Use of ltt protein to increase photosynthetic rate and / or increase salt tolerance in plants

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to application of LTT gene in improving photosynthetic rate of plants and / or improving salt tolerance of plants. The present application provides application of LTT protein in improving photosynthetic rate of plants and / or improving salt tolerance of plants, and the amino acid sequence of the LTT protein is shown as SEQ ID NO. 1. The present application obtains LTT gene of Arabidopsis thaliana through plant genome cloning, constructs LTT overexpression vector, and obtains LTT overexpression rice transgenic line by using the method of agrobacterium infection of rice callus. The present application finds that, compared with wild type rice, LTT overexpression rice has better salt tolerance through culture medium salt stress simulation experiment, and provides gene resources for crop salt tolerance molecular breeding. Meanwhile, the present application finds that overexpression of LTT in rice can improve photosynthesis efficiency of rice by using photosynthetic instrument determination, and provides technical support for molecular breeding and genetic modification of plants.

Description

Technical Field

[0001] The present 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 salination, is caused by the accumulation of salt from the ground surface on the soil surface through capillary action. When the water evaporates, the salt accumulates excessively in the soil. Salt stress can cause water to leak out of plants, exclude 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. It is well known that photosynthesis is crucial to plant growth, development and yield, so increasing food production by improving photosynthesis efficiency is also a reliable way.

[0003] The LTT protein is a six-transmembrane protein localized to the chloroplast envelope. LTT proteins lack known conserved domains. Literature reports indicate that LTT is a component of the chloroplast membrane Ycf2-FtsHi complex, which possesses ATP hydrolase activity and may function as a motor for chloroplast protein import. However, the function of LTT in chloroplast potassium transport remains unclear. Furthermore, LTT has been lost in the Poaceae family for unknown reasons, and its transgenic use in rice and its role in rice photosynthesis have not been reported. Summary of the Invention

[0004] To address the above problems, the present invention provides the use of LTT protein in increasing plant photosynthetic rate and / or improving plant salt tolerance. The present invention found that LTT protein can increase the photosynthetic rate and salt tolerance of rice, providing technical support for molecular breeding and genetic modification of plants.

[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 of LTT protein in increasing plant photosynthetic rate and / or improving plant salt tolerance. 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 salt tolerance concentration range is 5 mM-30 mM.

[0010] Preferably, 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, and the LTT gene is a gene encoding the LTT protein.

[0011] The present invention provides a biomaterial for improving plant photosynthetic rate and / or plant salt tolerance, comprising an expression vector containing an LTT gene and / or an engineered bacterium containing the 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 increasing plant photosynthetic rate and / or increasing plant salt tolerance, comprising the following steps: introducing the biological material described in the above technical solution into target plants to obtain transgenic plants with increased 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 the use of LTT protein or a biomaterial that increases the expression of LTT protein in increasing the photosynthetic rate and / or improving the salt tolerance of plants. 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-overexpressing rice transgenic line by infecting rice callus with Agrobacterium. The present invention finds through a culture medium salt stress simulation experiment that LTT-overexpressing rice has better salt tolerance than wild-type rice, providing gene resources for molecular breeding of salt-tolerant crops. At the same time, the present invention uses a photosynthetic instrument to determine that overexpressing the LTT gene in rice can increase the photosynthetic 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 for use in the embodiments are briefly introduced below.

[0019] Figure 1 Figure 1 shows the results of LTT-1303FLAG overexpression in rice. A shows the Western blotting results, with 1-21 representing the numbers of the tested plants. B shows the GUS staining results of LTT-FLAG seedlings, with the upper side showing the GUS staining results of LTT-FLAG and the lower side showing the CK staining results. The scale bar is 1 cm. C shows a diagram containing a protein marker, with I representing the number of the tested plant.

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

[0021] Figure 3 Figure 1 shows the results of agronomic characterization of LTT-overexpressing plants; A shows the phenotypes of LTT-FLAG and CK rice (scale: 10 cm); B shows the grain length of LTT-FLAG and CK; C shows the grain length of LTT-FLAG and CK; D to J show the panicle length, number of grains per panicle, plant height, 100-grain weight, grain width, number of effective tillers, and panicle length of CK and LTT-FLAG, respectively; n>=10, * indicates P<0.05, ** indicates P<0.01, and ns indicates no significant difference.

[0022] Figure 4 Figure 1 shows the results of LTT-overexpressing plants treated with different salt concentrations. A shows the rice phenotype after fourteen days of culture in rice nutrient solution after soaking the seeds (without KCl treatment). B shows the rice phenotype after twenty days of treatment with different concentrations of KCl in A. C shows the schematic diagram of the different rice groups in B. DETAILED DESCRIPTION

[0023] The present invention provides the use of LTT protein or a biomaterial that increases the expression of LTT protein in increasing plant photosynthetic rate and / or improving plant salt tolerance. 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 discovered that under normal growth conditions, rice plants in strains overexpressing the LTT gene have higher photosynthetic rates than wild-type plants. Furthermore, under salt stress, LTT-overexpressing plants exhibit higher tolerance to potassium salts than wild-type plants. Based on this function, plants with salt tolerance and high photosynthetic efficiency can be obtained through transgenic methods. Specifically, by introducing an LTT recombinant expression vector into a target plant, transgenic plants are obtained that exhibit higher salt tolerance and photosynthetic rates than 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, and electroporation, and the cells or tissues can be cultured into plants.

[0027] In 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 salt tolerance concentration range is 5mM-30mM.

[0030] In one embodiment, the biomaterial 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 increasing plant photosynthetic rate and / or increasing 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 increased 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 with reference to 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 thaliana genome sequence published on the TAIR official website (http: / / www.arabidopsis.org / ), we obtained the nucleotide sequence of the LTT gene (AT4G28210). The nucleotide sequence is 1301 bp long, consisting of 348 amino acids (SEQ ID NO. 1), with a molecular weight of approximately 38.34 kD. Specific primers were designed based on the LTT gene nucleotide sequence as follows:

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

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

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

[0045] 2. Functional identification of the LTT gene

[0046] 2.1 Construction of overexpression materials

[0047] 1) The commercially available pCAMBIA1303 vector was used for overexpression strain construction. First, it was modified to add the ubi promoter shown in 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] The amplification is performed using PhantaMax Super-Fidelity DNA Polymerase high-fidelity enzyme amplification (Vazyme), and the amplification system is as follows: 2x PhantaMax Buffer 25 μl, dNTPMix (10 mM each) 1 μl, upstream primer (10 μM) 2 μl, downstream primer (10 μM) 2 μl, Phanta Max Super-Fidelity DNA Polymerase 1 μl, template DNA 1 μl, and ddH2O is supplemented to 50 μl.

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

[0054] The amplification product is inserted between HindIII and BamHI of pCAMBIA1303 vector to obtain a modified pCAMBIA1303 vector.

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

[0056] 3) The LTT gene containing the enzyme digestion site is obtained by using PhantaMax Super-Fidelity DNA Polymerase high-fidelity enzyme amplification (Vazyme) with Arabidopsis thaliana cDNA as the template, and the amplification system and the amplification procedure are the same as above, and 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 enzyme cleavage site and the double-enzyme digestion vector were connected to obtain an LTT gene overexpression vector (denoted as LTT1-pCAMBIA1303 vector).

[0060] 5) The LTT1-pCAMBIA1303 vector was transformed into E. coli. The bacterial solution containing the LTT1-pCAMBIA1303 vector was then subjected to PCR identification using primer sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4. The correct bacterial solution was selected for plasmid extraction and sequencing.

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

[0062] The sequenced LTT1-pCAMBIA1303 vector constructed in step 2.1 was used to infect Nipponbare rice using the Agrobacterium strain EHA105. After receiving the T1 seeds, T2 breeding, sowing, and identification were performed. Western blotting was used to detect the expression of the target protein in the tagged transgenic plants. The results showed that 8 plants with expression were screened among the Flag-tagged overexpressing plants ( 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 determined whether it was homozygous or heterozygous. When the whole plant showed blue, it was homozygous. If part of the plant showed blue and the other part did not show blue, it was heterozygous. If none of the plants showed blue, it was wild type. Based on this staining result, a total of 5 homozygous transgenic rice plants ( Figure 1 Middle 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, actinic on). The parameters in brackets represent the environmental parameters when the photosynthetic rate was measured, namely, carbon dioxide density, leaf temperature, and light intensity in the leaf chamber.

[0065] The results showed that the photosynthetic rate of LTT overexpressing plants was slightly higher than that of CK plants at the same CO2 content, 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 restoration of LTT protein into rice has a certain effect on the growth and development of rice, the present invention statistically analyzed and investigated seven important agronomic traits of LTT overexpressing 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 LTT overexpression materials was less different from that of CK, and there was no significant difference in agronomic traits.

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

[0068] In order to analyze the tolerance of rice to different potassium salt concentrations, rice was treated and cultured using rice nutrient solutions containing different concentrations of potassium chloride, with potassium chloride concentrations of 0mM, 5mM, 10mM, 15mM, and 30mM. The tolerance experiment was conducted as follows: rice seeds were soaked for one week and then transferred to a rice hydroponic culture box. The hydroponic culture box 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 20 days after the treatment (as shown in the figure). 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 overexpression lines were significantly taller than the CK and wild-type plants. The average plant height of LTT-overexpressing line 1 was 16.46 cm, and that of LTT-overexpressing line 2 was 16.24 cm. The average plant height of the wild-type plant was 7.59 cm, and the average plant height of the CK was 9.45 cm. After 20 days of treatment with varying concentrations of potassium salt, the CK and wild-type plants showed lower tolerance to potassium salt than the LTT-overexpressing lines. This suggests that overexpressing the LTT gene in rice can increase the plant's photosynthetic rate and potassium salt tolerance.

[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 provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. Increased expression of LTT protein or use of a biomaterial that increases LTT protein expression in increasing 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; and the plant is Arabidopsis thaliana and / or rice.

2. The use according to claim 1, characterized in that The rice is Nipponbare rice.

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

4. The use according to claim 1, characterized in that The biological material for increasing the expression of LTT protein comprises LTT Gene expression vector and / or containing LTT Genetically engineered bacteria, LTT The gene encodes the LTT protein.

5. The use according to claim 4, characterized in that The engineering bacteria is Agrobacterium into which a recombinant expression vector is introduced; the recombinant expression vector contains LTT Gene expression vector; the Agrobacterium includes EHA105.

6. The use according to claim 5, characterized in that The said LTT The recombinant expression vector of the gene includes a basic vector and a LTT Gene; said recombinant expression vector LTT The promoter of the gene 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.

7. A method for increasing plant photosynthetic rate and / or increasing plant salt tolerance, characterized in that: The following steps are involved: The biological material is introduced into the target plant to obtain a transgenic plant with improved photosynthetic rate and / or salt tolerance; the biological material includes LTT Gene expression vector and / or containing LTT Genetically engineered bacteria, LTT The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 1; the target plant is Arabidopsis thaliana and / or rice.

8. The method according to claim 7, characterized in that The engineering bacteria is Agrobacterium into which a recombinant expression vector is introduced; the recombinant expression vector contains LTT Gene expression vector; the Agrobacterium includes EHA105.

9. The method according to claim 8, characterized in that The said LTT The recombinant expression vector of the gene includes a basic vector and a LTT Gene; said recombinant expression vector LTT The promoter of the gene 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.

10. The method according to claim 7, characterized in that The rice is Nipponbare rice.

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