CN112322635B - Coding sequence of larch growth and development regulation gene and application thereof - Google Patents
Coding sequence of larch growth and development regulation gene and application thereof Download PDFInfo
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Abstract
The invention discloses a coding sequence of a larch growth and development regulation gene and a method for cultivating fast-growing and high-yield transgenic plants by using the coding sequence of the gene, which comprises the following steps: extracting larch total RNA; synthesizing cDNA by reverse transcription of larch RNA; designing and synthesizing an amplification primer with enzyme cutting sites for the growth and development regulation gene of the larch; constructing an overexpression vector; genetic transformation; screening transgenic positive seedlings; and observing the growth and development traits of the transgenic plants. The observation results show that: compared with wild plants, the transgenic plants have obvious characteristics of fast growth and high yield, and the biomass of the overground parts of the transgenic plants is more than 20 percent higher than that of the wild plants under the same growth conditions. The method for cultivating fast-growing and high-yield transgenic plants by adopting the coding sequence of the larch growth and development regulation and control gene disclosed by the invention has important value for cultivating fast-growing and high-yield crops or economic plants such as forest trees and the like by adopting a genetic engineering means with high efficiency and low cost.
Description
Technical Field
The present invention relates to the field of biotechnology, and is especially the coding sequence of larch growth and development regulating gene and its application in breeding fast-growing and high-yield transgenic plant.
Background
Larch is a larch tree of larch genus of Pinaceae family, mainly distributed in cold and high mountainous areas, has good wood hardness, high compressive and bending strength and decay resistance, and has important application value in the aspects of building construction, civil engineering, processing and producing wood fiber industrial raw materials and the like. Meanwhile, the larch is tall and straight, has a developed root system, has strong adaptability to adverse environments such as cold and drought, has important environmental protection significance for afforestation and water and soil conservation in mountainous areas, and is an important afforestation tree species. In addition, the larch can be used as a raw material to prepare arabinogalactan and the like, and the method is widely applied to the fields of medicines and foods. However, larch grows slowly and has a long success period, and generally, at least 15 years are needed from the planting of the larch to the harvest and utilization. Therefore, although larch has economic and greening values, the slow growth of larch becomes the most important bottleneck for improving the comprehensive utilization value of larch. Hybrid breeding is a main means for obtaining fast-growing larch for a long time, but the larch has long growth period, huge genome and very complex genetic background, and is difficult to obtain hybrid seeds with excellent comprehensive properties by different hybrid combination screening like crops such as rice and the like and is used for afforestation practice. With the development of modern biotechnology, the separation and cloning of key genes related to the formation and regulation of the fast-growing traits of plants, and the cultivation of plants with high yield and fast-growing traits by means of genetic engineering such as transgenosis and the like are important directions for molecular breeding of agricultural and forestry crops. The method can realize rapid improvement or interference on the genetic characters of the agricultural and forestry crops, greatly reduce the cultivation period of the high-yield and high-quality agricultural and forestry crops, and has important application value and potential in the aspects of improving the scientific and technological level of agriculture in China and ensuring national food and ecological safety.
Disclosure of Invention
The present invention aims at providing a coding sequence of larch growth and development regulating gene, and provides a method for obtaining fast-growing and high-yield transgenic plants by using the coding sequence of the gene and adopting a genetic engineering means and an application thereof. In order to achieve the purpose, the invention discloses the following technical scheme:
<xnotran> , 5 'ATGGATTTCGAGATCGGACAACACTTGCGAGAAAGGTTTATCTCCATTCTTACTTAGTGCAACTTGCGAGGTTAAATGAGACTTGTGAGGTGGCACAGCGTCAAATCCACTGAGAATGCTAAACTCAATTTTCTGCGCCCAATGTTTTTAAATGATGCTGGCGGGCAGAAACTCTGGAACCAAGCTTAGGTTCTTCAAATGTATCTGGATGCAGCTCTTCAGAAGAGTCGAAGCCACAGGTAAAATTCAGGACATGAAATCTAGCGCAGGAAACGTCAAAGGAGCAAGCAATTTGATCATCAGGAACCATGGTTGATGCGTGGTGTTTATTTCAAAAACATGAAATGGCAAGCAGCCATCAAGCTCGAAAAGACACAAACTCATCTGTGAACAGTGCGTTCGCAAGAAGAGGCAGCTCATCTACATGATCGGGCAGCATTCATGTGTGGAAGGGAACCAAATTTTGAGCTTTCAGAAGAGGAGAAACAGGAACTCAGTCGCTTCAGCTGGGAACTGTTCCTCGAAATAACACGCAATGCAATTACAAGCAAAAAACACCAGATGAAGCTTGAGGCAAGAATGCGGAAAAAGCAGCTCAATCACGCTCAGGCCAATCAACAAGCAGTTAAGGCACAGGCACCTACAGCGCAGACAGCAGGGCACGAAGGATCCCAATTCTGA 3' (SEQ ID NO: 1). </xnotran>
The invention further discloses a method for cultivating fast-growing and high-yield transgenic plants by using the gene coding sequence, which comprises the following steps:
(1) Extracting larch total RNA: the method comprises the steps of purchasing larch seeds in a larch fine-breed forest farm seed garden of Qingyuan county of Fushun city of Liaoning province, processing the seeds at a low temperature of 4 ℃ for 14 days through a refrigerator, soaking the seeds in warm water at 40 ℃ for 12 hours, then sowing the seeds in moist sandy soil, harvesting overground parts of larch seedlings after 30 days, quickly freezing the seeds in liquid nitrogen, putting the larch seedling materials subjected to quick freezing in the liquid nitrogen into a mortar, adding the liquid nitrogen for grinding until the materials are ground into powder, quickly transferring 200 mg of the larch materials ground into the powder into a 2ml centrifuge tube, adding 700 mu L of lysis solution containing 5% CTAB +2% PVP +100 mmol/L Tris-HCl +2 mol/L NaCl +2% beta-mercaptoethanol +0.5% SDS, incubating in a water bath at 65 ℃ for 20 min, and shaking 5 times during the incubation. And then adding 700 mu L of water saturated phenol into the centrifuge tube, centrifuging at 12000 rpm for 10 min at room temperature, sucking the supernatant, adding chloroform with the same volume, slightly and uniformly mixing, centrifuging at 12000 rpm for 10 min at room temperature, sucking the supernatant, adding isopropanol with the same volume, slightly shaking, settling at-20 ℃, standing for 50 min, centrifuging at 12000 rpm for 10 min, and collecting the precipitate. Finally, 1 mL of precooled 75% ethanol is added for rinsing for 2 times, and each time is 500 muL; ethanol was discarded to obtain RNA precipitate, which was dissolved in 50. Mu.L of sterile DEPC water without RNase after natural drying. And taking 43 mu L of the RNA solution, adding 1 mu L of DNase I, 5 mu L of 10 XDNase I buffer solution and 1 mu L of RNase Inhibitor into the RNA solution, and carrying out water bath heat preservation at 37 ℃ for 30 min. And then adding 50 muL of chloroform into the reaction liquid, shaking for 3 times to enable the reaction liquid to be fully contacted with the chloroform, centrifuging for 5min at 12000 rpm at 4 ℃, taking the supernatant, transferring the supernatant into a new centrifuge tube, adding isopropanol with the same volume into the centrifuge tube, standing for 30 mm at-20 ℃, centrifuging for 5min at 12000 rpm at 4 ℃, discarding the supernatant, rinsing the precipitate with 75% ethanol for 2 times, completely sucking the liquid with a gun head, drying the RNA precipitate at room temperature, adding 30 muL to 50 muL of DEPC (diethyl phthalate) water to dissolve, and storing at-80 ℃.
(2) Synthesizing cDNA by reverse transcription of larch RNA: adding 2 mu g of larch RNA and Oligo d (T) into a 0.2 mL centrifuge tube in sequence 10 1 μ L (500 ng/. Mu.L) of RNA-free water and 1 μ L of RNA-free water; mixing, centrifuging for 5min at 75 deg.C, and immediately placing on ice for 5min; sequentially adding M-MLV (5 x Buffer) 6 muL, dNTP (2.5 muL), RNase Inhibitor (40U/muL) 1.25 muL, M-MLV (murine reverse transcriptase) (200U/muL) 2 muL and supplementing to 30 muL; lightly mixing, and keeping at 37 deg.C for 5 h; the enzyme activity was inactivated at 75 ℃ for 15 min and stored at-20 ℃.
(3) Designing and synthesizing an amplification primer with enzyme cutting sites for the larch growth and development regulation gene:
Larix-G-S:5'CCATGGATGGATTTCGAGATCGGACAA 3' (underlined indicatesNcoI cleavage site) (SEQ ID NO: 2) (ii) a
Ext> Larixext> -ext> Gext> -ext> Aext>:ext> 5 'GGTCACCTCCAGAATTGGGATCCTTCGTG 3' (underlined showsBstEII cleavage site) (SEQ ID NO: 3).
The primer sequence was synthesized by Shanghai bioengineering company.
(4) Construction of overexpression vector: the pCAMBIA3301 plasmid was usedNcoI andBstEII double digestion, 25. Mu.L digestion system containing 2. Mu.g (10. Mu.L) of plasmid, 10 XT buffer 2. Mu.L,Nco I（30 U/μL）1.0 μL、BstEII (100U/. Mu.L) 1.0. Mu.L, sterile water 11. Mu.L, and incubation at 37 ℃ for 6 h, followed by recovery of the cleaved pCAMBIA3301 vector using 0.8% agarose gel. By the same method for the components containingLarix Gmelini growth and development regulating gene obtained by amplification in step (3) andNcoi andBstand carrying out double enzyme digestion on the TA cloning vector of the EII site sequence, and recovering an enzyme digestion product. Connecting the larch growth and development regulation gene fragment with the same restriction enzyme cutting site with a recovered product of pCAMBIA3301 plasmid, wherein the connection system is as follows: to a 0.2 mL centrifuge tube were added 5. Mu.L of pCAMBIA3301 plasmid, 3.5. Mu.L of the target fragment, 1. Mu.L of 10 Xligase buffer, 0.5. Mu.L of Ligase and sterile water to make up 10. Mu.L, and the mixture was incubated at 16 ℃ for 16 hours in a water bath. The ligation products were transformed into E.coli DH 5. Alpha. Competent cells by heat shock method, spread on LB plate containing 150 mg/L Kan, and cultured overnight at 37 ℃. The positive clone is picked up and is subjected to amplification culture in an LB liquid culture medium containing 150 mg/L Kan, a positive recombinant plasmid is extracted and is transferred into agrobacterium LBA4404. The transformed Agrobacterium were spread on YEB plates (containing 100 mg/L Rif (rifampicin), 120mg/L Str (streptomycin) and 150 mg/L Kan (kanamycin)); culturing at 28 deg.C for 2d in inverted state. White clones were picked up in 1 mL YEB medium (containing 80mg/L Rif, 120mg/L Str and 150 mg/L Kan), cultured at 28 ℃ and 220 rpm for 1-2 days. Extracting agrobacterium tumefaciens positive plasmids for double enzyme digestion identification; and adding 500 mu L of the identified positive strain bacterial liquid into 500 mu L of 40% glycerol YEB culture medium, shaking, uniformly mixing, and storing at-20 ℃ for later use.
(5) Genetic transformation: inoculating the frozen agrobacterium tumefaciens liquid containing the recombinant plasmid into 5 mL YEB liquid culture medium containing 100 mg/L Rif, 120mg/L Str and 120mg/L Kan, and activating at 28 ℃ and 220 rpm for 1-2 d; inoculating 2.5 mL of activated bacterial liquid into 250 mL of Kan containing 80mg/L of Rif, 120mg/L of Str and 120mg/L of Kan, culturing at 28 ℃ and 220 rpm with shaking until OD 600 1.2 to 1.6;4000 Centrifuging at rpm for 10 min, collecting thallus, suspending the thallus in 500 mL of 5% sucrose solution, and mixing the transformed bacteria solution. Arabidopsis thaliana was genetically transformed by the floral dip method.
(6) Screening transgenic positive seedlings: harvesting transgenic Arabidopsis T 0 Seed generation, vernalization in refrigerator at 4 deg.C for 3d, sowing in nutrient soil, diluting 10% Basta mother liquor with tap water by 10000 times when Arabidopsis seedling grows to obtain true leaf, spraying twice a dayOne week later, the positive transformation strain containing the target gene grows normally, and the arabidopsis thaliana plant seedlings which are not successfully transformed gradually yellow and stop growing; transferring the positive seedling to newly sterilized nutrient soil for culture, and recording as T 1 And extracting DNA of the leaves to perform PCR molecular identification.
(7) And (3) observing growth and development traits of the transgenic plants: for T 3 The growth and development characters of the purified transgenic plants of the generation and the following generations are observed: under the same growth environment conditions, the transgenic plant has obvious fast growth characteristic compared with a wild type, and the overground part of the transgenic plant has the biomass more than 20 percent higher than that of a control.
The invention addresses the need for new varieties of fast-growing, high-yielding plants in breeding new varieties of crops or energy plants.
The larch RNA extraction lysate refers to a mixed liquid containing 5% CTAB, 2% PVP, 100 mmol/L Tris-HCl, 2 mol/L NaCl, 2% beta-mercaptoethanol and 0.5% SDS, and the mixed liquid needs to be prepared for use and cannot be stored for a long time.
The invention also discloses the application of the gene coding sequence (SEQ ID NO: 1) in intervening the growth and development of plants, in particular the formation of fast-growing and high-yield traits. The research result shows that: the transgenic plant has obvious fast-growing characteristic compared with a wild type, and the aboveground part biomass of the transgenic plant is more than 20 percent higher than that of the wild type under the same growth condition, which shows that the coding sequence of the larch growth and development regulation gene disclosed by the invention can play an important role in cultivating the fast-growing and high-yield transgenic plant.
Compared with the prior art, the method for obtaining the fast-growing and high-yield transgenic plant by utilizing the gene coding sequence and adopting a genetic engineering means has the positive effects that:
(1) The operation process is simpler, and the method can be carried out in a common laboratory only provided with simple molecular biological equipment and instruments;
(2) The cost is low, the applicability is strong, the method can be developed in gymnosperms such as larch and most dicotyledonous plants, and the industrial problem that the cultivation of the fast-growing and high-volume larch fine varieties is difficult is solved;
(3) High transformation efficiency, and can realize the acquisition of a large number of transgenic plants with the characteristics of fast growth and high yield in a short time.
Drawings
FIG. 1 shows the result of electrophoresis detection of RNA extraction from Larix Gmelini;
FIG. 2 (A, B, C, D) shows the construction result of the over-expression vector of the Larix Gmelini growth and development regulatory gene coding sequence. Wherein FIG. 2A shows the result of PCR amplification of the growth and development regulatory gene of Larix Gmelini of the present invention; FIG. 2B shows the result of double digestion of pCAMBIA3301 vector; FIG. 2C shows the double digestion result of the cloned larch growth and development regulatory gene TA; FIG. 2D shows the detection result of the combined primers after the growth and development regulatory gene of Larix Gmelini is connected with pCAMBIA3301 to form a recombinant expression vector;
FIG. 3 shows the result of screening positive transformants of transgenic Arabidopsis thaliana over-expressed by the growth and development regulatory gene of Larix Gmelini; in the figure, a green plant is a positive transformant obtained by Basta resistance screening;
FIG. 4 shows the molecular identification results of transgenic Arabidopsis transformants with the genes for regulating the growth and development of Larix Gmelini; the result in the figure is that the primer sequence on the pCAMBIA3301 carrier and the primer sequence of the transferred target gene are used for carrying out combined primer PCR amplification, if an amplification band with expected size is detected in a transformant, the target gene is successfully transferred into a plant, and lanes 5-14 in the figure show that the target gene is transferred into the plant;
FIG. 5 shows the observation result of the growth and development traits of transgenic Arabidopsis thaliana overexpressed from the Larix Gmelini growth and development regulatory gene.
Detailed Description
These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al, molecular cloning: conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989). All biochemical reagents, enzymes, vectors and strains can be purchased from various biological reagent companies, and primer sequences are synthesized by Shanghai bioengineering companies.
The sources of biochemical reagents, enzymes and the like required by the present invention are shown in the following table:
example 1
<xnotran> , 5 'ATGGATTTCGAGATCGGACAACACTTGCGAGAAAGGTTTATCTCCATTCTTACTTAGTGCAACTTGCGAGGTTAAATGAGACTTGTGAGGTGGCACAGCGTCAAATCCACTGAGAATGCTAAACTCAATTTTCTGCGCCCAATGTTTTTAAATGATGCTGGCGGGCAGAAACTCTGGAACCAAGCTTAGGTTCTTCAAATGTATCTGGATGCAGCTCTTCAGAAGAGTCGAAGCCACAGGTAAAATTCAGGACATGAAATCTAGCGCAGGAAACGTCAAAGGAGCAAGCAATTTGATCATCAGGAACCATGGTTGATGCGTGGTGTTTATTTCAAAAACATGAAATGGCAAGCAGCCATCAAGCTCGAAAAGACACAAACTCATCTGTGAACAGTGCGTTCGCAAGAAGAGGCAGCTCATCTACATGATCGGGCAGCATTCATGTGTGGAAGGGAACCAAATTTTGAGCTTTCAGAAGAGGAGAAACAGGAACTCAGTCGCTTCAGCTGGGAACTGTTCCTCGAAATAACACGCAATGCAATTACAAGCAAAAAACACCAGATGAAGCTTGAGGCAAGAATGCGGAAAAAGCAGCTCAATCACGCTCAGGCCAATCAACAAGCAGTTAAGGCACAGGCACCTACAGCGCAGACAGCAGGGCACGAAGGATCCCAATTCTGA 3' (SEQ ID NO: 1). </xnotran>
The method for cultivating fast-growing and high-yield transgenic plants by using the gene coding sequence comprises the following steps:
(1) Extracting larch total RNA: the method comprises the steps of purchasing larch seeds in a seed garden of a fine larch forest of Qingyuan county, fushun, liaoning, soaking the seeds in warm water at 40 ℃ for 12 hours after the seeds are subjected to low-temperature treatment for 14 days in a refrigerator at 4 ℃, then sowing the seeds in moist sandy soil, harvesting overground parts of larch seedlings in a collecting pipe after 30 days, quickly freezing the seeds by liquid nitrogen, putting the larch seedling materials subjected to quick freezing by the liquid nitrogen into a mortar, adding the liquid nitrogen into the mortar for grinding until the materials are ground into powder, quickly transferring 200 mg of the larch materials ground into the powder into a 2ml centrifuge tube, adding 700 mu L of a lysis solution containing 5% CTAB, 2% PVP, 100 mmol/L Tris-HCl, 2 mol/L NaCl, 2% beta-mercaptoethanol and 0.5% SDS, incubating in a water bath at 65 ℃ for 20 min, and shaking 5 times in the period. Then adding 700 mu L of water saturated phenol into the centrifuge tube, centrifuging at 12000 rpm at room temperature for 10 min, sucking supernatant, adding isometric chloroform, gently mixing uniformly at 12000 at room temperatureCentrifuging at rpm for 10 min, sucking supernatant, adding equal volume of precooled isopropanol, shaking gently, settling at-20 deg.C, standing for 50 min, centrifuging at 12000 rpm for 10 min, and collecting precipitate. Finally, 1 mL of precooled 75% ethanol is added for rinsing for 2 times, and each time is 500 muL; ethanol was discarded to obtain RNA precipitate, which was dissolved in 50. Mu.L of sterile DEPC water without RNase after natural drying. And taking 43 muL of the RNA solution, adding 1 muL of DNase I, 5 muL of 10 XDNase I buffer solution and 1 muL of RNase Inhibitor into the RNA solution, and preserving the temperature in a water bath at 37 ℃ for 30 min. Subsequently, adding 50 mu L of chloroform into the reaction liquid, shaking for 3 times to enable the reaction liquid to be fully contacted with the chloroform, centrifuging for 5min at 12000 rpm at 4 ℃, taking the supernatant, transferring the supernatant into a new centrifuge tube, adding isopropanol with the same volume into the centrifuge tube, standing for 30 mm at-20 ℃, centrifuging for 5min at 12000 rpm at 4 ℃, discarding the supernatant, rinsing the precipitate with 75% ethanol for 2 times, completely sucking the liquid with a gun head, drying the RNA precipitate at room temperature, dissolving with 30 mu L to 50 mu L of DEPC water, taking 2 mu L of RNA solution, detecting the integrity of the RNA by 1.0% agarose gel electrophoresis, taking 1 mu L of RNA, and utilizing NanDrop ® ND-1000 nucleic acid analyzer, OD value of extracted RNA is determined, and qualified RNA (figure 1) is stored at-80 deg.C.
(2) Reverse transcription of larch RNA to synthesize cDNA: adding 2 mu g of larch RNA and Oligo d (T) into a 0.2 mL centrifuge tube in sequence 10 (800 ng/. Mu.L) 1. Mu.L, RNA-free water 1. Mu.L; mixing, centrifuging for 5min at 75 deg.C, and immediately placing on ice for 5min; sequentially adding M-MLV (5 x Buffer) 6 muL, dNTP (2.5 muL), RNase Inhibitor (40U/muL) 1.25 muL, M-MLV (murine reverse transcriptase) (200U/muL) 2 muL and supplementing to 30 muL; mixing gently, and keeping at 37 deg.C for 5 hr; inactivating enzyme activity at 75 deg.C for 15 min, and storing at-20 deg.C.
(3) Designing and synthesizing an amplification primer with enzyme cutting sites for the larch growth and development regulation gene:
Larix-G-S：5’CCATGGATGGATTTCGAGATCGGACAA 3' (underlined representation)NcoI cleavage site) (SEQ ID NO: 2) (ii) a
Larix-G-A：5’GGTCACCTCAGAATTGGGATCCTTCGTG 3' (underlined representation)BstEII cleavage site) (SEQ ID NO: 3).
The primer sequence was synthesized by Shanghai bioengineering.
(4) Construction of overexpression vector: the pCAMBIA3301 plasmid was usedNcoI andBstEII double digestion, 25. Mu.L digestion system contains 2. Mu.g (10. Mu.L) of plasmid, 2. Mu.L of 10 XT buffer,Nco I（30U/μL）1.0 μL、BstEII (100U/. Mu.L) 1.0. Mu.L, sterile water 11. Mu.L, and incubation at 37 ℃ for 6 h, followed by recovery of the cleaved pCAMBIA3301 vector using 0.8% agarose gel. Using the same method for the gene containing the growth and development regulator of Larix Gmelini amplified in the step (3) andNcoi andBstTA cloning vector sequence of EII site sequenceNcoI andBstEII double-enzyme digestion and double-enzyme digestion, and recovering the digestion product (figure 2). Connecting the larch growth and development regulation gene fragment with the same restriction enzyme cutting site with a recovered product of pCAMBIA3301 plasmid, wherein the connection system is as follows: to a 0.2 mL centrifuge tube, 5. Mu.L of pCAMBIA3301 plasmid, 3.5. Mu.L of the target fragment, 1. Mu.L of 10 Xligase buffer, 0.5. Mu.L of Ligase and sterile water were added to make up to 10. Mu.L, and the mixture was incubated in a 16 ℃ water bath for 16 hours. The ligation products were transformed into E.coli DH 5. Alpha. Competent cells by heat shock method, spread on LB plate containing 150 mg/L Kan, and cultured overnight at 37 ℃. The positive clone is picked up, and is enlarged and cultured in LB liquid culture medium containing 150 mg/L Kan, the positive recombinant plasmid is extracted and is transferred into agrobacterium LBA4404. The transformed Agrobacterium was spread on YEB plates (containing 100 mg/L Rif (rifampicin), 120mg/L Str (streptomycin) and 150 mg/L Kan (kanamycin)); culturing at 28 deg.C for 2d in inverted state. White clones were picked up in 1 mL YEB medium (containing 80mg/L Rif, 120mg/L Str and 150 mg/L Kan), cultured at 28 ℃ and 220 rpm for 1-2 days. Extracting positive plasmid of agrobacteriumNcoI andBstEII double enzyme digestion identification; adding 500 mul of identified positive strain bacterial liquid into 500 mul of 40% glycerol YEB culture medium, shaking and mixing uniformly, and storing at-20 ℃ for later use;
(5) Genetic transformation: inoculating the cryopreserved positive agrobacterium tumefaciens strain liquid containing the larch growth and development regulating gene sequence amplified in the step (3) into 5 mL YEB liquid culture medium containing 100 mg/L Rif, 120mg/L Str and 120mg/L Kan, and 2Activating at 8 ℃ and 220 rpm for 1-2 d; inoculating 2.5 mL of activated bacterial liquid into 250 mL of Kan containing 80mg/L of Rif, 120mg/L of Str and 120mg/L of Kan, culturing at 28 ℃ and 220 rpm with shaking until OD 600 Is 1.6;4000 And centrifuging at the rpm for 10 min to collect thalli, suspending the thalli in 500 mL of 5% sucrose solution, and uniformly mixing the transformed bacteria solution. Genetically transforming arabidopsis by adopting a floral dip method;
(6) Screening transgenic positive seedlings: harvesting of transgenic Arabidopsis T 0 The seeds are used for generation, the seeds are sown in nutrient soil after being vernalized in a refrigerator at 4 ℃ for 3d, when the arabidopsis seedlings grow out of true leaves, 10 percent Basta mother liquor is diluted by 10000 times by tap water and then sprayed, and the spraying is carried out twice a day. After one week, the positive transformation strain containing the target gene grows normally, and the arabidopsis thaliana plant seedlings which are not successfully transformed gradually yellow and stop growing (figure 3); transferring the positive seedling to newly sterilized nutrient soil for culture, and recording as T 1 Next, DNA from leaves was extracted and subjected to PCR molecular characterization (FIG. 4).
(7) And (3) observing growth and development traits of the transgenic plants: for T 3 And (3) observing the growth and development characters of the purified transgenic plants of the generation and the following generations:
and (4) conclusion: under the same growth environment conditions, the transgenic plants have obvious fast-growing characteristics compared with wild type, and the overground part of the organisms of the transgenic plants are more than 20 percent higher than that of a control (figure 5).
SEQUENCE LISTING
<110> university of southern kayak
<120> coding sequence of Larix Gmelini growth and development regulatory gene and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 681
<212> DNA
<213> Artificial sequence
<400> 1
atggtcagca ttcgaaggcg caaacatttg gaaaggttta tctccattct tacttagtgc 60
aacttgcgag gttaaatgag acttgtgagg tggcacagcg tcaaatccac tgagaatgct 120
aaactcaatt ttctgcgccc aatgttttta aatgatgctg gcgggcagaa actctggaac 180
caagcttagg ttcttcaaat gtatctggat gcagctcttc agaagagtcg aagccacagg 240
taaaattcag gacatgaaat ctagcgcagg aaacgtcaaa ggagcaagca atttgatcat 300
caggaaccat ggttgatgcg tggtgtttat ttcaaaaaca tgaaatggca agcagccatc 360
aagctcgaaa agacacaaac tcatctgtga acagtgcgtt cgcaagaaga ggcagctcat 420
ctacatgatc gggcagcatt catgtgtgga agggaaccaa attttgagct ttcagaagag 480
gagaaacagg aactcagtcg cttcagctgg gaactgttcc tcgaaataac acgcaatgca 540
attacaagca aaaaacacca gatgaagctt gaggcaagaa tgcggaaaaa gcagctcaat 600
cacgctcagg ccaatcaaca agcagttaag gcacaggcac ctactgtgca gtcagcagat 660
gcaggtgtat caaatttctg a 681
<210> 2
<211> 27
<212> DNA
<213> Artificial sequence
<400> 2
ccatggatgg atttcgagat cggacaa 27
<210> 3
<211> 28
<212> DNA
<213> Artificial sequence
<400> 3
ggtcacctca gaattgggat ccttcgtg 28
Claims (4)
1. <xnotran> , 5 'ATGGATTTCGAGATCGGACAACACTTGCGAGAAAGGTTTATCTCCATTCTTACTTAGTGCAACTTGCGAGGTTAAATGAGACTTGTGAGGTGGCACAGCGTCAAATCCACTGAGAATGCTAAACTCAATTTTCTGCGCCCAATGTTTTTAAATGATGCTGGCGGGCAGAAACTCTGGAACCAAGCTTAGGTTCTTCAAATGTATCTGGATGCAGCTCTTCAGAAGAGTCGAAGCCACAGGTAAAATTCAGGACATGAAATCTAGCGCAGGAAACGTCAAAGGAGCAAGCAATTTGATCATCAGGAACCATGGTTGATGCGTGGTGTTTATTTCAAAAACATGAAATGGCAAGCAGCCATCAAGCTCGAAAAGACACAAACTCATCTGTGAACAGTGCGTTCGCAAGAAGAGGCAGCTCATCTACATGATCGGGCAGCATTCATGTGTGGAAGGGAACCAAATTTTGAGCTTTCAGAAGAGGAGAAACAGGAACTCAGTCGCTTCAGCTGGGAACTGTTCCTCGAAATAACACGCAATGCAATTACAAGCAAAAAACACCAGATGAAGCTTGAGGCAAGAATGCGGAAAAAGCAGCTCAATCACGCTCAGGCCAATCAACAAGCAGTTAAGGCACAGGCACCTACAGCGCAGACAGCAGGGCACGAAGGATCCCAATTCTGA 3' . </xnotran>
2. A method for producing fast-growing, high-yielding transgenic plants using the gene coding sequence of claim 1, characterized by the steps of:
(1) Extracting larch total RNA: treating larch seeds at low temperature of 4 ℃ in a refrigerator for 14 days, soaking the seeds in warm water at 40 ℃ for 12 hours, then sowing the seeds in moist sandy soil, harvesting the overground parts of larch seedlings after 30 days, placing the overground parts in a collecting pipe, quickly freezing the seedlings by using liquid nitrogen, placing the larch seedling materials subjected to quick freezing by using the liquid nitrogen into a mortar, adding the liquid nitrogen into the mortar for grinding until the materials are ground into powder, quickly transferring 200 mg of the larch seedling materials ground into 2ml of a centrifuge tube, adding 700 mu L of lysis solution containing 5% CTAB, 2% PVP, 100 mmol/L Tris-HCl, 2 mol/L NaCl, 2% beta-mercaptoethanol and 0.5% SDS into the centrifuge tube, incubating the mixture in a water bath at 65 ℃ for 20 min, and shaking the centrifuge tube for 5 times; adding 700 mu L of water saturated phenol into the centrifuge tube, centrifuging at 12000 rpm for 10 min at room temperature, sucking supernatant, adding chloroform with the same volume, slightly and uniformly mixing, centrifuging at 12000 rpm for 10 min at room temperature, sucking supernatant, adding isopropanol with the same volume for precooling, slightly shaking up, settling at-20 ℃, standing for 50 min, centrifuging at 4 ℃,12000 rpm for 10 min, collecting precipitate, and finally adding 1 mL of precooled 75% ethanol for rinsing for 2 times, wherein each time is 500 mu L; removing ethanol to obtain RNA precipitate, naturally drying, dissolving RNA in 50 muL of sterilized DEPC water without RNase, taking 43 muL of the RNA solution, adding 1 muL of DNase I, 5 muL of 10 XDNase I buffer solution and 1 muL of RNase Inhibitor into the RNA solution, preserving heat in 37 ℃ water bath for 30 min, then adding 50 muL of chloroform into the reaction solution, shaking for 3 times to fully contact the reaction solution with the chloroform, centrifuging for 5min at 4 ℃ and 12000 rpm, taking supernatant, transferring the supernatant into a new centrifuge tube, adding isopropanol with the same volume, standing for 30 mm at-20 ℃, standing for 4 ℃ and 12000 rpm for 5min, discarding the supernatant, rinsing the precipitate with 75% ethanol for 2 times, completely sucking the liquid with a gun head, drying the RNA precipitate at room temperature, adding 30 to 50 muL of DEPC water, dissolving, and storing at-80 ℃;
(2) Synthesizing cDNA by reverse transcription of larch RNA: adding 2 mu g of larch RNA and Oligo d (T) into a 0.2 mL centrifuge tube in sequence 10 1 μ L (500 ng/. Mu.L) of RNA-free water and 1 μ L of RNA-free water; mixing, centrifuging for 5min at 75 deg.C, and immediately placing on ice for 5min; sequentially adding M-MLV 5 Xbuffer 6. Mu.L, dNTP 2.5. Mu.L, RNase Inhibitor (40U/. Mu.L) 1.25. Mu.L and M-MLV; 2 muL (U/muL) to 30 muL; mixing gently, and keeping at 37 deg.C for 5 hr; inactivating enzyme activity at 75 deg.C for 15 min, and storing at-20 deg.C;
(3) Designing and synthesizing an amplification primer with enzyme cutting sites for the larch growth and development regulation gene:
Larix-G-S：5’CCATGGATGGATTTCGAGATCGGACAA 3’，SEQ ID NO：2；
Larix-G-A：5’GGTCACCTCAGAATTGGGATCCTTCGTG 3，SEQ ID NO：3；
(4) Construction of overexpression vector: the pCAMBIA3301 plasmid was usedNcoI andBstEII double digestion, 25. Mu.L digestion system containing 2. Mu.g (10. Mu.L) of plasmid, 10 XT buffer 2. Mu.L,Nco I（30 U/μL）1.0 μL、BstEII (100U/. Mu.L) 1.0. Mu.L, sterile water 11. Mu.L, incubating for 6 h at 37 ℃, and then recovering the pCAMBIA3301 vector after enzyme digestion by using 0.8% agarose gel; the same method is adopted to control the growth and development of the larch containing gene obtained by the amplification in the step (3) and the geneNco IAndBstcarrying out double enzyme digestion on the TA cloning vector of the EII site sequence, and recovering an enzyme digestion product; connecting the larch growth and development regulation gene fragment with the same restriction enzyme cutting site with a recovered product of pCAMBIA3301 plasmid, wherein the connection system is as follows: adding 5 muL of pCAMBIA3301 plasmid, 3.5 muL of target fragment, 1 muL of 10 XLigase buffer, 0.5 muL of Ligase and sterile water into a 0.2 mL centrifuge tube, and preserving heat for 16 h in a water bath at 16 ℃; subjecting the ligation product to hot stampingTransferring into Escherichia coli DH5 alpha competent cell by beating method, coating on LB plate containing 150 mg/L Kan, culturing overnight at 37 deg.C; selecting positive clones, carrying out amplification culture in an LB liquid culture medium containing 150 mg/L Kan, extracting positive recombinant plasmids, and transferring the positive recombinant plasmids into agrobacterium LBA4404; the transformed Agrobacterium was spread on YEB plates containing 100 mg/L rifrifampicin, 120mg/L Str streptomycin and 150 mg/L Kan kanamycin and cultured in inverted culture at 28 ℃ for 2 d; white clones were picked in 1 mL YEB medium: containing 80mg/L Rif, 120mg/L Str and 150 mg/L Kan, culturing at 28 deg.C and 220 rpm for 1-2 days, extracting Agrobacterium tumefaciens positive plasmid, and performing double enzyme digestion identification; adding 500 mul of identified positive strain bacterial liquid into 500 mul of 40% glycerol YEB culture medium, shaking and mixing uniformly, and storing at-20 ℃ for later use;
(5) Genetic transformation: inoculating the frozen agrobacterium tumefaciens liquid containing the recombinant plasmid into 5 mL YEB liquid culture medium containing 100 mg/L Rif, 120mg/L Str and 120mg/L Kan, and activating at 28 ℃ and 220 rpm for 1-2 days; inoculating 2.5 mL of activated bacterial liquid into 250 mL of Kan containing 80mg/L of Rif, 120mg/L of Str and 120mg/L of Kan, culturing at 28 ℃ and 220 rpm with shaking until OD 600 1.2-1.6;4000 Centrifuging at rpm for 10 min, collecting thallus, suspending the thallus in 500 mL of 5% sucrose solution, and mixing the transformed bacteria solution; genetically transforming arabidopsis by adopting a floral dip method;
(6) Screening transgenic positive seedlings: harvesting of transgenic Arabidopsis T 0 Seeds are generated, the seeds are sown in nutrient soil after being vernalized in a refrigerator at 4 ℃ for 3 days, 10 percent of Basta mother liquor is diluted by tap water by 10000 times and then sprayed when arabidopsis seedlings grow out of true leaves, the seeds are sprayed twice a day, after one week, a positive transformation strain containing a target gene grows normally, and the seedlings of the arabidopsis plants which are not successfully transformed gradually yellow and stop growing; transferring the positive seedling to newly sterilized nutrient soil for culture, and recording as T 1 Extracting DNA of leaves to carry out PCR molecular identification;
(7) And (3) observing growth and development traits of the transgenic plants: for T 3 And observing the growth and development characters of the purified transgenic plants of the generation and the later generation.
3. Use of the coding sequence of the gene of claim 1 for interfering with the development of larch and other plant organs; wherein the application of the intervention in the development of larch and other plant organs refers to: and (4) cultivating fast-growing and high-yield transgenic plants meeting the breeding target.
4. The lysis solution for the RNA extraction of Larix Gmelini as claimed in claim 2, which comprises a mixture of 5% CTAB +2% PVP +100 mmol/L Tris-HCl +2 mol/L NaCl +2% β -mercaptoethanol +0.5% SDS.
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