.3 Investigation of photosynthetic indices, agronomic traits and salt tolerance of homozygous LTT overexpressing rice plants

[0064] The present invention selected five homozygous and LTT overexpressing plants (LTT-FLAG) and five rice plants transformed with an empty vector (CK), and measured the photosynthetic rate at the same time (CO2 Mixer: CO2R->400μml, Tleaf->28.00C, LCFLamp: ParIn->1200μml, withblue=10percent, acting on), and the parameters in brackets represent the parameters of the environment when the photosynthetic rate was measured, which are carbon dioxide density, leaf temperature, and light intensity in the leaf chamber.

[0065] The results showed that at the same CO2 content, the photosynthetic rate of LTT overexpressing plants was slightly higher than that of CK plants, and the average photosynthetic rate was increased by 14% compared with CK ( Figure 2 ).

[0066] During the evolutionary process, LTT protein homologs were lost in rice. In order to determine whether the replenishment of LTT protein into rice has a certain effect on the growth and development of rice, the present invention statistically investigated and analyzed seven important agronomic traits of LTT overexpression materials and CK materials, including grain length, number of grains per panicle, plant height, 100-grain weight, grain width, number of effective tillers, and panicle length. The results are shown in Figure 3 The results showed that the overall phenotype of the LTT overexpression material was less different from that of the CK, and there was no significant difference in agronomic traits.

[0067] In order to analyze the sensitivity of wild-type rice strain (Nipponbare), CK (Nipponbare transformed with pCAMBIA1303 empty vector), LTT overexpression strain 1 (LTT-OE1) and LTT overexpression strain 2 (LTT-OE2) to potassium salt, the above strains were subjected to stress treatment as follows:

[0068] In order to count the tolerance of rice under different potassium salt concentrations, rice was treated and cultured with rice nutrient solution containing different concentrations of potassium chloride, and the potassium chloride concentrations were 0mM, 5mM, 10mM, 15mM, and 30mM, respectively. The tolerance experiment was as follows: After soaking the rice seeds for one week, they were transferred to a rice hydroponic box, which was cultured in a light box (light intensity 30000lx, 16h light / 8h dark, 28℃). After two weeks of culture in the hydroponic box, the rice was treated with different concentrations of potassium salt, and photographed twenty days after the treatment (such as Figure 4 ).

[0069] like Figure 4 As shown, the present invention divides the hydroponic box into four large compartments, which are CK, LTT overexpression strain 1 (LTT-OE1), LTT overexpression strain 2 (LTT-OE2), and wild type (Nipponbare) from left to right.

[0070] The results showed that after two weeks of cultivation, the height of the overexpression strains was significantly higher than that of the CK and wild type. The average height of LTT overexpression strain 1 was 16.46 cm, the average height of LTT overexpression strain 2 was 16.24 cm, the average height of the wild type was 7.59 cm, and the average height of CK was 9.45 cm. After being treated with different concentrations of potassium salt for 20 days, the tolerance of CK and wild type to potassium salt was lower than that of LTT overexpression strains. This shows that rice overexpressing the LTT gene can increase the photosynthetic rate of plants and their tolerance to potassium salt.

[0071] In summary, overexpression of the LTT gene in rice can significantly improve the salt stress tolerance of rice plants.

[0072] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of LTT protein or biological material with increased expression of LTT protein in improving plant photosynthetic rate and / or improving plant salt tolerance, wherein the amino acid sequence of the LTT protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The plants include Arabidopsis thaliana and / or rice.

3. The use according to claim 2, characterized in that: The rice is Nipponbare rice.

4. The use according to claim 1, characterized in that: The salt includes potassium chloride; the tolerance concentration range of the salt resistance is 5mM-30mM.

5. The use according to claim 1, characterized in that: The biological material for increasing the expression of LTT protein comprises an expression vector containing LTT gene and / or an engineered bacterium containing LTT gene, wherein the LTT gene is a gene encoding LTT protein.

6. A biological material for improving plant photosynthetic rate and / or improving plant salt tolerance, characterized in that: It comprises an expression vector containing an LTT gene and / or an engineered bacterium containing an LTT gene, wherein the amino acid sequence of the protein encoded by the LTT gene is shown as SEQ ID NO.

1.

7. The biomaterial according to claim 6, characterized in that The engineering bacteria is Agrobacterium into which a recombinant expression vector is introduced; the recombinant expression vector is an expression vector containing the LTT gene; and the Agrobacterium includes EHA105.

8. The biomaterial according to claim 6 or 7, characterized in that: The recombinant expression vector containing the LTT gene includes a basic vector and the LTT gene inserted into the basic vector; the promoter of the LTT gene on the recombinant expression vector is the ubi promoter; the nucleotide sequence of the ubi promoter is shown in SEQ ID NO.5; the basic vector includes the pCAMBIA1303 vector.

9. A method for increasing plant photosynthetic rate and / or increasing plant salt tolerance, characterized in that: The method comprises the following steps: introducing the biological material according to any one of claims 6 to 8 into a target plant to obtain a transgenic plant with improved photosynthetic rate and / or salt tolerance.

10. The use according to claim 9, characterized in that: The target plants include Arabidopsis thaliana and / or rice.

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