CN114774465A - Method for improving fruit setting rate of apricot trees - Google Patents
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- CN114774465A CN114774465A CN202210607166.4A CN202210607166A CN114774465A CN 114774465 A CN114774465 A CN 114774465A CN 202210607166 A CN202210607166 A CN 202210607166A CN 114774465 A CN114774465 A CN 114774465A
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Abstract
The invention discloses a method for improving the fruit setting rate of apricot trees, and relates to the technical field of biology. The method comprises the following steps: extracting total RNA from apple leaves, and performing reverse transcription to obtain cDNA; carrying out PCR amplification by using the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2 and the cDNA as a template to obtain an amplification product; and transforming the amplification product into apricot tree leaves, and performing tissue culture to obtain a recombinant apricot tree plant. The DHAR gene of the apple is transformed into apricot leaves by an agrobacterium-mediated method, and the recombinant apricot plant containing the DHAR gene is constructed by tissue culture. The recombinant apricot tree plant can reduce the harm of late frost and improve the fruit setting rate of the recombinant apricot tree under extremely cold conditions such as frost and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for improving the fruit setting rate of apricot trees.
Background
Apricot (with the academic name: Armeniaca vulgaris Lam.) deciduous tree is an important fruit tree in agricultural economy. The plants are hairless and are grown on the ground. Leaf intergrowth, broad-egg-shaped or round-egg-shaped leaf with blunt sawtooth at edge; two glands are arranged near the top of the petiole; pale red flowers alone or 2-3 syngeneic, white or reddish.
In actual agricultural planting, in the flower bud stage and the flower bud expansion stage of apricot trees, frost is generated when severe temperature reduction occurs, flowering delay is caused, or stamens and stamens are abnormally developed, so that fertilization and fruit setting are influenced. When the freezing is serious, pistils and stamens are browned and dried to be curled, and the pollination and fertilization capability is lost; the petal tissue is frozen and hardened, and when the temperature rises in the morning, the petals gradually turn from white to yellow brown, and then turn into brown to wither and fall off; when the ovaries are seriously frozen, the ovaries gradually wither and shrink, the basal parts of the pedicel generate abscission layers, and the abscission layers are the same in number and fall off; the young fruits are frozen, malformed fruits are produced mostly, the growth speed of the fruits is slowed down, and the fruits fall off finally; the damage of young leaves is manifested as leaf edge discoloration, softening of leaves, and even withering.
The apricot trees bloom in spring, the flowering time is early, the harm of late frost is serious, once frost is encountered in the flowering period, the yield is reduced for light people and the serious people are lost, so the late frost becomes an important factor for restricting the development of the apricot planting industry.
Disclosure of Invention
The invention aims to provide a method for improving the fruit setting rate of apricot trees, which aims to solve the problems in the prior art and enable the apricot trees to improve the fruit setting rate under the influence of frost and other cold conditions.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for improving the fruit setting rate of apricot trees, which comprises the following steps:
(1) extracting total RNA from apple leaves, and performing reverse transcription to obtain cDNA;
(2) carrying out PCR amplification by using the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2 and the cDNA as a template to obtain an amplification product;
(3) and (3) transforming the amplification product into apricot tree leaves, and performing tissue culture to obtain a recombinant apricot tree plant.
Further, in step (2), the amplification system of the PCR amplification is: 2. mu.L of 10mM dNTP, 10. mu.L of 10 XPCR Buffer, 5. mu.L of 10. mu.M forward primer, 5. mu.L of 10. mu.M reverse primer, 2. mu.L of template cDNA, 25mM MgSO410μL，5U/μL Taq DNApolymerase 0.2μL，ddH2O make up to 100. mu.L.
Further, in step (2), the amplification procedure of the PCR amplification is: pre-denaturation at 94 ℃ for 3 min; the cycle condition is 35 cycles of denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 30s and extension at 68 ℃ for 2 min; extension at 72 ℃ for 5 min.
Further, in the step (3), the amplification product is inserted into a plant transformation vector and then transformed into apricot leaves.
Further, the specific operation of inserting the amplification product into a plant transformation vector comprises: the pCAMBIA1303 plasmid and the pSB166 plasmid are firstly connected to obtain a recombinant plasmid pCSB, and the amplification product is inserted into the recombinant plasmid pCSB.
Further, the enzyme cutting system for connecting the pCAMBIA1303 plasmid and the pSB166 plasmid is as follows: plasmid 14. mu.L, EcoRI 2. mu.L, Hind III 2. mu.L, 10 XTango 2. mu.L.
Further, the enzyme cutting system for inserting the amplification product into the recombinant plasmid pCSB is as follows: plasmid/DNA 11 μ L, SalI 2 μ L, KpnI 2 μ L, 10 XTANGO 2 μ L, 1% BSA4 μ L.
Further, the method for transforming into apricot tree leaves is agrobacterium transformation.
Further, the specific operation of agrobacterium transformation comprises: taking terminal buds with 2-3 leaves on the apricot tree, scratching the back of the leaves, and dip-dyeing in the bacterial liquid of the agrobacterium containing the amplification product.
Further, the dip dyeing time is 5 min.
The invention discloses the following technical effects:
the DHAR gene of the apple is transformed into apricot leaves by an agrobacterium-mediated method, and the recombinant apricot plant containing the DHAR gene is constructed by tissue culture. The recombinant apricot tree plant can reduce the harm of late frost and improve the fruit setting rate of the recombinant apricot tree under extremely cold conditions such as frost and the like. The method provides a new technical idea for reducing the harm of late frost to apricot trees, and is beneficial to promoting the development of apricot planting industry.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated or intervening value in a stated range, and every other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
Example 1
First, experimental material
Apricot tree species: katy apricot
Coli strain e.coli DH5 α and agrobacterium strain EHA105 were purchased from shanghai virginia biotechnology limited, and plasmids pCAMBIA1303 and pSB166 were purchased from turba organisms.
Second, Experimental methods and results
2.1 extraction and reverse transcription of Total RNA
Weighing 0.1-0.2 g fresh apple leaves, grinding into powder in liquid nitrogen, adding into Tris saturated phenol preheated at 80 deg.C, and rapidly adding 800 μ L extraction buffer (50mmol/L Tris-HCl, pH 8.0; 10mmol/L EDTA-Na)2pH8.0; 1% SDS; 2% PVP; 81.9% NaCl) and 16. mu.L of beta-mercaptoethanol, shaking vigorously and mixing them uniformly, centrifuging at 4 ℃ and 12000r/min for 20min, taking the supernatant, adding the same volume of phenol, chloroform: isoamyl alcohol (volume ratio is 25: 24: 1), evenly mixing, centrifuging for 20min at 4 ℃ at 12000r/min, taking supernatant, adding chloroform with the same volume as the volume of the supernatant: isoamyl alcohol (volume ratio is 24: 1), evenly mixing, centrifuging at 4 ℃ and 12000r/min for 15min, taking supernatant, adding 2.5 times of anhydrous ethanol and 0.1 time of 3mol/LNaAc, keeping stand at pH 5.2-20 ℃ for precipitating for more than 3h, centrifuging at 4 ℃ and 12000r/min for 20min, discarding supernatant, washing precipitate with ethanol with volume fraction of 70%, centrifuging at 4 ℃ and 12000r/min for 5min, discarding supernatant, and airing until ethanol is dissolvedAnd (3) volatilizing the alcohol completely, dissolving the alcohol by using distilled water treated by 30-50 mu L of DEPC, and storing the solution at-70 ℃ for later use.
PrimeScript (Takara Co.) was usedTMThe RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit reverse transcribes the extracted RNA into cDNA.
2.2PCR amplification
According to DHAR gene (sequence number DQ322706), after conservative analysis, a specific primer is designed and synthesized:
the upstream primer P1: 5'-CAACCGTTCTGCCCCATTAC-3' (SEQ ID NO. 1);
the downstream primer P2: 5'-GCAACCAGCGGAGCTAATAC-3' (SEQ ID NO. 2).
PCR amplification was performed using the cDNA obtained in 2.1 as a template and P1 and P2 as primers to obtain an amplification product. The amplification system is shown in Table 1:
TABLE 1
And (3) amplification procedure: pre-denaturation at 94 ℃ for 3 min; the cycle condition is 35 cycles of denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 30s and extension at 68 ℃ for 2 min; extension at 72 ℃ for 5 min.
2.3 construction of expression vectors
2.3.1 plasmids pCAMBIA1303 and pSB166 were digested with HindIII/EcoRI, respectively.
The cleavage reaction system (20. mu.L) is shown in Table 2:
TABLE 2
After reacting at 37 ℃ for 2h, 1. mu.L of 10 × Loading Buffer was added to terminate the reaction, and agarose gel electrophoresis was performed, followed by recovery and purification using a gel recovery kit, to obtain linearized pCAMBIA1303 and pSB166 fragments.
2.3.2 ligation:
about 30ng of each of the pCAMIA1303 linearized plasmid and pSB166 fragment DNA recovered at 2.3.1, 1. mu.L of 10 XLigase buffer and 0.4. mu. L T4-DNAIgase were added sequentially on ice according to the instructions of T4-DNA Ligase kit, and the mixture was made up to a 10. mu.L system with sterile double distilled water. And (3) gently mixing uniformly, throwing to the bottom of the tube, reacting overnight at 16 ℃ and connecting to obtain the recombinant plasmid.
2.3.3 transformation
The recombinant plasmid prepared in 2.3.2 was introduced into competent cells of Escherichia coli containing 50 mg. multidot.L-1Resistant plasmids were screened on LB plates of Kan, and after identification, an intermediate expression vector containing the ED35s promoter, Omega element and TNOS terminator suitable for plant transformation was obtained and named pCSB.
2.3.4 construction of pCSB-DHAR
The amplification products from pCSB and 2.2 were double digested with SalI/KpnI (see Table 3), followed by T4And connecting the DNAligase kit, transforming the agrobacterium EHA105, extracting plasmids, and determining that the recombinant pCSB-DHAR plasmid is successfully introduced into the agrobacterium EHA105 after identification.
TABLE 3
2.4 tissue culture of apricot by Agrobacterium tumefaciens mediated transformation
Experimental groups: taking terminal buds with 2-3 leaves on an apricot tree, scratching the back of the leaves, dip-dyeing in 2.3.4 bacterial liquid of agrobacterium EHA105 introduced with pCSB-DHAR for 5min, and then performing subsequent tissue culture by using an MS culture medium containing 25 mu g/mL hygromycin according to a conventional method to obtain a regeneration plant. The upstream primer P1 and the downstream primer P2 are used for carrying out PCR detection on the regenerated plants, and 5 DHAR positive plants are obtained through detection.
Two regeneration plants (marked as X1 and X2) with good growth vigor are selected and respectively grafted on the rootstock, and the two regeneration plants are planted in a sunlight greenhouse. At the beginning of the third year of flowering (3 months and 10 days), the temperature in the planting greenhouse was lowered to-6 ℃ for 4 hours, simulating frost, and then the planting experiment was performed according to normal planting conditions.
Meanwhile, the unloaded agrobacterium EHA105 bacterial liquid is used as a positive control, and agrobacterium conversion to a negative control is not carried out, and experiments are respectively carried out according to the method.
In the above experiments, the fruit setting rate was counted at 15 days after the simulated frost, and the results are shown in table 4. The results shown in table 4 prove that the construction of the recombinant apricot plant containing DHAR by using the method of the present invention can improve fruit setting rate of apricot under sudden cold conditions such as frost.
TABLE 4
| Group of | Investigation flower | Percentage of fruit set (%) |
| Experimental group X1 | 500 | 15.8 |
| Experimental group X2 | 500 | 14.0 |
| Positive control | 500 | 0 |
| Negative control | 500 | 0.6 |
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> Zhang Kouzhou agricultural science institute (alpine crop research institute in Hebei province)
<120> method for improving fruit setting rate of apricot trees
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
caaccgttct gccccattac 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gcaaccagcg gagctaatac 20
Claims (10)
1. The method for improving the fruit setting rate of the apricot trees is characterized by comprising the following steps:
(1) extracting total RNA from apple leaves, and performing reverse transcription to obtain cDNA;
(2) carrying out PCR amplification by using the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2 and the cDNA as a template to obtain an amplification product;
(3) and (3) transforming the amplification product into apricot tree leaves, and performing tissue culture to obtain a recombinant apricot tree plant.
2. The method for improving the fruit setting rate of the apricot tree according to claim 1, wherein in the step (2), the amplification system of the PCR amplification is: 2. mu.L of 10mM dNTP, 10. mu.L of 10 XPCR Buffer, 5. mu.L of 10. mu.M forward primer, 5. mu.L of 10. mu.M reverse primer, 2. mu.L of template cDNA, 25mM MgSO 24 10μL，5U/μL Taq DNA polymerase 0.2μL，ddH2O make up to 100. mu.L.
3. The method for improving fruit setting rate of apricot trees according to claim 1, wherein in the step (2), the amplification procedure of the PCR amplification is: pre-denaturation at 94 ℃ for 3 min; the cycle condition is 35 cycles of denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 30s and extension at 68 ℃ for 2 min; extension at 72 ℃ for 5 min.
4. The method for improving the fruit setting rate of the apricot according to claim 1, wherein in the step (3), the amplification product is inserted into a plant transformation vector and then transformed into apricot leaves.
5. The method for improving fruit setting rate of apricot trees according to claim 4, wherein the specific operation of inserting the amplification product into the plant transformation vector comprises: the pCAMBIA1303 plasmid and the pSB166 plasmid are firstly connected to obtain a recombinant plasmid pCSB, and the amplification product is inserted into the recombinant plasmid pCSB.
6. The method for improving the fruit setting rate of the apricot trees as claimed in claim 5, wherein the enzyme cutting system for connecting the pCAMBIA1303 plasmid and the pSB166 plasmid is as follows: plasmid 14. mu.L, EcoRI 2. mu.L, Hind III 2. mu.L, 10 XTango 2. mu.L.
7. The method for improving the fruit setting rate of the apricot trees as claimed in claim 5, wherein the enzyme cutting system for inserting the amplification product into the recombinant plasmid pCSB is as follows: plasmid/DNA 11. mu.L, SalI 2. mu.L, KpnI 2. mu.L, 10 XTango 2. mu.L, 1% BSA 4. mu.L.
8. The method for improving fruit set percentage of the apricot tree according to claim 4, wherein the method for transforming into leaves of the apricot tree is agrobacterium transformation.
9. The method for improving the fruit setting rate of the apricot trees according to claim 8, wherein the specific operations of agrobacterium transformation comprise: taking terminal buds of 2-3 leaves on the apricot tree, scratching the back of the leaves, and dip-dyeing in the bacterial liquid of the agrobacterium containing the amplification product.
10. The method for improving the fruit setting rate of the apricot trees according to claim 9, wherein the dip dyeing time is 5 min.
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| US20120180165A1 (en) * | 2009-09-25 | 2012-07-12 | Basf Plant Science Company Gmbh | Plants Having Enhanced Yield-Related Traits and a Method for Making the Same |
| CN103525860A (en) * | 2013-10-29 | 2014-01-22 | 泰安市泰山林业科学研究院 | Apricot cold-resist gene transformation method |
| US20140245493A1 (en) * | 2011-08-25 | 2014-08-28 | Kyungpook National University Industry-Academic Coopeeration Foundation | Use of plant-derived dhar or mdhar gene as a modulator for crop yield and environmental stress |
| CN114480416A (en) * | 2022-01-18 | 2022-05-13 | 广西壮族自治区药用植物园 | Application of tsaoko AtDRM2 gene in improving cold resistance of plants |
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| US20120180165A1 (en) * | 2009-09-25 | 2012-07-12 | Basf Plant Science Company Gmbh | Plants Having Enhanced Yield-Related Traits and a Method for Making the Same |
| US20140245493A1 (en) * | 2011-08-25 | 2014-08-28 | Kyungpook National University Industry-Academic Coopeeration Foundation | Use of plant-derived dhar or mdhar gene as a modulator for crop yield and environmental stress |
| CN103525860A (en) * | 2013-10-29 | 2014-01-22 | 泰安市泰山林业科学研究院 | Apricot cold-resist gene transformation method |
| CN114480416A (en) * | 2022-01-18 | 2022-05-13 | 广西壮族自治区药用植物园 | Application of tsaoko AtDRM2 gene in improving cold resistance of plants |
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