CN110257401B - Application of PtrMYB119 gene of Chinese white poplar in improvement of drought tolerance of tobacco - Google Patents
Application of PtrMYB119 gene of Chinese white poplar in improvement of drought tolerance of tobacco Download PDFInfo
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Abstract
The invention provides application of a PtrMYB119 gene of Chinese littleleaf poplar in improving the drought tolerance of tobacco, belonging to the technical field of plant genetic engineering. The research of the invention finds that the PtrMYB119 gene of the Chinese hairy poplar is transferred into tobacco leaves, so that the anthocyanin content in the obtained transgenic tobacco is obviously improved, and the drought resistance of the tobacco can be effectively improved.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of a PtrMYB119 gene of Chinese white poplar in improving the drought resistance of tobacco.
Background
Drought can cause the water content of plant cells to be reduced, active oxygen radicals to burst, membrane systems of the plant cells to be damaged, and the plants grow and develop slowly and even die, so that the yield of the plants is reduced. At present, more than one third of the land in the world belongs to arid and semiarid regions, wherein drought is one of the main reasons influencing the agricultural development, and the range, degree and frequency of drought disasters are increased, so that the growth and development of crops are seriously hindered, and the yield of the crops is reduced to cause huge economic loss. Improving the drought resistance of plants has become one of the key problems in modern plant research work, and the research of the drought resistance mechanism is the basis of drought resistance breeding and is also one of the key factors of breeding. Therefore, the excavation of the gene with the drought resistance effect has important significance for cultivating the drought-resistant plant.
Anthocyanin is a plant pigment with natural biological activity, the synthesis and accumulation of which in nutritive organs are important for the plant to adapt to and resist severe environmental conditions, and can enhance the resistance of the plant to different biotic stresses and abiotic stresses, such as drought, high temperature, low temperature salt stress, plant diseases and insect pests and the like. More researches on the color development mechanism and the biosynthesis regulation of anthocyanin in plant tissues are carried out, and less researches on plant resistance are carried out, and more researches show that the anthocyanin plays an important role in the plant response to abiotic stress and biotic stress.
Disclosure of Invention
In view of the technical problems in the background art, the invention aims to provide the application of the PtrMYB119 gene of populus tomentosa in improving the drought resistance of tobacco.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a PtrMYB119 gene of populus tomentosa in improving the drought resistance of tobacco.
Preferably, the application comprises the following steps:
(1) extracting total RNA of the populus trichocarpa, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain a PtrMYB119 gene;
(2) inserting the PtrMYB119 gene into an original vector to obtain a recombinant plant expression vector;
(3) transforming the recombinant plant expression vector into agrobacterium tumefaciens to obtain a recombinant agrobacterium tumefaciens transformant;
(4) infecting the recombinant agrobacterium tumefaciens transformant on tobacco leaves to obtain T0 generation transgenic plants.
Preferably, the primer pair for PCR amplification in the step (1) is PtrMYB119-F and PtrMYB 119-R; the nucleotide sequence of the PtrMYB119-F is shown as SEQ ID No. 1; the nucleotide sequence of the PtrMYB119-R is shown in SEQ ID No. 2.
Preferably, the reaction system for PCR amplification in step (1) is 20 μ L: 1. mu.L of cDNA template, 0.4. mu.L (10. mu.M) of each of the upstream and downstream primers, 0.1. mu.L of 10 XBuffer 2. mu. L, dNTPs 1.6.6. mu. L, rTaq enzyme, ddH2O14.5μL。
Preferably, the procedure of PCR amplification in step (1) is as follows: 94 ℃ for 3 min; 30 cycles of 94 ℃, 30s, 60 ℃, 30s, 72 ℃ and 1min each; final extension at 72 ℃ for 10 min.
Preferably, the original vector in step (2) is pCAMBIA2300, pCAMBIA1300, pCAMBIA1301 or pCAMBIA 3300.
Preferably, the transformation in step (3) and/or the transfection in step (4) further comprises a positive identification step.
Preferably, after the transgenic plants of T0 generation are obtained in step (4), the method further comprises the step of transplanting the transgenic plants of T0 generation into a greenhouse.
Furthermore, the invention also provides application of the recombinant plant expression vector containing the populus tomentosa PtrMYB119 gene in improving the drought resistance of tobacco.
Furthermore, the invention also provides application of the recombinant agrobacterium tumefaciens transformant containing the populus trichocarpa PtrMYB119 gene in improving the drought resistance of tobacco.
Has the advantages that: the invention provides application of a PtrMYB119 gene of populus tomentosa in improving the drought resistance of tobacco. The research of the invention finds that the PtrMYB119 gene of the Chinese hairy poplar is transferred into tobacco leaves, so that the anthocyanin content in the obtained transgenic tobacco is obviously improved, and the drought resistance of the tobacco can be effectively improved.
Description of the drawings:
FIG. 1 shows the identification result of the transgenic plant according to example 1 of the present invention, wherein FIG. 1-A shows the identification result of the dyeing; FIG. 1-B shows the results of PCR identification.
FIG. 2 is a graph showing the phenotypic changes of transgenic tobacco under drought treatment, wherein FIG. 2-A is the phenotype at day 0 of drought treatment; FIG. 2-B is the phenotype at 10 days of drought treatment; FIG. 2-C is the phenotype at 30 days of drought treatment; FIG. 2-D is a statistical result of root length at 30 days of drought treatment; FIG. 2-E is the root growth phenotype at 30 days of drought treatment; FIG. 2-F is the phenotype of the stem at 30 days of drought treatment; FIGS. 2-G are phenotypes of stem cross-sections at 30 days of drought treatment.
FIG. 3 is the change of anthocyanin and chlorophyll contents under the drought treatment of transgenic tobacco, wherein, FIG. 3-A is the change of anthocyanin contents; FIG. 3-B shows the change in chlorophyll content.
FIG. 4 shows the change of ABA content and MDA content of transgenic tobacco under drought treatment, wherein FIG. 4-A shows the change of ABA content; FIG. 4-B shows the change in MDA content.
FIG. 5 shows the change in POD, SOD and CAT activities under drought treatment of transgenic tobacco, wherein FIG. 5-A shows the change in POD activity; FIG. 5-A shows the change in SOD activity; FIG. 5-C shows the change in CAT activity.
FIG. 6 is the relative expression levels of antioxidant genes, polyamine biosynthesis genes and drought response genes under drought treatment of transgenic tobacco, wherein FIG. 6-A is the relative expression level of antioxidant genes; FIG. 6-B shows relative expression levels of polyamine biosynthesis genes; FIG. 6-C is the relative expression levels of drought response genes.
Detailed Description
The invention provides application of PtrMYB119 gene (with the sequence number of Potri.017G125600) of Chinese white poplar in improving the drought tolerance of tobacco. The research of the invention finds that the PtrMYB119 gene of the Chinese hairy poplar is transferred into tobacco leaves, so that the anthocyanin content in the obtained transgenic tobacco is obviously improved, and the drought resistance of the tobacco can be effectively improved.
In the present invention, the application preferably comprises the steps of:
(1) extracting total RNA of the populus trichocarpa, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain a PtrMYB119 gene;
(2) inserting the PtrMYB119 gene into an original vector to obtain a recombinant plant expression vector;
(3) transforming the recombinant plant expression vector into agrobacterium tumefaciens to obtain a recombinant agrobacterium tumefaciens transformant;
(4) infecting the recombinant agrobacterium tumefaciens transformant on tobacco leaves to obtain T0 generation transgenic plants.
The invention firstly extracts the total RNA of the populus trichocarpa, obtains cDNA through reverse transcription, and obtains the PtrMYB119 gene through PCR amplification by taking the cDNA as a template. In the invention, the primer pair for PCR amplification is preferably PtrMYB119-F and PtrMYB 119-R; the nucleotide sequence of the PtrMYB119-F is shown as SEQ ID No. 1; the nucleotide sequence of the PtrMYB119-R is shown in SEQ ID No. 2. In the present invention, the reaction of PCR amplification in step (1)The system should preferably be 20 μ L: 1. mu.L of cDNA template, 0.4. mu.L (10. mu.M) of each of the upstream and downstream primers, 0.1. mu.L of 10 XBuffer 2. mu. L, dNTPs 1.6.6. mu. L, rTaq enzyme, ddH2O14.5. mu.L. The PCR amplification procedure in step (1) is preferably as follows: 94 ℃ for 3 min; 30 cycles of 94 ℃, 30s, 60 ℃, 30s, 72 ℃ and 1min each; final extension at 72 ℃ for 10 min.
After obtaining the PtrMYB119 gene, the invention inserts the PtrMYB119 gene into an original vector to obtain a recombinant plant expression vector. In the present invention, the original vector is preferably pCAMBIA2300, pCAMBIA1300, pCAMBIA1301 or pCAMBIA 3300; more preferably pCAMBIA 2300.
The recombinant plant expression vector is transformed into agrobacterium tumefaciens to obtain a recombinant agrobacterium tumefaciens transformant. In the present invention, the Agrobacterium tumefaciens is preferably Agrobacterium tumefaciens EHA 105. The invention preferably performs positive identification on the recombinant agrobacterium tumefaciens transformant. In the present invention, the positive identification is preferably carried out by picking a white single colony (recombinant Agrobacterium tumefaciens transformant), inoculating it in LB liquid medium containing 100mg/L kanamycin, shake-culturing at 250rpm and 28 ℃ for 4 hours, and PCR-detecting the positive clone with primers PtrMYB119-F and PtrMYB 119-R.
After the recombinant agrobacterium tumefaciens transformant is obtained, the recombinant agrobacterium tumefaciens transformant is infected into a tobacco leaf to obtain a T0 generation transgenic plant. In the present invention, the infestation is preferably carried out using the dipping method: and soaking the cut tobacco leaves into LB liquid culture medium containing the recombinant agrobacterium tumefaciens transformant for 1-3 min. The T0 generation transgenic plant is preferably positively identified by the invention. In the present invention, the positive identification is preferably performed by a staining method or a PCR method.
After transgenic plants of the T0 generation are obtained, the present invention preferably further comprises the step of transplanting the transgenic plants of the T0 generation into a greenhouse.
The related gene PtrMYB119 for anthocyanin biosynthesis is cloned from populus trichocarpa, an overexpression vector is constructed, and then the overexpression vector is transferred into tobacco leaves by an agrobacterium transformation method. The invention is characterized in that the gene is over-expressed in tobacco strains, and the function of the gene is analyzed. The results show that: the over-expression of the PtrMYB119 gene can improve the contents of anthocyanin and ABA in tobacco, the activity of antioxidant enzyme and the expression quantity of related genes, and can reduce the concentration of MDA, so that the drought tolerance of transgenic tobacco is improved.
The invention also provides a recombinant plant expression vector containing the PtrMYB119 gene of the Chinese white poplar and application of a recombinant agrobacterium tumefaciens transformant containing the PtrMYB119 gene of the Chinese white poplar in improving the drought resistance of tobacco. The invention does not specially limit the specific application mode of the recombinant plant expression vector containing the PtrMYB119 gene of the Chinese hairy poplar and the recombinant agrobacterium tumefaciens transformant containing the PtrMYB119 gene of the Chinese hairy poplar.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1:
(1) extracting total RNA of the hairy fruit poplar, carrying out reverse transcription to obtain cDNA, and amplifying the PtrMYB119 gene coding sequence from the poplar cDNA by using a PCR (polymerase chain reaction) technology. The specific process is as follows: the following nucleotide sequences are taken as primers: PtrMYB 119-F: 5'-CTAAGGAAGTGCGTTGAGAA-3', PtrMYB 119-R: 5'-GCCAAGCAACTTGTGTAGTC-3', PCR amplification was performed. The amplification conditions were 94 ℃ for 3 min; 30 cycles of 94 ℃, 30s, 60 ℃, 30s, 72 ℃ and 1min each; final extension at 72 deg.C for 10 min; the reaction system is 20 μ L: 1. mu.L of cDNA template, 0.4. mu.L (10. mu.M) of each of the upstream and downstream primers, 0.1. mu.L of 10 XBuffer 2. mu. L, dNTPs 1.6.6. mu. L, rTaq enzyme, ddH2O14.5. mu.L. And (3) performing agarose gel electrophoresis on the PCR product, performing gel cutting recovery by using a gel extraction kit, and storing the recovered product at the temperature of-20 ℃ after the concentration of the recovered product is determined, or directly performing reactions such as enzyme digestion, connection and the like.
(2) The obtained PtrMYB119 gene is inserted into a vector pCAMBIA2300 to obtain a plant expression vector pCAMBIA2300-PtrMYB 119. The specific process is as follows: the amplification product of the gene PtrMYB19 obtained in step 1 was cloned into an overexpression vector pCAMBIA2300 (kanamycin-resistant) by a Gateway recombination system to obtain a Plant expression vector pCAMBIA2300-PtrMYB119 (reference: Karimi, M., Inze, D., Depicker, A., Gateway vectors for Agrobacterium-mediated transformation. trends Plant Sci.2002 May:7(5): 193-195).
(3) The resulting plant expression vector was transformed into Agrobacterium tumefaciens EHA 105. The specific process is as follows:
firstly, taking agrobacterium tumefaciens competent cells stored at the temperature of minus 80 ℃, receiving a palm piece, melting part of the cells, and inserting the cells into ice when the cells are in an ice-water mixed state;
secondly, adding 0.01-1.0 mu g of plasmid into 100 mu L of competent cells, tapping the bottom of the tube with fingers, mixing uniformly, and standing for 5min on ice, in liquid nitrogen, in a water bath at 37 ℃ and in an ice bath in sequence;
thirdly, adding a sterile LB liquid culture medium, and performing shake culture at 28 ℃ for 2-3 hours;
fourthly, centrifuging at 6000rpm for 1min to collect bacteria, taking 50 mu L of supernatant, slightly blowing and beating the heavy suspension bacteria block, coating the heavy suspension bacteria block on an LB (Langmuir-Blodgett) flat plate containing 100mg/L kanamycin, and inversely placing the heavy suspension bacteria block on a shaking table at 28 ℃ for culturing for 2-3 days.
Detecting positive clones: and (3) selecting a white single colony, inoculating the white single colony in an LB liquid culture medium containing 100mg/L kanamycin, shaking the colony at 250rpm and 28 ℃ for culture for 4 hours, carrying out PCR (polymerase chain reaction) detection on positive clones by using primers, and after identifying, shaking the colony for later use in transformation of K326 common tobacco.
(4) And infecting flue-cured tobacco K326 tobacco leaves with the obtained agrobacterium transformant to obtain T0 generation transgenic plants. The specific process is as follows: the correctly identified Agrobacterium transformant pCAMBIA2300-PtrMYB119 was inoculated into 30mL of LB liquid medium (containing 50mg/mL kanamycin) and cultured in a shaker at 28 ℃ until OD600And (3) 0.5-0.8, centrifugally collecting thalli, re-suspending the agrobacterium with 30ml of transformation medium MSo, transforming tobacco leaves by adopting a soaking method, taking fresh and tender leaves, cutting into small pieces, and soaking in an agrobacterium culture medium for about two minutes. Taking out the leaves, placing the leaves on an MS solid culture medium without antibiotics for dark culture at 28 ℃ for 2-3 days, transferring the leaves to an MS differentiation culture medium containing kanamycin after 2-3 days for waiting for differentiation, changing the culture medium once a week, and detecting after resistant buds grow out from the leaves after the leaves are differentiated.
Identification of transgenic plants:
firstly, dyeing and identifying: cutting 0.2cm leaf of the test-tube seedling, placing in GUS dye solution, and dyeing at 37 deg.C for more than 12 hr. Then, absolute ethyl alcohol is used for decoloring. The blue color of the leaf indicates the successful integration of the reporter gene into the plant genome, and the plant is a transgenic plant (FIG. 1A).
Secondly, PCR identification: PCR verification of the transgenic plants was performed on blue-stained plants using a PCR kit produced by Baori Biotechnology (Beijing) Ltd. (FIG. 1B). The following nucleotide sequences are taken as primers: PtrMYB 119-F: 5'-CTAAGGAAGTGCGTTGAGAA-3', PtrMYB 119-R: 5'-GCCAAGCAACTTGTGTAGTC-3', performing PCR amplification under the conditions of 94 ℃ for 3 min; 30 cycles of 94 ℃, 30s, 60 ℃, 30s, 72 ℃ and 1min each; final extension at 72 deg.C for 10 min; the reaction system is 20 μ L: 1. mu.L of DNA template, 0.4. mu.L (10. mu.M) of each of the upstream and downstream primers, 0.1. mu.L of 10 XBuffer 2. mu. L, dNTPs 1.6.6. mu. L, rTaq enzyme, ddH2O14.5μL。
(5) And transplanting the transgenic plant into a greenhouse to obtain the transgenic tobacco with improved anthocyanin content and improved drought resistance. The specific process is as follows: three over-expressing transgenic lines (A, B and C) each having 5 plants, and wild-type plants were selected for drought treatment. After the plants are finely cultured in a tissue culture bottle for 20 days, the plants are placed in a light incubator for hardening seedlings for 7 days, and the plants are transplanted into a plastic flowerpot in a greenhouse for culture after the hardening seedlings are finished. Greenhouse conditions: the temperature is 28 ℃, and the light-dark period is 16h/8 h. After the plants are fully watered and the growth vigor of the plants is consistent, drought treatment is started, sampling and photographing are carried out once every 10 days, and 3 times of continuous sampling are carried out.
(6) The method for measuring the anthocyanin content and the drought resistance of the transgenic tobacco comprises the following steps:
the transgenic tobacco overexpressing PtrMYB119 and wild tobacco are used as controls, the anthocyanin content in the plant body is measured through spectrophotometer emission, and the result shows that: under drought treatment, the anthocyanin content of the transgenic tobacco overexpressing PtrMYB119 is obviously higher than that of wild tobacco.
Under drought treatment, the drought resistance of the transgenic tobacco is verified by methods of observing phenotypes, measuring root length, leaf wilting ratio and stem cross section, measuring ABA and MDA contents, measuring antioxidant enzyme activity, measuring relative expression of antioxidant genes, polyamine biosynthesis genes and drought response genes and the like, and the results are shown in FIGS. 2 to 6. Fig. 2 to 6 show that: compared with wild tobacco, the transgenic tobacco can be remarkably improved in drought resistance by over-expressing PtrMYB 119.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of plant of Chinese academy of sciences of Jiangsu province
Application of PtrMYB119 gene of populus tomentosa in improvement of drought resistance of tobacco
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gccaagcaac ttgtgtagtc 20
Claims (10)
1. The PtrMYB119 gene of populus tomentosa is applied to the improvement of the content of abscisic acid in tobacco and the promotion of the expression of tobacco polyamine biosynthesis genes and drought response genes; the polyamine biosynthesis genes are ERD10D, ADC1 and SAMDC; the drought response genes are NCED3 and NAC/RD 26.
2. Use according to claim 1, characterized in that it comprises the following steps:
(1) extracting total RNA of the populus trichocarpa, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain a PtrMYB119 gene;
(2) inserting the PtrMYB119 gene into an original vector to obtain a recombinant plant expression vector;
(3) transforming the recombinant plant expression vector into agrobacterium tumefaciens to obtain a recombinant agrobacterium tumefaciens transformant;
(4) infecting the recombinant agrobacterium tumefaciens transformant on tobacco leaves to obtain T0 generation transgenic plants.
3. The use of claim 2, wherein the primer pair for PCR amplification in step (1) is PtrMYB119-F and PtrMYB 119-R; the nucleotide sequence of the PtrMYB119-F is shown as SEQ ID No. 1; the nucleotide sequence of the PtrMYB119-R is shown in SEQ ID No. 2.
4. The use of claim 3, wherein the reaction system of the PCR amplification of step (1) is 20 μ L: mu.L of cDNA template, 0.4. mu.L of each of 10. mu.M upstream and downstream primers, 0.1. mu.L of 10 XBuffer 2. mu. L, dNTPs 1.6.6. mu. L, rTaq enzyme, and 14.5. mu.L of ddH 2O.
5. The use of claim 4, wherein the PCR amplification procedure of step (1) is as follows: 94 ℃ for 3 min; 30 cycles of 94 ℃, 30s, 60 ℃, 30s, 72 ℃ and 1min each; final extension at 72 ℃ for 10 min.
6. The use of claim 2, wherein the original vector of step (2) is pCAMBIA2300, pCAMBIA1300, pCAMBIA1301 or pCAMBIA 3300.
7. The use of claim 2, wherein said transformation of step (3) and/or said transfection of step (4) is followed by a positive identification step.
8. The use of claim 2, wherein the transgenic plant of T0 generation obtained in step (4) further comprises the step of transplanting the transgenic plant of T0 generation into a greenhouse.
9. The application of the recombinant plant expression vector containing the PtrMYB119 gene of the populus tomentosa in improving the content of the tobacco abscisic acid and promoting the expression of tobacco polyamine biosynthesis genes and drought response genes; the polyamine biosynthesis genes are ERD10D, ADC1 and SAMDC; the drought response genes are NCED3 and NAC/RD 26.
10. The recombinant agrobacterium tumefaciens transformant containing the PtrMYB119 gene of the populus trichocarpa is applied to the improvement of the content of the tobacco abscisic acid and the promotion of the expression of tobacco polyamine biosynthesis genes and drought response genes; the polyamine biosynthesis genes are ERD10D, ADC1 and SAMDC; the drought response genes are NCED3 and NAC/RD 26.
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