Application and method for promoting photosynthesis of tobacco by enhancing MT2c gene expression through double promoters

Abstract

The invention relates to application and a method for promoting photosynthesis of tobacco by enhancing MT2c gene expression through double promoters and belongs to the field of plant genetic engineering, the MT2c gene is overexpressed in the tobacco by constructing a gene expression cassette pActin-MT2c-OCS, or gene expression cassettes pActin-MT2c-OCS and p35S-MT2c-T35S are respectively constructed, and the MT2c gene is respectively driven to be overexpressed in the tobacco by a strong promoter 35S and an Actin promoter, so that the photosynthesis of the tobacco is promoted. The photosynthetic efficiency, the transpiration speed and the stomatal conductance of the tobacco can be obviously improved, and the photosynthesis of the tobacco is promoted; and the promotion effect is not limited to resistance to abiotic stress, and the photosynthetic yield of plants can be effectively improved in a pollution-free environment without abiotic stress.

Description

Applications and methods of dual promoter enhancement of MT2c gene expression to promote tobacco photosynthesis Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the application and method of using dual promoters to enhance MT2c gene expression and promote tobacco photosynthesis. Background Technology

[0002] Photosynthesis, the core physiological process by which plants convert light energy into chemical energy, directly determines crop yield and biomass accumulation. Tobacco is an important economic crop in my country, and the efficiency of leaf photosynthesis directly determines its appearance quality, making it a key indicator for evaluating the quality of flue-cured tobacco. Against the backdrop of rapid development in molecular biology techniques, regulating photosynthetic pathways through genetic engineering has become an important direction for modern agricultural improvement. Currently, strategies to improve plant photosynthetic efficiency mainly include: modifying key enzymes in the Calvin cycle (such as Rubisco), optimizing the light reaction center complex, regulating stomatal development and function, and improving the chloroplast electron transport chain. However, these strategies mostly focus on components directly related to photosynthesis, and research on indirectly affecting photosynthetic efficiency through regulating cellular ion homeostasis remains relatively limited.

[0003] Metallothioneins are rich in cysteine, which can chelate various metal ions such as zinc, cadmium, and copper, and scavenge reactive oxygen species (ROS). Studies have shown that overexpression of metallothionein MT2c in rice can improve rice's tolerance to the heavy metal cadmium. Current research on the application of the MT2c gene is mostly limited to its protective function under abiotic stress; there is no technology yet to use MT2c overexpression as a routine method to optimize plant cultivation under non-stress conditions. Summary of the Invention

[0004] One of the technical problems to be solved by this invention is to promote tobacco photosynthesis, and to provide an application of the metallothionein MT2c gene in promoting tobacco photosynthesis.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: The MT2c gene is overexpressed in tobacco by constructing the gene expression cassette pActin-MT2c-OCS, or by constructing gene expression cassettes pActin-MT2c-OCS and p35S-MT2c-T35S respectively, with the strong promoters 35S and Actin driving the overexpression of the MT2c gene in tobacco, thereby improving the photosynthetic efficiency of tobacco. The nucleotide sequence of the coding region of the MT2c gene is shown in SEQ ID NO. 1.

[0006] The aforementioned promotion of tobacco photosynthesis is specifically manifested in: improving the photosynthetic efficiency, transpiration rate, and stomatal conductance of tobacco through overexpression of the MT2c gene.

[0007] The second technical problem to be solved by this invention is to provide an application of the metallothionein MT2c gene in the cultivation of new varieties of plants with strong photosynthesis, using single or double promoters to drive the overexpression of the MT2c gene in plants to obtain transgenic plants with strong photosynthesis.

[0008] The third technical problem to be solved by this invention is to provide a method for promoting plant photosynthesis, wherein the MT2c gene is inserted into the vector CactF to construct the recombinant expression vector CactF-MT2c, and the Actin promoter of the vector CactF is used to drive the overexpression of the MT2c gene in plants; or the two expression frames of the MT2c gene, p35S-MT2c-T35S and pActin-MT2c-OCS, are linked into the vector CactF to construct the recombinant expression vector CactF-MT2c-MT2c; the 35S strong promoter and the Actin promoter enhance the overexpression of the MT2c gene in plants.

[0009] Specifically, the steps include: (1) Selecting the plant expression vector CactF, designing primers based on the multiple cloning site sequence between the promoter Actin and the terminator OCS in the vector CactF and the MT2c gene, amplifying the MT2c gene, and ligating the MT2c gene into CactF that has been double-digested with SpeI and PstI to obtain the vector CactF-MT2c; (2) Selecting the intermediate vector D1 with the promoter P2×35S, amplifying the MT2c gene based on the KpnI and XbaI restriction sites between the promoter P2×35S and the terminator T35S in the intermediate vector D1; ligating the MT2c gene into the intermediate vector D1 that has been double-digested with KpnI and XbaI to obtain the expression cassette P35S-OsMT2c. -T35s vector D1-MT2c; (3) According to the KpnI restriction site before the Actin promoter on the vector CactF-MT2c, the expression cassette P35S-OsMT2c-T35s on the vector D1-MT2c is amplified and linked into the vector CactF-MT2c after KpnI single restriction enzyme digestion to obtain the recombinant vector CactF-MT2c-MT2c with dual promoter enhanced expression; (4) The recombinant vector CactF-MT2c-MT2c obtained in step (3) is transformed into Agrobacterium EHA105 by freeze-thaw method; (5) The recombinant vector CactF-MT2c-MT2c is transformed into plant callus tissue by Agrobacterium-mediated transformation and resistant transgenic plants are obtained by tissue culture.

[0010] The plant in question is tobacco.

[0011] The beneficial effects of this invention are as follows: This invention constructs a plant expression vector for enhanced expression of metallothionein MT2c with a single or dual promoter, and transforms plants using Agrobacterium-mediated transformation to obtain transgenic plants with enhanced MT2c expression. This significantly improves the photosynthetic efficiency, transpiration rate, and stomatal conductance of plants, thus promoting plant photosynthesis. Moreover, this promoting effect is not limited to resistance to abiotic stress; it can also effectively increase the photosynthetic yield of plants in pollution-free environments without abiotic stress.

[0012] The transgenic tobacco with enhanced MT2c dual-promoter expression provided by this invention can significantly increase the growth and photosynthetic efficiency of tobacco leaves. This can be used as a routine method to optimize plant cultivation under non-stress conditions. It also improves tobacco's tolerance to abiotic stresses and can be used for cultivation under abiotic stress conditions. This invention will provide more theoretical basis and application value for regulating leaf growth and improving photosynthetic efficiency in tobacco and other leaf-harvesting economic crops. Attached Figure Description

[0013] Figure 1 is a schematic diagram of the recombinant plant expression vector CactF-MT2c-MT2c.

[0014] Figure 2 shows the screening of positive MT2c transgenic tobacco lines with enhanced expression of dual promoters.

[0015] Figure 3 is a phenotypic analysis diagram of genetically modified tobacco.

[0016] Figure 4 shows a comparison of tobacco growth status.

[0017] Figure 5 is a graph showing the photosynthetic efficiency analysis of genetically modified tobacco. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. The specific contents listed in the following embodiments are not limited to the technical features necessary to solve the technical problem of the present invention. Furthermore, the listed embodiments are merely a part of the present invention, and not all embodiments.

[0019] This invention promotes plant photosynthesis and cultivates new plant varieties with strong photosynthetic capacity by overexpressing the MT2c gene of rice in plants. The specific implementation plan is as follows: I. Sequence analysis of the rice OsMT2c gene Sequence source: The OsMT2c gene (AB002800) sequence was obtained from the National Center for Biotechnology Information (NCBI) in the United States. The nucleotide sequence of its coding region is shown in the sequence listing SEQ ID NO: 01, and the sequence length is 243bp.

[0020] II. Construction of the Recombinant Expression Vector for MT2c: The plant expression vector CactF, containing the Actin promoter, was selected. Primers were designed based on the multiple cloning site sequence between the Actin promoter and the OCS terminator in the CactF vector, as well as the rice OsMT2c gene sequence. The OsMT2c gene sequence was amplified by PCR using primers OsMT2c-Ca-F and OsMT2c-Ca-R, as shown in SEQ ID NO: 02 and SEQ ID NO: 03. CactF was double-digested with SpeI and PstI. The OsMT2c gene sequence was then ligated into CactF using a one-step recombinant cloning kit to obtain the vector CactF-MT2c.

[0021] The D1 vector containing the P2×35S promoter was selected as the intermediate vector. Primers were designed based on the KpnI and XbaI restriction sites between the P2×35S promoter and the T35S terminator, as well as the OsMT2c gene. The rice OsMT2c gene was amplified by PCR, and OsMT2c was ligated into the KpnI and XbaI double-digested vector D1 to obtain D1-MT2c. The primers used were MT2c-D1-F and MT2c-D1-R, with sequences shown in SEQ ID NO: 04 and SEQ ID NO: 05.

[0022] Based on the KpnI restriction site preceding the Actin promoter on the CactF-MT2c vector, the expression cassette P35S-OsMT2c-T35s on the D1-MT2c vector was amplified by PCR using primers Ca-MT2c-MT2c-F and Ca-MT2c-MT2c-R, with sequences shown in SEQ ID NO: 06 and SEQ ID NO: 07 of the sequence listing. The amplified expression cassette P35S-OsMT2c-T35s was then ligated into the KpnI-digested vector CactF-MT2c to obtain the recombinant vector CactF-MT2c-MT2c with enhanced dual-promoter expression.

[0023] The constructed recombinant vector CactF-MT2c-MT2c is shown in Figure 1, which includes two gene expression cassettes: p35S-MT2c-T35S and pActin-MT2c-OCS. HPT is a hygromycin resistance gene used for later screening of transgenic plants.

[0024] III. Obtaining Transgenic Plants The correctly sequenced recombinant vectors CactF-MT2c and CactF-MT2c-MT2c were transformed into Agrobacterium EHA105 using the freeze-thaw method. The plant callus tissue was then infected using the Agrobacterium-mediated transformation method. After pre-differentiation, differentiation, and rooting and strengthening of the resistant callus, transgenic plants M2 (driven by a single promoter) and M2M2 (driven by a dual promoter) were obtained.

[0025] Genomic DNA was extracted from the leaves of T0 generation transgenic plants using the CTAB method. Transgenic plants were screened by PCR using primers Ca-M2M2-F and Ca-M2M2-R, with sequences shown in SEQ ID NO: 08 and SEQ ID NO: 09 in the sequence listing. Figure 2 shows the screening results of positive lines of MT2c transgenic tobacco with enhanced dual-promoter expression.

[0026] IV. Phenotypic Identification of Transgenic Plants Tobacco seedlings of uniform size were selected and cultured in Hoagland nutrient solution for 10 days, followed by treatment with 0 or 100 μmol / L CdCl2 for 7 days, with the nutrient solution changed every 2 days. Culture conditions were controlled as follows: alternating 22℃ light environment for 14 hours and 18℃ dark environment for 10 hours, relative humidity of 75%, and light intensity of 360 µmol / L. −2 s −1 As shown in Figure 3, under both normal and cadmium stress conditions, the leaf area of ​​transgenic plants was significantly higher than that of wild-type plants, by as much as 3-6 times. The transgenic tobacco with enhanced expression of the dual promoter was superior to that with single-gene expression. The growth-promoting effect of overexpression of the MT2c gene is not limited to alleviating cadmium stress; it can also promote growth in the absence of cadmium stress.

[0027] Transgenic and wild-type plants were planted in plastic pots (15 cm in diameter and 10 cm deep) filled with sand and peat moss (volume ratio 1:1). The transgenic M2, M2M2, and wild-type treatment groups all included a normal cultivation group and a cadmium stress cultivation group. The normal cultivation group was irrigated every 5 days with 1 / 2 Hoagland nutrient solution, while the cadmium stress cultivation group received an additional 100 μmol / L Cd in the 1 / 2 Hoagland nutrient solution. Phenotypic identification was performed after 30 days of outdoor cultivation.

[0028] Figure 4 shows a comparison of the growth status of tobacco plants without Cd addition. It can be seen that the transgenic M2 and M2M2 plants are more robust than the wild-type WT, with the transgenic M2M2 showing a more pronounced improvement. Photosynthetic-related indicators were measured using a portable Li-6800 photosynthesis system during a clear, cloudless morning (9:30-11:30) to ensure stable light intensity. The results are shown in Figure 5. The results indicate that, under both normal and cadmium stress conditions, the photosynthetic efficiency (Pn), transpiration rate (Tr), and stomatal conductance (Gs) of the transgenic plants were significantly higher than those of the wild-type plants, increasing by 200-400%, 300-500%, and 300-500%, respectively, demonstrating excellent growth-promoting characteristics. The transgenic tobacco with dual-promoter enhanced expression showed better results than the single-promoter driven gene expression. These results indicate that overexpression of the MT2c gene can not only reduce photosynthetic damage by alleviating cadmium stress but also improve plant photosynthetic performance through other mechanisms.

[0029] Experiments show that enhancing MT2c gene expression through dual promoters can effectively promote plant photosynthesis, even in uncontaminated general growing environments, thus increasing photosynthetic yield. MT2c overexpression can be used as a routine method to optimize plant cultivation under non-stress conditions. Based on this result, the overexpression vector of this gene can be used to cultivate transgenic crops or traditional Chinese medicinal herbs with leaves as harvest organs, thereby improving their photosynthetic efficiency and yield.

[0030] The above description of specific embodiments is only for the purpose of helping to understand the technical concept and core idea of ​​the present invention. Although specific preferred embodiments have been used to describe and illustrate the technical solutions, they should not be construed as limiting the present invention itself. Those skilled in the art can make various changes in form and detail without departing from the technical concept of the present invention. These easily conceived changes or substitutions should all be covered within the protection scope of the present invention.

Claims

1. Application of the metallothionein MT2c gene in promoting tobacco photosynthesis.

2. The application as described in claim 1, characterized in that: By constructing the gene expression box pActin-MT2c-OCS, the MT2c gene was overexpressed in tobacco to improve the photosynthetic efficiency of tobacco; the nucleotide sequence of the coding region of the MT2c gene is shown in SEQ ID NO.

1.

3. The application as described in claim 1, characterized in that: Gene expression frames pActin-MT2c-OCS and p35S-MT2c-T35S were constructed, and the MT2c gene was overexpressed in tobacco by the strong promoters 35S and Actin, respectively, to improve the photosynthetic efficiency of tobacco; the nucleotide sequence of the coding region of the MT2c gene is shown in SEQ ID NO.

1.

4. The application as described in any one of claims 1-3, characterized in that: Overexpression of the MT2c gene can improve the photosynthetic efficiency, transpiration rate and stomatal conductance of tobacco.

5. The application of the metallothionein MT2c gene in breeding new varieties of plants with strong photosynthetic capabilities, characterized by: Transgenic plants with strong photosynthesis were obtained by overexpressing the MT2c gene in plants using single or dual promoters; the nucleotide sequence of the coding region of the MT2c gene is shown in SEQ ID NO.

1.

6. A method for promoting plant photosynthesis, characterized in that: The MT2c gene was inserted into the vector CactF to construct the recombinant expression vector CactF-MT2c. The Actin promoter of the vector CactF was used to drive the overexpression of the MT2c gene in plants. The nucleotide sequence of the coding region of the MT2c gene is shown in SEQ ID NO.

1.

7. The method as described in claim 6, characterized in that: Specifically, the following steps are included: (1) Select the plant expression vector CactF, design primers based on the multiple cloning site sequence between the promoter Actin and the terminator OCS in the vector CactF and the MT2c gene, amplify the MT2c gene, and ligate the MT2c gene into CactF that has been double-digested with SpeI and PstI to obtain the recombinant expression vector CactF-MT2c; (2) Transform the recombinant expression vector CactF-MT2c obtained in step (1) into Agrobacterium EHA105 by freeze-thaw method; (3) Transform the recombinant expression vector CactF-MT2c into plant callus tissue by Agrobacterium-mediated transformation, and obtain resistant transgenic plants by tissue culture.

8. A method for promoting plant photosynthesis, characterized in that: The two expression cassettes of the MT2c gene, p35S-MT2c-T35S and pActin-MT2c-OCS, were ligated into the vector CactF to construct the recombinant expression vector CactF-MT2c-MT2c. The overexpression of the MT2c gene in plants was enhanced by the 35S strong promoter and the Actin promoter. The nucleotide sequence of the coding region of the MT2c gene is shown in SEQ ID NO.

1.

9. The method as described in claim 8, characterized in that: Specifically, the following steps are included: (1) Select the plant expression vector CactF, and design primers based on the multiple cloning site sequence between the promoter Actin and the terminator OCS in the vector CactF and the MT2c gene to amplify the MT2c gene. Then, ligate the MT2c gene into CactF that has been double-digested with SpeI and PstI to obtain the vector CactF-MT2c; (2) Select the intermediate vector D1 with the promoter P2×35S, and amplify the MT2c gene based on the KpnI and XbaI restriction sites between the promoter P2×35S and the terminator T35S in the intermediate vector D1. (2) The MT2c gene was ligated into the intermediate vector D1, which was double-digested with KpnI and XbaI, to obtain the vector D1-MT2c with expression cassette P35S-OsMT2c-T35s; (3) Based on the KpnI restriction site before the Actin promoter on the vector CactF-MT2c, the expression cassette P35S-OsMT2c-T35s on the vector D1-MT2c was amplified and ligated into the vector CactF-MT2c, which was single-digested with KpnI, to obtain the recombinant vector CactF-MT2c-MT2c with enhanced expression of dual promoters; (4) The recombinant vector CactF-MT2c-MT2c obtained in step (3) was transformed into Agrobacterium EHA105 by freeze-thaw method; (5) The recombinant vector CactF-MT2c-MT2c was transformed into plant callus tissue by Agrobacterium-mediated transformation, and resistant transgenic plants were obtained by tissue culture.

10. The method as described in claim 8, characterized in that: The plant in question is tobacco.

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