Application of overexpressed CwPHL7 in lemon

Abstract

The invention relates to the technical field of gene engineering, in particular to application of overexpressed CwPHL7 in lemons. The invention provides application of overexpressed CwPHL7 in lemons, which comprises the following steps: introducing a CwPHL7 gene into hairy roots of lemons by using an agrobacterium rhizogenes vacuum filtration method to obtain CwPHL7 overexpressed lemon plants, simulating drought stress and natural drought stress on the CwPHL7 overexpressed lemon plants, and measuring and analyzing the change of plant morphological phenotypes and physiological indexes. It is determined that overexpression of CwPHL7 in lemons improves the drought resistance of lemons.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to the application of overexpressing CwPHL7 in lemon. Background Art

[0002] Citrus wilsonii Tanaka, also known as Zhique, is a plant of the genus Citrus in the Rutaceae family and is a unique local hybrid variety in southern Shaanxi. Citrus wilsonii Tanaka shows significant resistance to iron deficiency chlorosis in production, which is superior to the widely used Poncirus trifoliata rootstock at present and is an important excellent rootstock resource in southern Shaanxi. At the same time, Citrus wilsonii Tanaka is also an important citrus medicinal plant, which can be used to treat symptoms such as cough, inflammation, and excessive phlegm. It is one of the sources of Citrus medica L. in the Chinese Pharmacopoeia and has extremely high economic and medicinal value. In addition, Citrus wilsonii Tanaka also shows significant stress resistance traits such as drought resistance and salt tolerance, providing a valuable resource for exploring stress resistance genes. In previous studies, we have successfully cloned this gene and constructed an overexpression vector pK7WG2D-CwPHL7. To deeply analyze the function of the CwPHL7 gene, in this study, this gene was transformed into lemon, and its regulatory effect on the drought resistance of lemon was systematically evaluated through drought stress treatment.

[0003] Lemon is native to southeastern and southern Asia and is cultivated in the areas south of the Yangtze River in China. Now it is widely cultivated in tropical and subtropical regions. Lemon prefers warm weather and is not cold-tolerant. It has no strict requirements for soil, but it is best to use slightly acidic soil with deep, loose, rich in organic matter, strong moisture and fertility retention, good drainage, and low groundwater level. Lemon is mostly propagated by grafting. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an application of overexpressing CwPHL7 in lemon in view of the above-mentioned deficiencies of the prior art. By using the method of vacuum filtration of Agrobacterium rhizogenes, the CwPHL7 gene is introduced into lemon hairy roots to obtain CwPHL7 overexpressing lemon plants, and the CwPHL7 overexpressing lemon plants have strong drought resistance.

[0005] The present invention provides an application of overexpressing CwPHL7 in lemon, and overexpressing CwPHL7 in lemon improves the drought resistance of lemon.

[0006] According to the application of overexpressing CwPHL7 in lemon provided by the present invention, the method of overexpressing CwPHL7 in lemon is to introduce the CwPHL7 gene into lemon hairy roots.

[0007] According to the application of overexpressing CwPHL7 in lemon provided by the present invention, the nucleotide sequence of the CwPHL7 gene is as shown in SEQ ID NO:1.

[0008] Application of overexpressing CwPHL7 in lemon provided by the present invention. The upstream and downstream primers for PCR amplification of the CwPHL7 gene are pKPH-F / pKPH-R or GFP-F / GFP-R. The nucleotide sequence of pKPH-F is as shown in SEQ ID NO:2, the nucleotide sequence of pKPH-R is as shown in SEQ ID NO:3, the nucleotide sequence of GFP-F is as shown in SEQ ID NO:4, and the nucleotide sequence of GFP-R is as shown in SEQ ID NO:5.

[0009] Application of overexpressing CwPHL7 in lemon provided by the present invention. The reaction system for PCR amplification of the CwPHL7 gene is 25 μL of P525 Mix, 1 μL of lemon hairy root DNA, 2 μL of pKPH-F / GFP-F, 2 μL of pKPH-R / GFP-R, and 20 μL of ddH2O;

[0010] The PCR amplification program is: 95°C for 5 min; 95°C for 15 sec; 60°C for 15 sec; 72°C for 1 min (35 cycles); 72°C for 5 min.

[0011] The present invention has the following advantages compared with the prior art:

[0012] The present invention provides an application of overexpressing CwPHL7 in lemon. By using the method of vacuum filtration of Agrobacterium rhizogenes, the CwPHL7 gene is introduced into lemon hairy roots to obtain CwPHL7 overexpressing lemon plants. Simulated drought stress and natural drought stress are carried out on them, and the changes of plant morphological phenotypes and physiological indexes are measured and analyzed to determine that overexpressing CwPHL7 in lemon improves the drought resistance of lemon. Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0014] Figure 1 It is a sample diagram of preparing plant materials;

[0015] Figure 2 It is a detection result diagram of colony PCR;

[0016] Figure 3 It is a diagram of screening and PCR identification of CwPHL7 overexpressing lemon;

[0017] Figure 4It is a comparison diagram of the morphological characteristics of lemon with overexpression of CwPHL7;

[0018] Figure 5 It is a diagram for measuring the physiological indexes of the leaves of WT and lemon with overexpression of CwPHL7 after drought stress;

[0019] Figure 6 It is a diagram of the histochemical staining results of the leaves of WT and lemon with overexpression of CwPHL7 under drought stress;

[0020] Figure 7 It is a diagram for analyzing the relative expression levels of drought-related genes in lemon with overexpression of CwPHL7 after drought stress. Detailed implementation mode

[0021] Example 1

[0022] This example provides a trifoliate orange CwPHL7 gene, and the nucleotide sequence of this gene is shown in SEQ ID NO:1;

[0023] This example provides a method for overexpressing CwPHL7 in lemon, and the specific steps are as follows:

[0024] S1. Prepare plant materials and strains

[0025] Collect mature fresh lemon fruits, peel the seeds, wash them clean with distilled water, soak them in 0.5 mol / L NaOH solution to remove the pectin on the seed surface, wash them clean with distilled water, remove the outer seed coat, perform ripening treatment in a water bath at 45 °C for 3 - 4 h, then lay them flat on a clean and wet gauze, place them in an incubator at 30 °C for germination for 7 d, and take them out for infection after the young roots of the seeds germinate to 1 - 2 cm in length.

[0026] The test strain is Agrobacterium rhizogenes K599, as Figure 1 shown, Figure A is a diagram of a lemon fruit, Figure B is a cross-section of a lemon fruit, Figure C is a diagram of a lemon seed, and Figure D is a diagram of the germinated young root of a lemon seed with a length of 1 - 2 cm;

[0027] S2. Extract and transform the plasmid

[0028] Extract the plasmid from the pK-PHL7 Escherichia coli bacterial solution and transform it into Agrobacterium rhizogenes K599. Take the competent cells of K599 and let them stand at room temperature or in the palm of the hand for a moment until they partially melt. When in an ice-water mixture state, insert them into ice and continue to melt the Agrobacterium competent cells; add 1 μg of plasmid to 100 μL of Agrobacterium competent cells, incubate on ice for 30 min, in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and on ice for 2 min; add 700 μL of antibiotic-free LB liquid medium, incubate at 28 °C with shaking at 200 rpm for 3 h; pipette 50 μL of the bacterial solution and spread it on a plate; incubate upside down at 28 °C for 2 d; pick positive clones for colony PCR identification and then store the glycerol bacteria for subsequent experiments;

[0029] S3. Genetic transformation of lemon hairy roots

[0030] According to the "Method for Genetic Transformation of Citrus Hairy Roots Using Seeds as Materials" disclosed in CN2023111109806, use the vacuum infiltration method to perform genetic transformation on lemon seeds. Pick single colonies of Agrobacterium rhizogenes K599 containing the empty vector pK7WG2D and the recombinant expression vector pK-CwPHL7, and inoculate them into 3 mL of LB liquid medium containing 50 μg / mL spectinomycin respectively. Incubate with shaking at 28 °C and 200 rpm for 16 h for the first activation. Take 1 mL of the bacterial solution and transfer it to 50 mL of LB liquid medium containing 50 μg / mL spectinomycin, and continue to culture at 28 °C and 200 rpm until OD 600 = 0.8 - 1.0 for the second activation;

[0031] After centrifuging and collecting the activated bacterial solution, resuspend it with the infiltration solution (10 mmol / L MES, 10 mmol / L MgCl2, 100 μmol / L acetosyringone, pH = 5.6) to OD 600 = 0.6 - 0.8. Soak the seeds with 1 - 2 cm long young roots in the Agrobacterium rhizogenes infiltration solution, infiltrate at a vacuum of 0.08 - 0.09 MPa for 10 min, take out the seeds and incubate them in the dark in a 28 °C constant temperature incubator for 3 d, then plant them in vermiculite substrate and transfer them to the culture room for continued cultivation, keeping the environment moist to make them root. The culture room conditions are: light intensity 4000 lx, photoperiod 16 h / 8 h (day / night), temperature 21 ± 2 °C, humidity 60% - 70%;

[0032] S4. Screening and identification of positive lemon hairy root seedlings

[0033] S41. Screening of positive lemon hairy root seedlings

[0034] After the infected seeds are planted for 15 d, observe and detect them using a portable fluorescent protein observation lamp, and screen positive materials based on whether the hairy roots can emit green fluorescence;

[0035] S42. Positive Identification of Lemon Hairy Roots

[0036] According to the proteins and protein sequences present on the vector sequence, 2 pairs of primers were designed respectively, which could amplify the target fragment and the vector fragment. The primers included pKPH-F and pKPH-R. The nucleotide sequence of pKPH-F is shown in SEQ ID NO:2, and the nucleotide sequence of pKPH-R is shown in SEQ ID NO:3;

[0037] The primers for green fluorescent protein included GFP-F and GFP-R. The nucleotide sequence of GFP-F is shown in SEQ ID NO:4, and the nucleotide sequence of GFP-R is shown in SEQ ID NO:5;

[0038] Use a plant DNA extraction kit to extract the DNA of hairy roots with green fluorescence and use it as a template for PCR amplification;

[0039] The PCR amplification system was: P525 Mix 25 μL, lemon hairy root DNA 1 μL, pKPH-F / GFP-F 2 μL, pKPH-R / GFP-R 2 μL, ddH2O 20 μL;

[0040] The PCR amplification program was: 95°C for 5 min; 95°C for 15 sec; 60°C for 15 sec; 72°C for 1 min (35 cycles); 72°C for 5 min;

[0041] S5. Analysis of Drought Resistance Function of Transgenic Lemons

[0042] S51. Comparison of Morphological Characteristics of CwPHL7 Overexpressing Lemons

[0043] Transplant WT and lemons transformed with pK and pK-CwPHL7 into nutrient soil (peat soil: vermiculite = 2:1) and place them in the culture room for continued cultivation. Observe and photograph the plant height and root length of lemons every 30 days during the cultivation period. Observe 3 plants each time and repeat 3 times. Observe the density of oil gland dots under light irradiation, with a range of 1 cm each time as a field of view. Observe the stomatal density under an electron microscope, with a range of 1 mm each time as a field of view. Observe 3 fields of view and perform significance analysis after repeating 3 times. 2 range as a field of view, and observe the stomatal density under an electron microscope, with a range of 1 mm 2 range as a field of view, observe 3 fields of view, and perform significance analysis after repeating 3 times.

[0044] S52. Drought Stress of CwPHL7 Overexpressing Lemons

[0045] Cultivate lemon seedlings normally until they are 2 months old. Select plants with consistent growth and strong growth for natural drought and simulated drought stress treatments, and photograph and record the changes of the plants;

[0046] Prepare Hoagland nutrient solution containing 10% sorbitol and hydroponically culture lemon seedlings of the treatment group WT and overexpressing CwPHL7. Use RO water to hydroponically culture lemon seedlings of WT and overexpressing CwPHL7 in the control group. Change the water every 2 days. After 5 days, select several lemon leaves and roots of WT and overexpressing CwPHL7 in the treatment group and the control group, put them into self-sealing bags and store them at -80 °C for subsequent determination of the relative expression level of CwPHL7 and physiological indexes. Set 3 biological replicates for each treatment;

[0047] S6. Determination of physiological indexes of CwPHL7 overexpressing lemon

[0048] Measure the relative water content, relative conductivity, chlorophyll content and MDA content of the control group and the treatment group of WT and overexpressing CwPHL7 lemon under osmotic stress respectively;

[0049] S7. O2 -. and histochemical staining of H2O2

[0050] Collect Arabidopsis thaliana leaves of WT and overexpressing CwPHL7 after drought stress respectively. After rinsing them with distilled water, immerse them in NBT (nitroblue tetrazolium chloride) and DAB (diaminobenzidine) staining solutions respectively and store them in the dark for 8 h. Place them in the decolorizing solution and decolorize them in a boiling water bath for 1 h. Take out the leaves and take pictures for preservation after they become transparent. The solution preparation methods are as follows: NBT staining solution: 0.2 g NBT, made up to 100 mL with 50 mM Tris buffer (PH = 7.5); DAB staining solution: 1.15 g Tris, 0.25 g DAB, made up to 250 mL with distilled water (pH = 5.8); Decolorizing solution: ethanol: acetic acid: glycerol = 3:1:1.

[0051] S8. RT-qPCR real-time fluorescence quantitative analysis

[0052] Carry out RT-qPCR real-time fluorescence quantitative analysis on the roots of WT and CwPHL7 overexpressing lemon after drought stress collected;

[0053] S81. RNA extraction of trifoliate orange

[0054] Use the RNA-prep pure polysaccharide polyphenol plant total RNA extraction kit. First, grind the roots of the control group and the drought group of WT and lemon transformed with pK-CwPHL7 into powder with liquid nitrogen, and then extract the total RNA according to the kit instructions respectively. Use a micro ultraviolet spectrophotometer to detect the RNA concentration and purity;

[0055] S82. cDNA synthesis

[0056] Using the extracted RNA as a template, synthesize cDNA with the Evo M-MLV Reverse Transcription Kit. Prepare Reverse Transcription System 1 according to Table 1, react at 42°C for 2 min to remove genomic DNA, prepare Reverse Transcription System 2 according to Table 2, first react at 37°C for 15 min, then react at 85°C for 5 s, and store at 4°C.

[0057] Table 1

[0058] Reaction components Dosage 5×gDNAClean Reaction Mix 2 μL Total RNA 1 μg RNase free water UP to 10 μL

[0059] Table 2

[0060] Reaction components Usage amount Reaction solution of System 1 10 μL 5×Evo M-MLV RT Reaction Mix 4 μL RNase free water 6 μL

[0061] S83, RT-qPCR determination of relative expression

[0062] By consulting the prior art, 4 genes that have been reported to positively regulate citrus drought stress were obtained, namely CsWRKY47, CsMYB96, ClMYB15, and ClWRKY75. Design primers using Primer 5 software. The primers for the reference gene and the internal reference gene are Actin-F, Actin-R, qCwPHL7-F, qCwPHL7-R, qCsWRKY47-F, qCsWRKY47-R, qCsMYB96-F, qCsMYB96-R, qClMYB15-F, qClMYB15-R, qClWRKY75-F, and qClWRKY75-R in sequence.

[0063] The nucleotide sequences of the above primers are as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 in sequence.

[0064] The RT-qPCR reaction system is shown in Table 3. Three technical replicates are designed for each sample, and the 2−ΔΔCT method is used to calculate the relative expression levels of drought-related genes in WT and CwPHL7-overexpressing lemons after drought stress. -ΔΔCT The reaction program: 95°C for 3 min; 95°C for 30 sec, 58°C for 30 sec, 72°C for 80 sec (35 cycles); 72°C for 5 min

[0065] Table 3

[0066] Component name Usage amount 2×SYBR Green Pro Taq HS Premix 10 μL cDNA 2 μL F 0.4 μL R 0.4 μL RNase free water 7.2 μL

[0067] Reaction program: 95°C 3 min; 95°C 30 sec, 58°C 30 sec, 72°C 80 sec (35 cycles); 72°C 5 min

[0068] S9. Data Processing and Analysis

[0069] Data statistical analysis was carried out using Excel 2010 and SPSS22, and plate production was carried out using GraphPad Prism5 and Photoshop software.

[0070] Example 2

[0071] This example provides a result and analysis of Example 1.

[0072] ① Transformation of Agrobacterium rhizogenes K599 with pK-CwPHL7

[0073] The overexpression vector pK-CwPHL7 was transformed into Agrobacterium rhizogenes K599. The results of colony PCR showed that the bands were all correct at 811 bp. The PCR results are as Figure 2 shown, proving that the pK-CwPHL7 overexpression vector was successfully transferred into Agrobacterium.

[0074] ② Screening and identification of CwPHL7-overexpressing lemons

[0075] The infected lemon seeds were sown in vermiculite and continued to be cultured. After 15 days, they were screened using a portable fluorescent protein observation lamp. Compared with WT lemons, the roots of lemons transformed with the pK empty vector and pK-CwPHL7 had obvious green fluorescence, while the WT roots had no green fluorescence. After 30 days, the green fluorescence in the roots was more obvious, as Figure 3 shown in Figure A in the middle. Figure A is the green fluorescence screening of CwPHL7-overexpressing lemons.

[0076] The lemon positive seedlings with green fluorescence were transplanted into nutrient soil. After growing for 30 days, the roots and leaves were taken for PCR verification. The results of root PCR verification showed that the wild-type (WT) lemons in lane 1 failed to amplify the CwPHL7 and GFP protein sequences; the Arabidopsis thaliana transformed with the empty vector in lanes 2 and 3 could only amplify the GFP protein sequence on the vector; the Arabidopsis thaliana transformed with the CwPHL7 gene overexpression vector in lanes 4-10 could amplify the CwPHL7 and GFP sequences, and the band sizes were 812 bp and 439 bp respectively, as Figure 3 shown in Figures B and C in the middle. Figure B is the PCR identification of the target gene, and Figure C is the PCR identification of the GFP protein gene; the PCR band results of the leaves did not amplify the CwPHL7 and GFP sequences;

[0077] The PCR identification results showed that the lemons with green fluorescence in the roots were all chimeric positive plants;

[0078] ③ Morphological analysis of CwPHL7-overexpressing lemon plants

[0079] Morphological comparison and significance analysis (P≤0.05) were performed on WT lemons grown for 30 days and lemons transformed with pK and pK-CwPHL7. The comparison found that, compared with WT lemons, lemon plants transformed with pK and pK-CwPHL7 had shorter internodes and were characterized by plant dwarfing and root growth, as Figure 4 shown in Figure A in the middle, and the plant height was significantly reduced by 1.88 times, as Figure 4 shown in Figure D in the middle, and the root length was significantly increased by 1.6 times, as Figure 4 shown in Figure E in the middle; Observation of the oil gland density and stomatal density of the leaves of three groups of lemons found that the oil gland density of the leaves of lemon plants transformed with pK-CwPHL7, as Figure 4 shown in B and F in the middle, and the stomatal density was significantly reduced by 3.32 times and 1.38 times compared with the leaves of WT and pK-transformed lemons, respectively, as Figure 4 shown in C and G in the middle;

[0080] Figure 4 In Figure A in the middle is the morphological comparison of plants, B is the comparison of the oil gland density of leaves, C is the comparison of stomatal density, D is the significance analysis of plant height differences, E is the significance analysis of root length differences, F is the significance analysis of oil gland density differences, and G is the significance analysis of stomatal density differences;

[0081] ④ Determination of physiological indexes of CwPHL7 overexpressing lemons after drought stress

[0082] Physiological indexes of the leaves of WT and CwPHL7 overexpressing lemons after 5 days of drought stress were measured. After drought stress with 10% sorbitol, the overall color of the WT leaves became darker compared with those of CwPHL7 overexpressing lemons, and the degree of yellowing at the edges was more serious, as Figure 5 shown in Figure A in the middle.

[0083] The results of the physiological index determination showed that after drought stress with 10% sorbitol, the relative water content and chlorophyll content of the leaves of CwPHL7 overexpressing lemons were significantly (P≤0.05) 4.10 and 1.45 times higher than those of WT lemon leaves, as Figure 5 shown in B and C in the middle, where B is the relative water content of the leaves and C is the chlorophyll content of the leaves;

[0084] The MDA content and relative conductivity of the leaves of CwPHL7 overexpressing lemons were significantly (P≤0.05) 1.15 and 1.12 times lower than those of WT lemon leaves, as Figure 5 shown in D and E in the middle, where D is the MDA content of the leaves and E is the relative conductivity of the leaves, indicating that the drought stress damage suffered by CwPHL7 overexpressing lemons was smaller than that of WT;

[0085] ⑤ Histochemical staining of leaves of CwPHL7 overexpressing lemons after drought stress

[0086] The accumulation levels of reactive oxygen species H2O2 and O2 in cells can be reflected by NBT and DAB histochemical staining, as -. shown below. Figure 6 shown.

[0087] The staining results showed that, compared with the control group, the area distribution of H2O2 and O2 in WT lemon leaves after drought stress was larger and the staining was deeper, while the staining degree of CwPHL7-overexpressing lemon leaves was lighter, with only a small amount of distribution at the leaf margin, petiole and leaf veins. The results indicated that the ROS accumulation level in CwPHL7-overexpressing lemon plants was lower than that in WT after drought stress; -. The staining results showed that, compared with the control group, the area distribution of H2O2 and O2 in WT lemon leaves after drought stress was larger and the staining was deeper, while the staining degree of CwPHL7-overexpressing lemon leaves was lighter, with only a small amount of distribution at the leaf margin, petiole and leaf veins. The results indicated that the ROS accumulation level in CwPHL7-overexpressing lemon plants was lower than that in WT after drought stress;

[0088] ⑥ Expression analysis of drought-related genes in CwPHL7-overexpressing lemons

[0089] Relative expression levels of drought-related genes in WT and CwPHL7-overexpressing lemons after drought stress were analyzed by RT-qPCR technology, as Figure 7 shown below.

[0090] The RT-qPCR results showed that, compared with WT, the relative expression levels of CwPHL7 and drought stress-related genes (WRKY47, MYB96, MYB15, WRKY75) in the roots of CwPHL7-overexpressing lemons were significantly (P<0.05) up-regulated by 2.60-fold, 3.28-fold, 1.68-fold, 2.80-fold and 2.87-fold, respectively. The results indicated that the expression levels of drought-related genes in CwPHL7-overexpressing lemons were significantly higher than those in WT after drought stress.

[0091] According to the detection results of this example, it can be known that the lemons overexpressing CwPHL7 have strong drought resistance.

[0092] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes and equivalent changes made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An application of overexpressing CwPHL7 in lemon, characterized in that: Overexpression of CwPHL7 in the lemon improves the drought resistance of the lemon.

2. The use of overexpressing CwPHL7 in lemon according to claim 1, characterized in that: The method for overexpressing CwPHL7 in lemon is to introduce the CwPHL7 gene into the lemon hairy roots.

3. The use of overexpressing CwPHL7 in lemon according to claim 2, characterized in that: The nucleotide sequence of the CwPHL7 gene is shown in SEQ ID NO:

1.

4. The use of overexpressing CwPHL7 in lemon according to claim 2, characterized in that: The upstream and downstream primers for PCR amplification of the CwPHL7 gene are pKPH-F / pKPH-R or GFP-F / GFP-R, the nucleotide sequence of the pKPH-F is shown in SEQ ID NO:2, the nucleotide sequence of the pKPH-R is shown in SEQ ID NO:3, the nucleotide sequence of the GFP-F is shown in SEQ ID NO:4, and the nucleotide sequence of the GFP-R is shown in SEQ ID NO:

5.

5. The use of overexpressing CwPHL7 in lemon according to claim 4, characterized in that: The reaction system for PCR amplification of the CwPHL7 gene is P525 Mix 25 μL, lemon hairy root DNA 1 μL, pKPH-F / GFP-F 2 μL, pKPH-R / GFP-R 2 μL, ddH2O 20 μL; The PCR amplification program was: 95°C for 5 min; 95°C for 15 sec; 60°C for 15 sec; 72°C for 1 min (35 cycles); 72°C for 5 min.

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