CN116854794A - Ginkgo MYC2 transcription factor, and coding gene and application thereof - Google Patents
Ginkgo MYC2 transcription factor, and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a ginkgo MYC2 transcription factor, and a coding gene and application thereof. The amino acid sequence of the MYC2 transcription factor is shown in a sequence table SEQ ID NO:1, the plant is transferred into a model plant Arabidopsis thaliana, and through drought and bemisia tabaci stress experiments, the plant Arabidopsis thaliana is found to have certain drought resistance and insect resistance functions, and a theoretical basis is laid for developing new varieties of drought resistance and insect resistance plants.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a ginkgo MYC2 transcription factor, and a coding gene and application thereof.
Background
Drought is a main factor of crop yield reduction in China, and the water deficiency of plant tissues under drought conditions influences the absorption and transportation of nutrients, so that the concentration of the nutrients of each tissue is reduced. Drought tolerance genes in plants are mainly divided into two types, wherein one type is a functional protein gene, such as a gene related to encoding a permeation regulating substance; the other is a transcription factor which has regulation and control function on drought-enduring gene expression, such as DREB and the like. The regulation and control of the transcription factors have obvious effects, and a plurality of plant transcription factors are reported to be used for regulating drought resistance at present, and as disclosed in patent 201010161188.X, novel genes of rice zinc finger protein transcription factors and drought resistance and salt tolerance application are disclosed; patent 202210026086.X discloses a MYB transcription factor for improving drought resistance of mountain new poplar and application thereof; patent 201910627653.5 discloses a transcription factor NtMYB44b capable of improving drought resistance of tobacco, and a site-directed mutagenesis method and application thereof; patent 201811421792.4 discloses the use of coptis SDIR transcription factor in improving drought resistance of plants; drought-resistant transcription factors are found in various plants, but currently, drought-resistant transcription factors are not found in ginkgo.
Insect disaster is also a main factor of crop yield reduction in China, bemisia tabaci is a worldwide pest, bemisia progressive insects, and the individual development of the bemisia tabaci is divided into 3 stages of eggs, nymphs and adults. Nymphs of 3 years old, the pupae formed after the nymphs of 3 years old are often called pseudopupae or pseudopupae. Adults are mainly parasitic on She Beimian, are light yellow and white in body, are 2 pairs of wings, are white, have no spots by wax powder, and are smaller than greenhouse white flies. Bemisia tabaci presents different hazard symptoms to different plants, leaf vegetables such as cabbage and cauliflower suffer leaf atrophy, yellowing and wilting; root vegetables such as radish are damaged and show color whitening, smell-less and weight-reduced; fruit and vegetable such as tomato suffer damage and the fruit is unevenly ripe. Bemisia tabaci is of various biotypes. According to investigation on crops such as cotton, soybean and the like, the distribution of bemisia tabaci on host plants has a tendency of gradually transferring from the middle part to the lower part to the upper part, adults are mainly concentrated on the lower part, the numbers of eggs and 1-2-year-old nymphs are gradually increased from bottom to top, and the numbers of 3-4-year-old nymphs and pupa shells are gradually reduced. The plant gene for resisting the bemisia tabaci is found, the bemisia tabaci is resisted by the plant itself, the pesticide use is reduced, and the method has great significance for green agriculture development.
Disclosure of Invention
The invention aims to provide a gingko MYC2 transcription factor, and a coding gene and application thereof.
A gingko MYC2 transcription factor, the amino acid sequence of which is shown in a sequence table SEQ ID NO: 1.
The gene sequence of the MYC2 transcription factor is shown in a sequence table SEQ ID NO: 2.
A vector containing the MYC2 transcription factor gene.
A host bacterium containing the vector of the MYC2 transcription factor gene.
And amplifying a primer of any fragment of the MYC2 transcription factor gene.
Application of Ginkgo MYC2 transcription factor in plant stress resistance is provided.
The stress resistance is drought resistance.
Application of Ginkgo MYC2 transcription factor in plant pest resistance is provided.
The invention has the beneficial effects that: the invention clones a transcription factor gene GbMYC2 in ginkgo leaf, transfers the gene into model plant Arabidopsis thaliana, and discovers that the gene has certain drought resistance and insect resistance through drought and bemisia tabaci stress experiments, thereby laying a theoretical foundation for developing new varieties of drought resistance and insect resistance plants.
Drawings
FIG. 1 is a schematic diagram of the structure of a pBI121-GbMYC2 plant expression vector.
FIG. 2 is a phenotype of WT, L13, L29, L33 genotype Arabidopsis under drought stress.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1
The ginkgo leaf is used as a material, and total RNA is extracted and reversely transcribed into cDNA. RNA extraction of ginkgo leaf material using Trizol method (Invitrogen); digestion of DNA in RNA using DNase I kit (Promega Co.); RNA was reverse transcribed into cDNA using the M-MLV Reverse Transcriptase kit (Promega). The steps of RNA extraction, DNA digestion treatment and RNA reverse transcription are referred to the relevant company specifications.
According to the gene sequence of the ginkgo MYC2 transcription factor published by NCBI https:// www.ncbi.nlm.nih.gov/protein/WFD53488.1, a primer with enzyme cutting sites XbaI and KpnI is designed, and the gene sequence of the MYC2 transcription factor is amplified.
MYC2 primer F: TGC (TGC)TCTAGAATGGCCGAACAAACCC；
MYC2 primer R: TCGC (TCGC)GGTACCTCAAATACTAGGATC。
The cDNA is used as a template to amplify and obtain the full-length GbMYC2 gene, and the full-length GbMYC2 gene is cloned to a pMD18-T vector. The pMD18-T vector and pBI121 empty vector containing full length GbMYC2 were digested with XbaI and KpnI, respectively, and GbMYC2 was ligated into pBI121 to finally obtain pBI121-GbMYC2 plant expression vector (FIG. 1), and the pBI121-GbMYC2 plant expression vector was transferred into Agrobacterium and stored at-20 ℃.
Example 2
Inoculating agrobacterium tumefaciens bacteria solution containing target gene recombinant plasmid stored at-20 ℃ into 10mL YEB liquid culture medium (containing 80mg/L Rif, 120mg/L Str and 150mg/L Kan), and activating at 28 ℃ for 2d at 200 rpm; according to the following steps of 1:100 proportion 2.5mL bacterial liquid is inoculated in 250mL YEB liquid culture medium (containing 80mg/LRif, 120mg/L Str and 150mg/L Kan), and cultured at 28 ℃ at 200rpm overnight until OD600 is 1.2-1.6; centrifuging at 5,000rpm for 10min, collecting thallus, suspending the thallus in 500mL 5% sucrose solution, mixing, and transferring into a conversion cup as conversion solution; pouring the arabidopsis thaliana to be transformed for 2 days before water penetration, removing the pod, inverting the arabidopsis thaliana in a transformation cup, gently shaking the transformation liquid, removing plants after 30s of dip dyeing, shaking off the redundant transformation liquid, wrapping the plants with a preservative film, and placing the plants in a tray at the side of the tray to avoid light for 1d.
Taking out the T0 generation seeds and the wild seeds of the transgenic arabidopsis thaliana which are vernalized in a refrigerator at the temperature of 4 ℃, respectively and uniformly sowing the T0 generation seeds and the wild seeds on the surface of sterilized nutrient soil, and coating the seeds; and after the seeds germinate, removing the preservative film. After a week, when the true leaves grow out, the Basta herbicide is sprayed (10% of Basta mother liquor is diluted 10000 times by tap water), the wild type Arabidopsis thaliana is sprayed with the same concentration of Basta herbicide and tap water respectively as a control, and the spraying is carried out twice a day. Screening effect was observed after one week: the wild type and non-positive seedlings gradually yellow and grow stagnantly, while the positive seedlings have emerald green leaves and grow normally like the wild type Arabidopsis sprayed with tap water; transferring positive seedlings to newly sterilized nutrient soil for culture, marking as T1 generation, and taking leaf extract DNA for PCR molecular identification when more leaves grow out.
And (5) continuing planting seeds harvested on the transgenic arabidopsis positive to the PCR identification for generation-adding propagation. PCR identification is carried out on each generation of Arabidopsis single plant, and selfing is carried out on positive plants, and 3 transgenic Arabidopsis homozygous lines, namely GbMYC2-13 (L13), gbMYC2-29 (L29) and GbMYC2-33 (L33), are detected in the T3 generation.
WT, L13, L29 and L33 genotype Arabidopsis are stopped watering for 10 days after the soil grows normally for 4 weeks, so that the Arabidopsis is naturally drought, and the growth states of the 4 genotypes are similar and have no obvious difference under the normal growth state. When drought hypochondriac force drop is temporary, compared with WT plants, L13, L29 and L33 genotype Arabidopsis plants grow well and have luxuriant leaves (figure 2).
The change in water content of WT, L13, L29, L33 genotype arabidopsis before and after drought was measured, and the measurement results are shown in table 1:
TABLE 1
Note that: * Representing P <0.05 compared to WT group.
100mg of leaves of wild type Arabidopsis thaliana and transgenic Arabidopsis thaliana lines L13, L29, L33 after drought were respectively ground into fine powder. Soluble sugar content determination using a plant soluble sugar content kit (a 145); the total protein content adopts a total protein quantitative kit (A045-2); POD activity was measured using the peroxidase kit (A084-3).
Statistical analysis using SPSS 24.0 software for metering data results(mean Standard deviation) means that data normalization test is performed by using a Kolmogorov-Smirnov test method, and for data conforming to normal distribution, the mean difference between two groups is compared by using a t test, so that the data is P<A difference of 0.05 is statistically significant.
The measurement results are shown in Table 2:
TABLE 2
Note that: * Representing P <0.05 compared to WT group.
Example 3
The bemisia tabaci used in the experiment is Bemisiatabaci MEAM (mtCOI, genBank number is MF 579701) which is presented by the institute of microbiology of the national academy of sciences, the bemisia tabaci is fed into an insect cage, cotton is used as a host plant for growth and reproduction, the humidity is 65%, and the illumination period is 12h illumination/12 h darkness.
Firstly, growing wild arabidopsis plants, L13, L29 and L33 arabidopsis plants on a culture dish for 21 days, then moving the plants to soil for 14 days, and adopting a leaf cage method to perform a bemisia tabaci bioassay experiment: in the bemisia tabaci oviposition experiment, 3 female worms and 3 male worms are placed in leaf cages, and the number of eggs is counted after 10 days. In experiments of the development of the bemisia tabaci pseudo pupae, placing 16 female insects into leaf cages, and after the female insects lay eggs for 2 days, completely removing the bemisia tabaci adults, and keeping eggs continuously developing in the leaf cages; the number of pseudo pupae was counted after 20 days. The experiment was repeated 3 times for 8 strains per line, and the results were averaged.
Statistical analysis using SPSS 24.0 software for metering data results(mean Standard deviation) means that data normalization test is performed by using a Kolmogorov-Smirnov test method, and for data conforming to normal distribution, the mean difference between two groups is compared by using a t test, so that the data is P<A difference of 0.05 is statistically significant.
The measurement results are shown in Table 3:
TABLE 3 Table 3
Note that: * Representing P <0.05 compared to WT group.
As can be seen from table 3, the number of oviposition and pseudo pupae of bemisia tabaci on the L13, L29, L33 arabidopsis plants was significantly reduced compared to the wild type arabidopsis plants. The result shows that the pest resistance of the transgenic GbMYC2 Arabidopsis plant is obviously improved, and the expression of the GbMYC2 gene is shown to improve the pest resistance of the plant.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (8)
1. The ginkgo MYC2 transcription factor is characterized in that the amino acid sequence of the MYC2 transcription factor is shown in a sequence table SEQ ID NO: 1.
2. The MYC2 transcription factor of claim 1, wherein the gene sequence of the MYC2 transcription factor is as set forth in the sequence listing SEQ ID NO: 2.
3. A vector comprising the MYC2 transcription factor gene of claim 2.
4. A host bacterium comprising the vector of MYC2 transcription factor gene according to claim 3.
5. A primer for amplifying any fragment of the MYC2 transcription factor gene of claim 2.
6. Application of Ginkgo MYC2 transcription factor in plant stress resistance is provided.
7. The use of MYC2 transcription factor according to claim 6 in stress tolerance, which is drought resistance, of plants.
8. Application of Ginkgo MYC2 transcription factor in plant pest resistance is provided.
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