CN116790627A - Poplar MYBS2 gene and application thereof - Google Patents
Poplar MYBS2 gene and application thereof Download PDFInfo
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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Abstract
The invention relates to a poplar MYBS2 gene and application thereof, and belongs to the technical field of genetic engineering. The invention provides a poplar MYBS2 gene, and the nucleotide sequence of the poplar MYBS2 gene is shown as SEQ ID NO. 1. After the RNAi technology is used for reducing the MYBS2 gene expression of the poplar, the height, the stem circumference, the branch number, the dry weight and the fresh weight of the poplar are all improved; the change in growth status of wild-type poplar plants is more pronounced than transgenic poplar lines under drought and salt stress conditions, where RNAi transgenic poplar plants grow faster than wild-type plants under drought stress conditions. The expression level of the poplar MYBS2 gene is inversely related to the biomass and stress resistance of the poplar, so that a new poplar variety with high yield and strong stress resistance can be obtained by interfering the expression of the poplar MYBS2 gene, and the poplar has good market prospect and economic value.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a poplar MYBS2 gene and application thereof.
Background
The poplar (Populus trichocarpa) is used as a fast-growing tree species with the most extensive planting area in the global scope, is one of important artificial forests in China, and brings great ecological and social benefits while creating great economic value for people. Previous studies have found that MYBS2 plays an important role in rice growth, stress tolerance, grain weight and yield. However, related studies of MYBS2 gene in woody plants have not been reported so far.
The present invention has been made based on this.
Disclosure of Invention
The invention aims to provide a poplar MYBS2 gene and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a poplar MYBS2 gene, and the nucleotide sequence of the poplar MYBS2 gene is shown as SEQ ID NO. 1.
The invention also provides a primer group for amplifying and interfering the poplar MYBS2 gene, wherein the forward primer of the primer group is shown as SEQ ID NO. 2;
the reverse primer of the primer group is shown as SEQ ID NO. 3.
The invention also provides an interference fragment obtained by amplifying the primer group, and the interference fragment is shown as SEQ ID NO. 4.
The invention also provides application of the interference fragment in improving the biomass and stress resistance of poplar.
The invention also provides an RNAi expression vector for interfering the poplar MYBS2 gene, wherein the RNAi expression vector comprises the interference fragment and an empty vector;
the empty vector is a pBWA (V) KS vector containing a 35S promoter.
The invention also provides a construction method of the RNAi expression vector, which comprises the following steps:
(6.1) using wild poplar cDNA as a template, and amplifying by using the primer group to obtain the interference fragment;
(6.2) connecting the interference fragment obtained in the step (6.1) with an empty carrier by using T4 ligase to obtain a connection product;
and (6.3) converting the ligation product into escherichia coli competence, then performing a plaque PCR identification test, selecting bacterial liquid corresponding to a positive band, coating the bacterial liquid into a resistance culture medium containing kanamycin, and extracting the plasmid, namely an RNAi expression vector after the sequencing is correct.
The invention also provides application of the RNAi expression vector or the RNAi expression vector constructed by the construction method in improving the biomass and stress resistance of poplar.
The invention also provides a recombinant bacterium, which comprises the RNAi expression vector and a receptor strain;
the recipient strain is agrobacterium LBA4404.
The invention also provides application of the recombinant bacterium in improving the biomass and stress resistance of poplar.
The invention also provides a breeding method for improving the biomass and stress resistance of poplar, which comprises the following steps:
(10.1) placing the poplar aseptic seedling leaves and/or stem segments into the bacterial suspension of the recombinant bacteria, and soaking for 6-10 min to obtain the poplar explant carrying the bacteria;
(10.2) placing the poplar explant carrying the thalli in a co-culture medium, culturing for 2-3 d at the temperature of 23-27 ℃, and transferring to a screening medium for culturing to obtain a resistant bud;
(10.3) cutting off the resistant buds, transferring the resistant buds to a rooting culture medium, and continuing to culture until the root system grows completely to obtain transgenic poplar seedlings to be transplanted;
the co-culture medium takes water as a solvent and comprises the following components in concentration:
4.0-5.0 g/LMS, 28-32 g/L sucrose, 1.5-2.5 mg/L zeatin, 0.8-1.2 mg/L naphthylacetic acid, 7.0-8.0 g/L agar;
the pH value of the co-culture medium is 5.8-6.0;
the screening culture medium takes water as a solvent and comprises the following components in concentration:
4.0-5.0 g/LMS, 28-32 g/L sucrose, 1.5-2.5 mg/L zeatin, 0.8-1.2 mg/L naphthylacetic acid, 180-220 mg/L terliptin, 80-120 mg/L kanamycin and 7.0-8.0 g/L agar;
the pH value of the screening culture medium is 5.8-6.0;
the rooting culture medium takes water as a solvent and comprises the following components in concentration:
2.0 to 2.5g/LMS, 28 to 32g/L sucrose, 0.08 to 0.12mg/L naphthylacetic acid, 80 to 120mg/L terliptin, 80 to 120mg/L kanamycin and 5.5 to 6.5g/L agar;
the pH value of the rooting culture medium is 5.8-6.0 。
The invention provides a poplar MYBS2 gene and application thereof. After the RNAi expression vector interferes with MYBS2 expression, the height, stem circumference, branch number, dry weight and fresh weight of poplar are all improved, and the fresh weight and dry weight of the RNAi poplar are 1.2 times that of a wild plant; under drought and salt stress conditions, the change in growth status of wild type poplar plants is more pronounced than that of RNAi transgenic poplar plants, wherein transgenic Yang Shuzhu lines Ri-2, ri-3 under drought stress conditions grow faster than wild type plants, and it is possible that poplar plants increase its drought tolerance by stimulating SOD enzyme activity, promoting MDA, SP and inhibiting accumulation of Pro content. Poplar with stronger drought tolerance has higher CAT enzyme activity, lower POD enzyme activity and lower SS content. The reason for the low SS content may be that poplar resists damage to cells by drought stress by consuming SS.
Drawings
FIG. 1 shows RNAi expression vectors (A shows RNAi expression vector patterns, and B shows plasmid restriction enzyme electrophoresis patterns).
FIG. 2 shows genetic transformation and identification of transgenic poplar (A represents co-cultivation of poplar, B represents differentiation of resistant buds of transgenic poplar, C represents before transplanting of Ri-1 transgenic poplar, D represents PCR identification result of Ri-1 transgenic poplar, maker DL 1200 marker, P represents positive control, WT-1 represents wild type plant, and Ri-1 represents positive plant of first batch of transgenic poplar).
FIG. 3 shows the growth status of Ri-2, ri-3, ri-4, ri-5, ri-6 transgenic poplar before transplanting and PCR identification results.
FIG. 4 shows the growth of Ri-1 transgenic poplar and wild poplar (A before transplanting; B for the phenotype of day 107 of transplanting; C for the root system of day 107 of transplanting).
FIG. 5 shows the agronomic trait measurements of Ri-1 transgenic poplar and wild poplar (A represents the plant height of the transgenic plant; B represents the diameter of the transgenic plant; C represents the number of branches of the transgenic plant; D represents the internode number of the transgenic plant).
FIG. 6 shows the relative expression levels of MYBS2 in roots, stems and leaves of transgenic poplar and wild poplar (A shows the relative expression level of MYBS2 in roots; B shows the relative expression level of MYBS2 in stems; C shows the relative expression level of MYBS2 in leaves).
FIG. 7 shows the growth phenotype and plant height of transgenic and wild type plants after drought treatment for 20d (A shows the phenotype before and after treatment; B shows the plant height before and after treatment).
FIG. 8 shows the growth phenotype and plant height of transgenic and wild type plants treated with salt for 20d (A shows the phenotype before and after treatment; B shows the plant height before and after treatment).
FIG. 9 shows SOD, CAT, POD activity of drought treatment 20d of transgenic and wild type plants (A represents SOD activity; B represents CAT activity; C represents POD activity).
FIG. 10 is MDA content of drought treated 20d for transgenic and wild type plants.
FIG. 11 shows the SS, SP, pro content of drought treatment 20d for transgenic and wild type plants (A for SS; B for SP; C for Pro).
FIG. 12 shows SOD, CAT, POD activity of transgenic plants and wild type plants treated with salt for 20d (A indicates SOD activity; B indicates CAT activity; C indicates POD activity).
FIG. 13 shows MDA content of salt treatment of transgenic plants and wild type plants for 20 d.
FIG. 14 shows the SS, SP, pro content of transgenic plants and wild type plants treated with salt for 20d (A shows the SS content; B shows the SP content; C shows the Pro content).
FIG. 15 shows the relative expression levels of MYBS2 in roots, stems and leaves after 20d drought treatment of transgenic and wild type plants (A shows the relative expression levels of MYBS2 in roots; B shows the relative expression levels of MYBS2 in stems; C shows the relative expression levels of MYBS2 in leaves).
FIG. 16 shows the relative expression levels of MYBS2 in roots, stems and leaves after 20d salt treatment of transgenic and wild type plants (A shows the relative expression levels of MYBS2 in roots; B shows the relative expression levels of MYBS2 in stems; C shows the relative expression levels of MYBS2 in leaves).
Detailed Description
The poplar used in the examples of the present invention is a aseptic seedling of populus tomentosa.
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
Obtaining transgenic poplar seedlings
(1) Designing a primer group for amplifying the interference poplar MYBS2 gene:
the nucleotide sequence of the MYBS2 gene is shown as SEQ ID NO. 1.
SEQ ID NO.1：
ATGGTAAAAGAGGCAGCAAGAAAGTGCTCCCATTGTGGCCAAAATGGCCATAACTCAAGAACATGTACTAAGGATTGTATCAAATTGTTTGGGGTTAGCATTGAAAAACGTGAACAAACAATTAAAGGAAGTGCTAGCTTAGATAACATAGCGTCCTTAGATGACATTCACGGTGCACATCATGTCGATCCCGGCTATAGTTCCGACGGCGTTATCGGTTCTAAGAGAGGCAGAACAGCGTATACACGGAAGAAAGGCAAGCCATGGACCGAGGAGGAGCATAGAACATTTTTATCCGGTCTAAGCAATCTTGGCAAGGGCGATTGGAGAGGCATTTCGAAGAAATTTGTGATTACTAGGACCCCAAGTCAGGTTGCTAGTCATGCACAGAAGTATTTTCTGAGACAGCAGGCTTCAAATGAGAAGAAGAAACGTAGATCAAGCCTCTTTGACATGACCTTCAAAGGAACTGATCTGGCTTCTCATCAGGATGCTCCGAAACTGCCTTTAATTAAGACTTGTGGGAGTTCATCACAGGCTAGCACCTCTTCAGCTTCACCATTGAGGAAAGCTGGCGAGGATATCCCATCACAAGCTATCAGCCCATTACACCTCATCAACCAATTTCCTTTGCTTTGCTTGCACAATCCCCAGGTCATGAGTCCTACTGTCGCAGCCGGTACTGGCGTTTCAAACTACAATCCTTGCATGCAACGAGTCCTTGCCAATGGACGGCGGAGCTTTCCGGCAAGTAAAGCAGCACCCTTTGTCTCCATGATGAACTATCCAAGGGCCTACCATCCTTACATGCTTAACAGCCCTGCAAGCCTGGCTGGTTGCGCACCTTGTATTGCCCATCAACCATCCGGTATCCCTTCACCAAGTTCATTTCCTCAGAGCTTTTCTCCACAAGGTGCTTCAACTTCATTAGCAAAAATGGAAGACCCTCTTGAGCTTAAAATTGGACAACCCCCTAAATCCCCCCAAGGAGCAAATATATCATCCCCAGCATCTGGTGCCATTAGTGTTATATGA。
Bsal and Eco31I cleavage sites are added to both ends of the forward primer, bsal and Eco31I cleavage sites are also added to both ends of the reverse primer, and CGAT protecting bases are added to both 5' ends of the primer. The specific primer sequences are shown in SEQ ID NO. 2-3.
The sequence of SEQ ID NO.2 is: CTCGTCAAGAAGGCGATAGAAG;
the sequence of SEQ ID NO.3 is: CGTTGGCTACCCGTGATATT.
(2) Construction of RNAi expression vectors
The DNA of wild aspen is used as a template (https:// phytozome. Jgi. Doe. Gov/pz/portal. Html), the primers of SEQ ID NO. 2-3 obtained by the above arrangement are used for amplification, and 1.5% agarose gel electrophoresis detection is carried out, so that 204bp amplification product is obtained, the amplification product is an interference fragment, and the interference fragment is shown as SEQ ID NO. 4. After the interference fragments are dissolved in water, the interference fragments are connected with a pBWA (V) KS vector containing a 35S promoter, the connection products are transformed into escherichia coli competent, kan (kanamycin) resistance plates are coated on the transformation, and then plaque PCR identification experiments are carried out, wherein the target bands are 744bp fragments. And (3) selecting bacterial liquid corresponding to a positive strip, coating the bacterial liquid into an LB culture medium containing 8mLKan resistance, and after the sequencing is correct, obtaining the extracted plasmid which is the pBWA-35S-MYBS2RNAi expression vector, and preserving at the temperature of minus 20 ℃ for later use. RNAi expression vector is shown in FIG. 1A, and plasmid restriction map is shown in FIG. 1B.
SEQ ID NO.4：CAGCAAGAAAGTGCTCCCATTGTGGCCAAAATGGCCATAACTCAAGAACATGTACTAAGGATTGTATCAAATTGTTTGGGGTTAGCATTGAAAAACGTGAACAAACAATTAAAGGAAGTGCTAGCTTAGATAACATAGCGTCCTTAGATGACATTCACGGTGCACATCATGTCGATCCCGGCTATAGTTCCGACGGCGTTATC。
(3) Acquisition of transgenic poplar
The pBWA-35S-MYBS2RNAi expression vector is transferred into agrobacterium LBA4404 to obtain recombinant bacteria. The poplar is genetically transformed by agrobacterium-mediated method, kanamycin is used for screening. The specific results are as follows:
recombinant bacteria were inoculated onto YEP solid medium (20 mg/L Rif, 100 mg/LKan) and the plates were placed upside down in a 28℃incubator for 2d. Inoculating single colony with sterile gun head into 10mLYEP liquid culture medium (20 mg/L Rif, 100mg/LKan, shake culturing at 28deg.C and 200rpm for 24 hr, sucking 1mL of the above bacterial liquid, inoculating into 20mLYEP liquid culture medium (20 mg/L Rif, 100 mg/LKan), and continuing expansion culturing at 28deg.C and 200rpm 600 Value, OD 600 Sub-packaging the bacterial liquid of 0.3-0.5 into a centrifuge tube (10 mL), centrifuging at 4 ℃ and 5000rpm for 10min, pouring out supernatant, and re-suspending bacterial body by using re-suspension to obtain bacterial suspension of recombinant bacteria.
Rif represents rifampicin (weighing 5.0g of RIF powder, placing in a small beaker, adding a small amount of methanol and NaOH (10 mol/L) solution for full dissolution, fixing the volume of deionized water to 50mL, and sub-packaging in a sterile centrifuge tube for refrigeration at-20 ℃ for later use);
the heavy suspension is as follows: 4.432g/L MS+30g/L sucrose+100. Mu. Mol/L acetosyringone, pH was adjusted to 5.90 with acid-base
The leaves and stem segments of the aseptic seedlings of populus tomentosa are used as explants, the leaves are cut into small blocks of 0.5cm multiplied by 0.5cm by a sterile scalpel, the small blocks are immersed in bacterial suspension of recombinant bacteria, the small blocks are soaked for 8min (continuous shaking is carried out during the period to enable bacterial liquid to fully contact with the leaves), then bacterial liquid on the leaves of populus tomentosa is sucked to be dry by using sterile absorbent paper, the leaf backs are tightly arranged on a co-culture medium, the co-culture is carried out as shown in figure 2A, and the co-culture is carried out in a dark incubator at 25 ℃ after marking and sealing by a sealing film. Leaves and stem segments without infection of recombinant bacteria were left as controls, and the culture conditions were unchanged.
Transferring the dark cultured poplar leaves and stem segments into a screening culture medium, and culturing in a 25 ℃ illumination tissue culture chamber; the frequency of medium replacement is that the medium is replaced once in 14 days, and the polluted medium is timely taken away; the resistant buds growing to about 1-2 cm are cut off and transferred to rooting medium for continuous culture, and the differentiation of the resistant buds in rooting medium is shown in figure 2B. And hardening off the seedlings for 2-3 d after root systems of the poplar tissue culture seedlings are complete in development, so that the transgenic poplar seedlings to be transplanted are obtained, and the growth condition before transplanting is shown in figure 2C.
The co-culture medium takes water as a solvent and comprises 4.43g/LMS, 30g/L sucrose, 2.00mg/L Zeatin (ZT), 1mg/L Naphthalene Acetic Acid (NAA) and 7.5g/L agar powder. The pH of the co-culture medium was 5.9.
The screening culture medium takes water as a solvent and comprises 4.43g/LMS, 30g/L sucrose, 2.00mg/L Zeatin (ZT), 1mg/L naphthylacetic acid (NAA), 200mg/L terliptin, 100mg/L Kan and 7.5g/L agar powder. The pH of the screening medium was 5.9.
The rooting culture medium takes water as a solvent and comprises 2.21g/LMS, 30g/L sucrose, 0.1mg/LNAA 100mg/L terlipressin, 100mg/LKan and 6.0g/L agar powder. The pH of the rooting medium was 5.90.
(4) PCR method for detecting transgenic poplar
The poplar DNA is extracted by using a CTAB method, and PCR identification is carried out by using kanamycin sequence design primers, so that 351bp transgenic plants are obtained. The PCR primer sequences, reaction systems and reaction procedures are shown in tables 1, 2 and 3, respectively:
TABLE 1 PCR primer sequences
| Primer name | Primer sequence (5 '-3') |
| Forward | GAATCGGGAGCGGCGATACC |
| Reverse | CCACCAAGCGAAACATCGCAT |
Forward is shown as SEQ ID No.5, and Reverse is shown as SEQ ID No. 6.
TABLE 2 PCR amplification reaction System
TABLE 3PCR amplification reaction procedure
The PCR results are shown in figures 2-3, ri-1 is the transgenic poplar obtained in the first batch; the transgenic poplar obtained in the second batch is: ri-2, ri-3, ri-4, ri-5, ri-6.
As can be seen from the results of FIGS. 2-3, there is no difference between Ri-1 and Ri-2, ri-3, ri-4, ri-5, ri-6. MYBS2 genes in Ri-1, ri-2, ri-3, ri-4, ri-5 and Ri-6 are interfered.
Example 2
Determination of agronomic traits of transgenic poplar
Transplanting the obtained transgenic poplar seedlings to be transplanted, measuring agronomic characters of the obtained Ri-1 transgenic poplar and wild type plants every 7 days from the 30 th day after transplanting, measuring the plant height (cm) of the transgenic poplar and wild type plants by using a metric ruler, measuring the stem thickness (mm) of the plants at the position of 10cm by using a vernier caliper, and recording the internode number and the branch number. The obtained Ri-1 transgenic poplar and wild type plant were cut down at 107d, and the fresh weights of the roots, stems and leaves were measured (the roots were washed with water before weighing, and then dried at 80℃for 60 hours in a constant temperature oven, and when the weights of the respective tissues were constant, the dry weights of the roots, stems and leaves were measured).
TABLE 4 Dry and fresh weight of RNAi transgenic poplar
FIGS. 4-5 and Table 4 show that the Ri-1 transgenic poplar lines obtained were short relative to the wild type plants prior to transplanting, but the heights of the transgenic poplar lines exceeded the wild type plants on day 65; the stem thickness at 10cm exceeded the wild type plants on day 51; branching occurs on the 39 th day of transplanting of the wild type plants, branching occurs on the 42 th day of transplanting of the transgenic lines, and the number of branches of the transgenic lines is more than that of the wild type plants; the fresh and dry weight of the transgenic poplar are 1.2 times that of the wild type plant. This suggests that interfering with expression of the MYBS2 gene may increase the height, stem circumference, branch number, dry weight and fresh weight of transgenic poplar lines.
Example 3
Quantitative PCR detection
Collecting the leaves, stem segments and root systems of wild poplar and transgenic poplar respectively, quick freezing in liquid nitrogen, and extracting the total RNA of poplar by adopting a CTAB method.
Synthesis of cDNA:
the RNA obtained by extraction was reverse transcribed into cDNA by the first strand cDNA synthesis kit for later quantitative analysis, and the reaction system is shown in Table 5.
TABLE 5 Synthesis of first strand cDNA
| Reagent(s) | Volume (mul) |
| Total RNA | 50ng-5μg |
| Oligo(dT)18(0.5μg/μl) | 4μl |
| 5×TRUE Reaction Mix | 4μl |
| RNase free H 2 O to final volume | 20μl |
Gently mixing, incubating at 42℃for 20min, and heating at 85℃for 5min to inactivate TRUEscript H-RTase. The obtained cDNA product is put into a refrigerator with the temperature of minus 80 ℃ for standby.
Real terliptin e-PCR reaction:
MYBS2 gene quantitative amplification primers are designed through on-line software IDT, and the populus tomentosa actin gene (actin) is used as an internal reference gene. The primer sequences are shown in Table 6 and SEQ ID NOS.7 to 10. Fluorescent quantitative PCR experiments were performed on a Bio Rad CFX ConnectTM real-time quantitative PCR instrument. The fluorescent quantitative PCR was performed using a general high-sensitivity dye method quantitative PCR kit for Nanjinopran, and a 10. Mu.l system was prepared according to the instructions on the kit, and the fluorescent quantitative PCR reaction procedure was as shown in Table 7 below and Table 8 below. 3 replicates of each cDNA sample were run and the relative expression was calculated using 2 -ΔΔCt . The results are shown in FIG. 6.
TABLE 6 fluorescent quantitative PCR primer sequences
TABLE 7 fluorescent quantitative PCR reaction System
| Component (A) | Volume (mul) |
| cDNA | 2 |
| Primer F | 0.4 |
| Primer R | 0.4 |
| EvaGreen 2×qPCR Master Mix | 10 |
| Nuclease-free H 2 O | 7.2 |
| Total volume of | 20 |
TABLE 8 fluorescent quantitative PCR reaction procedure
| Temperature (temperature) | Time | Number of cycles |
| 95℃ | 3min | 1 |
| 95℃ | 10sec | 40 |
| 60℃ | 20sec | 40 |
| 72℃ | 30sec | 40 |
FIG. 6 shows that the relative expression levels of MYBS2 genes in roots, stems and leaves of transgenic plants were down-regulated compared to wild type plants. Wherein the relative expression amount of MYBS2 in the roots, stems and leaves of the wild type plants is 1.7 times, 2.1 times and 2.4 times that of the roots, stems and leaves of the Ri-1 strain respectively, and the relative expression amount difference in the roots is obvious (0.01 < P < 0.05).
The experimental results show that: by inhibiting the expression of MYBS2 genes, the relative expression quantity of MYBS2 genes in transgenic plants can be reduced.
Example 4
Abiotic stress treatment and physiological index measurement
Transgenic poplar Ri-2, ri-3, ri-4, ri-5 transplanted 56d and wild type lines transplanted 56d were selected and divided into 2 groups, drought and salt stress treatment groups. Each group included 1 wild type and 2 transgenic plants. A specific method for drought and salt stress treatment is to irrigate 300mL of 25% PEG6000 solution and 150mM NaCl solution per pot, once every 5 d. The plant height before and after the treatment was measured, and leaves at the same position were extracted at 20d of the treatment, and activities or contents of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), malondialdehyde (MDA), soluble Sugar (SS), soluble Protein (SP), and proline (Pro) were measured. Specific results are shown in FIGS. 7 to 14.
Figures 7-8 show that transgenic poplar plants grew better than wild-type plants after 20d drought and salt treatment. Leaf curl occurs in both wild type plants and transgenic plants, but leaf curl is more severe in wild type plants, and Ri-2 is more severe than Ri-3 and Ri-4 is more severe than Ri-5 in transgenic plants with leaf curl. Measurement of plant height before and after treatment found that transgenic lines Ri-2 and Ri-3 grew faster than wild type under drought stress conditions, with Ri-3 growing slightly faster than Ri-2; under salt stress conditions, transgenic lines Ri-4 and Ri-5 grew slower than the wild type.
The experimental results show that: transgenic poplar plants are more tolerant to drought and high salt environments than wild-type plants, wherein Ri-3 is more tolerant to drought environments than Ri-2; ri-5 is more resistant to high salt environments than Ri-4. Whereas transgenic lines Ri-4 and Ri-5 grew slower than the wild type strain, probably because Ri-4 and Ri-5 grew less well before treatment.
FIGS. 9-11 show that transgenic plants Ri-2, ri-3 have higher SOD enzyme activity than wild type after 20d drought treatment; the CAT enzyme activity of the Ri-3 strain is slightly higher than that of the wild type, the CAT enzyme activity of the Ri-2 strain is lower than that of the wild type, and the differences are obvious (0.01 < P < 0.05); the POD enzyme activity of the Ri-2 strain is higher than that of the wild type, the Ri-3 strain is lower than that of the wild type, and the differences are also obvious (0.01 < P < 0.05). The MDA content of the transgenic plants Ri-2 and Ri-3 is increased relative to that of the wild plants, and the difference is obvious (0.01 < P < 0.05). The SS content of the transgenic plant Ri-2 is higher than that of the wild type, the SS content of the plant Ri-3 is lower than that of the wild type, the SP content is higher than that of the wild type, and the differences are obvious (0.01 < P < 0.05); pro content of both Ri-2 and Ri-3 plants was lower than wild type and the differences were very significant (P < 0.01).
FIGS. 12-14 show that after 20d salt treatment, the transgenic plants Ri-4 had higher SOD enzyme activity than the wild type and the plants Ri-5 had lower SOD enzyme activity than the wild type; CAT enzyme activities of Ri-4 and Ri-5 plants are lower than that of wild type, and the difference is very remarkable (P < 0.01); POD enzyme activities of Ri-4 and Ri-5 were higher than wild type and differed significantly (0.01 < P < 0.05). The MDA content of the transgenic plant Ri-4 is increased, the MDA content of the plant Ri-5 is reduced, and the difference is obvious (0.01 < P < 0.05). The SS and SP contents of the transgenic plants Ri-4 and Ri-5 are higher than those of the wild plants, and the differences are obvious (0.01 < P < 0.05) and extremely obvious (P < 0.01) respectively; pro content of Ri-4 plants was higher than that of wild type plants, and Ri-5 plants were lower than that of wild type plants.
The experimental results show that: under drought stress conditions, inhibition of MYBS2 gene expression may affect SOD, CAT and POD antioxidant enzyme activity, cell membrane permeable MDA content, SS, SP and Pro osmotic regulating substance regulation pathways, and plants may stimulate SOD enzyme activity during stress to improve their drought tolerance. Plants with higher drought tolerance have higher CAT enzyme activity and lower POD enzyme activity, promote MDA and SP content and inhibit accumulation of Pro content to improve the drought tolerance of the plants. Plants with greater drought tolerance have lower SS content, probably because plants resist damage to themselves by drought stress through consumption of SS. Under salt stress conditions, inhibition of MYBS2 gene expression may affect SOD, CAT and POD antioxidant enzyme activity, cell membrane permeable MDA content, SS, SP and Pro osmotic regulator regulation pathways, and plants may stimulate POD enzyme activity and inhibit CAT enzyme activity during stress to increase their salt tolerance. Plants with higher salt tolerance have smaller reduction of SOD enzyme activity. Plants with higher salt tolerance have lower MDA content. Greatly promotes the accumulation of the SS and SP content to improve the salt tolerance, and the accumulation of the SS and SP content in plants with higher salt tolerance is less. The Pro content of the plant with higher salt tolerance is reduced, and the Pro content of the plant with lower salt tolerance is slightly increased.
Example 5
Quantitative PCR detection
The procedure of example 5 was followed by the steps of the reaction system, the reaction procedure, etc. according to the procedure of example 3. The detection results are shown in FIGS. 15 to 16.
Fig. 15 to 16 show: after 20d drought treatment, the relative expression levels of MYBS2 in both transgenic poplar Ri-2, ri-3 roots, stems, leaves were reduced compared to wild type plants, wherein the relative expression levels in the stems were very different (P < 0.01). After salt treatment for 20d, the relative expression level of MYBS2 in both transgenic poplar Ri-4, ri-5 roots, stems, leaves was reduced compared to wild type plants, wherein the relative expression level difference in roots was very significant (P < 0.01) and in stems and leaves was significant (0.01 < P < 0.05).
The experimental results show that: under drought and salt stress conditions, the tolerance of the transgenic poplar plant is stronger than that of a wild plant, and the relative expression quantity of MYBS2 genes in roots, stems and leaves of the transgenic poplar plant is lower.
From the above examples, the present invention provides a poplar MYBS2 gene and its application. After the RNAi expression vector interferes with MYBS2 expression, the height, stem circumference, branch number, dry weight and fresh weight of poplar are all improved, and the fresh weight and dry weight of the RNAi poplar are 1.2 times that of a wild plant; under drought and salt stress conditions, the change in growth status of wild type poplar plants is more pronounced than that of RNAi transgenic poplar plants, wherein transgenic Yang Shuzhu lines Ri-2, ri-3 under drought stress conditions grow faster than wild type plants, and it is possible that poplar plants increase its drought tolerance by stimulating SOD enzyme activity, promoting MDA, SP and inhibiting accumulation of Pro content. Poplar with stronger drought tolerance has higher CAT enzyme activity, lower POD enzyme activity and lower SS content. The reason for the low SS content is probably that poplar resists damage to cells by drought stress through SS consumption, and provides basis for the preparation of transgenic poplar in later period.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The poplar MYBS2 gene is characterized in that the nucleotide sequence of the poplar MYBS2 gene is shown as SEQ ID NO. 1.
2. A primer group for amplifying and interfering with the poplar MYBS2 gene of claim 1, wherein the forward primer of the primer group is shown as SEQ ID NO. 2;
the reverse primer of the primer group is shown as SEQ ID NO. 3.
3. The interference fragment obtained by amplification of the primer set of claim 2, wherein the interference fragment is shown in SEQ ID NO. 4.
4. Use of the interference fragment of claim 3 for increasing poplar biomass and stress resistance.
5. An RNAi expression vector that interferes with the poplar MYBS2 gene of claim 1, wherein the RNAi expression vector comprises the interfering fragment of claim 3 and an empty vector;
the empty vector is a pBWA (V) KS vector containing a 35S promoter.
6. A method for constructing an RNAi expression vector according to claim 5, comprising the steps of:
(6.1) amplifying the wild poplar cDNA as a template by using the primer set of claim 2 to obtain the interference fragment of claim 3;
(6.2) connecting the interference fragment obtained in the step (6.1) with an empty carrier by using T4 ligase to obtain a connection product;
and (6.3) converting the ligation product into escherichia coli competence, then performing a plaque PCR identification test, selecting bacterial liquid corresponding to a positive band, coating the bacterial liquid into a resistance culture medium containing kanamycin, and extracting the plasmid, namely an RNAi expression vector after the sequencing is correct.
7. The RNAi expression vector of claim 5 or the RNAi expression vector constructed by the construction method of claim 6 is applied to improving the biomass and stress resistance of poplar.
8. A recombinant bacterium comprising the RNAi expression vector and the recipient strain of claim 5;
the recipient strain is agrobacterium LBA4404.
9. The use of the recombinant bacterium of claim 8 for increasing poplar biomass and stress resistance.
10. A breeding method for improving poplar biomass and stress resistance, comprising the steps of:
(10.1) placing the aseptic seedling leaves and/or stem segments of poplar into the bacterial suspension of the recombinant bacteria in claim 8, and soaking for 6-10 min to obtain the poplar explant carrying the bacteria;
(10.2) placing the poplar explant carrying the thalli in a co-culture medium, culturing for 2-3 d at the temperature of 23-27 ℃, and transferring to a screening medium for culturing to obtain a resistant bud;
(10.3) cutting off the resistant buds, transferring the resistant buds to a rooting culture medium, and continuing to culture until the root system grows completely to obtain transgenic poplar seedlings to be transplanted;
the co-culture medium takes water as a solvent and comprises the following components in concentration:
4.0-5.0 g/LMS, 28-32 g/L sucrose, 1.5-2.5 mg/L zeatin, 0.8-1.2 mg/L naphthylacetic acid, 7.0-8.0 g/L agar;
the pH value of the co-culture medium is 5.8-6.0;
the screening culture medium takes water as a solvent and comprises the following components in concentration:
4.0-5.0 g/LMS, 28-32 g/L sucrose, 1.5-2.5 mg/L zeatin, 0.8-1.2 mg/L naphthylacetic acid, 180-220 mg/L terliptin, 80-120 mg/L kanamycin and 7.0-8.0 g/L agar;
the pH value of the screening culture medium is 5.8-6.0;
the rooting culture medium takes water as a solvent and comprises the following components in concentration:
2.0 to 2.5g/LMS, 28 to 32g/L sucrose, 0.08 to 0.12mg/L naphthylacetic acid, 80 to 120mg/L terliptin, 80 to 120mg/L kanamycin and 5.5 to 6.5g/L agar;
the pH value of the rooting culture medium is 5.8-6.0.
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