Application of ZmSCL14 gene in improving plant stress resistance

Abstract

The present invention discloses the application of ZmSCL14 gene in improving plant stress resistance. The present invention proves through experiments that the ZmSCL14 gene has the ability to improve corn salt stress resistance and drought stress. The present invention obtains an overexpression strain of the ZmSCL14 gene through genetic engineering technology, and finally obtains a homozygous strain of overexpression of the ZmSCL14 gene after screening. The homozygous strain has the ability to significantly improve plant salt stress resistance and drought stress, can not only increase the seed germination rate, but also can improve the root development of plants under salt stress in the seedling stage. The present invention confirms through experiments that the ZmSCL14 gene has potential application value in improving plant salt stress resistance and drought stress, and lays a good theoretical and application foundation for cultivating salt-tolerant, drought-tolerant and high-yield corn varieties using the ZmSCL14 gene.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of ZmSCL14 gene in improving plant stress resistance. Background Art

[0002] Corn is a major food crop and cash crop in China. In recent years, under the combined effects of adverse natural conditions (such as changes in climate factors such as less rainfall), artificial conditions (such as excessive use of land resources and unreasonable fertilization and irrigation), and farming patterns that are not adapted to local conditions, the salinization problem of cultivated land in some of my country's main corn-producing areas has become increasingly serious, which is not suitable for the normal growth of corn and cannot guarantee corn yields. Therefore, it is of great theoretical and practical significance to systematically analyze the molecular genetic basis of corn drought and salt tolerance, deeply explore the potential of corn drought and salt tolerance, and cultivate salt-tolerant and drought-tolerant corn varieties.

[0003] The ZmSCL14 gene belongs to the LISCL subfamily of the GRAS transcription factor family, and studies have analyzed other members of this subfamily. Arabidopsis GRAS protein SCL14 can interact with class II TGA transcription factors, and it is determined that the AtSCL14 gene regulates the induced expression of genes involved in the detoxification of harmful chemicals and is a transcriptional co-activator of TGA transcription factors. In wheat, TaSCL14 can be induced to express under strong light stress, confirming that TaSCL14 is very sensitive to strong light stress, and the silencing of TaSCL14 will inhibit plant growth and reduce the photosynthetic capacity of plants. However, to date, no reports on the ZmSCL14 gene, abiotic stress and plant root morphology have been found. Summary of the invention

[0004] The present invention provides an application of the ZmSCL14 gene in improving plant stress resistance. The present invention proves through experiments that the ZmSCL14 gene has the function of improving corn salt stress resistance and drought stress resistance.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0006] The invention provides application of ZmSCL14 gene in improving plant stress resistance.

[0007] Furthermore: the CDS nucleotide sequence of the ZmSCL14 gene is shown in SEQ ID NO.1.

[0008] Further: the stress resistance includes salt stress resistance and drought stress resistance.

[0009] Further: the plant is corn.

[0010] Furthermore: Under salt stress, the plant seed germination rate of the ZmSCL14 gene overexpression strain was significantly higher than that of the wild-type strain.

[0011] Furthermore: Under salt stress, the plant growth of the ZmSCL14 gene overexpression strain was significantly better than that of the wild-type strain.

[0012] Furthermore: the plant growth conditions include main root length, longest seminal root length, number of adventitious roots, lateral root density, chlorophyll content, and seedling length.

[0013] Furthermore: under salt stress, the main root length and the longest seminal root length of the ZmSCL14 gene overexpression strain were longer than those of the wild-type strain; the number of its adventitious roots and lateral root density were greater than those of the wild-type strain; and the seedling height and chlorophyll content were higher than those of the wild-type strain.

[0014] Furthermore: Under drought stress, the plant seed germination rate of the ZmSCL14 gene overexpression strain was significantly higher than that of the wild-type strain.

[0015] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0016] The present invention obtains an overexpression vector pUbi::ZmSCL14 of the ZmSCL14 gene through genetic engineering technology, and finally obtains a homozygous strain of corn ZmSCL14 gene overexpression after screening. Under salt stress, the homozygous strain has the ability to significantly improve plant salt stress resistance and drought stress, which can not only increase the seed germination rate, but also improve the root development of plants under salt stress in the seedling stage. The present invention confirms through experiments that the ZmSCL14 gene has potential application value in improving plant salt stress resistance and drought stress, and lays a good theoretical and application foundation for cultivating salt-tolerant, drought-tolerant, and high-yield corn varieties using the ZmSCL14 gene; it can also prove that corn ZmSCL14 is an important transcription factor involved in regulating plant adaptation to adversity, and has great application potential in molecular breeding of corn stress tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electrophoresis diagram for the identification of homozygous strains overexpressing the ZmSCL14 gene.

[0018] Figure 2 This is the result of expression level analysis of ZmSCL14 gene overexpression homozygous strain.

[0019] Figure 3 is the seed germination rate of wild type WT and ZmSCL14 gene overexpression homozygous strains OE-1 and OE-3; Figure 3 A is the seed germination phenotype of the strain under normal conditions and under salt stress and drought stress; Figure 3 B is the statistical result of germination rate.

[0020] Figure 4 Seedling phenotypes of root development of wild type WT and ZmSCL14 gene overexpression homozygous line OE-1

[0021] Figure 5 Statistical results of root development of wild type WT and ZmSCL14 gene overexpression homozygous line OE-1. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0023] The reagents used in the following experiments were purchased from biological companies such as TAKARA, Sigma, ABclonal, and Hushi; the culture matrix is ​​a commercially available product.

[0024] Example 1: Acquisition and identification of homozygous strains overexpressing ZmSCL14 gene

[0025] The CDS sequence of the maize ZmSCL14 gene was obtained from the NCBI database. The CDS sequence is 1860 bp in length and encodes a protein containing 645 amino acids. The nucleotide sequence and amino acid sequence of the ZmSCL14 gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0026] 1. Obtaining homozygous strains of ZmSCL14 gene overexpression

[0027] 1. Obtaining ZmSCL14 gene overexpression strain

[0028] (1) Based on the sequence of the ZmSCL14 gene obtained above, gene-specific primers were designed, and the sequences were as follows: ZmSCL14-F: 5'-ATGGATCCATGATAATGGACCCTCGTCC-3' (SEQ ID NO. 3); ZmSCL14-R: 5'-ATGAGCTCCTAGCTAGTCCATGCTGAAA-3' (SEQ ID NO. 4).

[0029] The ZmSCL14 gene was amplified by PCR technology, and the amplification system (50 μL system) was: ZmSCL14-F primer (5 μmol / μL) 2 μL, ZmSCL14-R primer (5 μmol / μL) 2 μL, 2×PhantaMax polymerase mix (final concentration 1×) 25 μL, template DNA 1 μL, and ddH2O 20 μL.

[0030] The PCR program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 58°C for 30 s; extension at 72°C (2×PhantaMax polymerase 1 Kb / 30 s) for 2 min; (denaturation-annealing-extension for 35 cycles); post-extension at 72°C for 7 min; and storage at 16°C.

[0031] After that, 50 μL of the reaction solution was placed in a 1% agarose gel for electrophoresis, the target band was cut out, and the gel was recovered using a full-form gold gel recovery kit. The T4 DNA ligase used for connection was purchased from Takara. This experiment used a 10 μL system for connection: 1 μL of T4 DNA ligase, 1 μL of T4 Buffer, 4 μL of gel recovery product (fragment), and 4 μL of gel recovery product (vector). After adding the sample and centrifuging, it was blown with a pipette and reacted at 16°C overnight. After the connection was completed, Escherichia coli DH5α was transformed to obtain the pUbi::ZmSCL14 recombinant plasmid. After sequencing confirmation, it was handed over to the company for overexpression corn material transformation.

[0032] 2. Identification of homozygous strains overexpressing ZmSCL14

[0033] After transformation, the ZmSCL14 gene overexpression maize material received was germinated, samples were taken, DNA was extracted, and PCR was performed for identification. The amplification system (50 μL system) was: OESCL14-F primer (5 μmol / μL) 2 μL, OESCL14-R primer (5 μmol / μL) 2 μL, 2×PhantaMax polymerase mix (final concentration 1×) 25 μL, template DNA 1 μL, ddH2O 20 μL. The amplification program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension (2×PhantaMax polymerase 1Kb / 30s) 1 min; (denaturation-annealing-extension 35 cycles); 72℃ post-extension for 7 min; 16℃ storage.

[0034] Afterwards, 50 μL of the reaction solution was placed in a 1% agarose gel for electrophoresis. Figure 1 As shown: B104 is the WT control without the target band, while OE1 and OE3 all have the target band of 750bp, which is identified as a possible homozygous strain.

[0035] Amplification primers are as follows:

[0036] OESCL14-F: 5'-CTCGTCTTCAAGAAGGAACTG-3' (SEQ ID NO.5);

[0037] OESCL14-R: 5'-GCAGCATCTATTCATATGCTC-3' (SEQ ID NO. 6).

[0038] 3. RT-PCR detection of expression

[0039] RNA was extracted from seeds of ZmSCL14 gene overexpression homozygous strains OE1 and OE3, and reverse transcription was performed. The RNA reverse transcription kit cDNA was purchased from Kangwei Reagent Co., Ltd., and the experimental methods were all referred to the kit instructions. For the convenience of the experiment, the concentration of RNA used as a reverse transcription template in the experiment was 200ng / μL. The reverse transcription system (10μL) is as follows: Total RNA 5μL, Oligo (dT) 0.5μL, ddH2O 0.5μL, 5×Buffer 2μL, 10mM dNTP Mix 1μL, inhibitor 0.5μL, Reversetranscriptases 0.5μL. The reverse transcription program is: 42℃, 15min; 85℃, 5min. The concentration of the cDNA sample obtained in the experiment was adjusted to 300 ng / μL for fluorescence quantitative PCR experiments. The kit used for fluorescence quantitative PCR was purchased from AG Bioreagent Co., Ltd. The system was as follows: SYBR Green 10 μL, cDNA added to 1000 ng, ZmSCL14-RT-F primer (5 μmol / μL) 0.4 μL, ZmSCL14-RT-R primer (5 μmol / μL) 0.4 μL, ddH2O added to 20 μL. The instrument used for fluorescence quantitative PCR was QuantStudio6plex real-time fluorescence quantitative PCR instrument produced by ABI. RT-PCR experiments were performed.

[0040] ZmSCL14-RT-F: 5'-GCGAATGCCTTACGGATGGATG-3' (SEQ ID NO. 7);

[0041] ZmSCL14-RT-R: 5'-CCCTGAGGTTTCTCATCTGCCA-3' (SEQ ID NO. 8).

[0042] The experimental results are as follows Figure 2 As shown, it was shown that the expression level of ZmSCL14 gene was increased in the homozygous strains.

[0043] Example 2: Effect of ZmSCL14 gene on seed germination under salt stress and drought stress

[0044] Seeds of the wild-type strain B104 and the two homozygous strains of ZmSCL14 gene overexpression OE1 and OE3 obtained in Example 1 were used as treatment materials. Experimental corn seeds of the same size and full grains were selected at the same time, and the surface of the seeds was disinfected with sodium hypochlorite for 15 minutes. And washed with distilled water. The seeds were evenly placed in a sterilized glass culture dish, and the same volume of treatment solution was added to each culture dish and sealed with paraffin film. The experimental group was treated with 100mM NaCl, 150mM NaCl, and 12% PEG-6000, and the control group was treated with the same volume of water. Each culture dish was a replicate, and the experiment was repeated three times. Put it in an incubator and culture it until germination, treat it for five days, and observe and record the statistical phenotype.

[0045] Phenotypic results Figure 3 As shown in A, under normal conditions, there was no significant difference in germination between wild-type B104 and overexpression plants (Bar = 7.5 cm); however, under 100 mM NaCl and 150 mM NaCl salt stress conditions and 12% PEG-6000 simulated drought stress conditions, the germination of OE1 and OE3 plants was significantly better than that of wild-type B104 plants. Figure 3 As shown in B, during the same period, there was no significant change in the seed germination rate of the wild-type plant B104 in the control group and the overexpression plant, but in the salt stress group and the simulated drought treatment group, the seed germination rate of the overexpression line was significantly higher than that of the wild-type line B104. The above phenotypic experimental results show that the overexpression of the ZmSCL14 gene can significantly improve the salt stress resistance and drought stress resistance of corn, increase the germination rate of corn seeds, and alleviate the salt stress and drought stress suffered by seeds.

[0046] Example 3: Effect of ZmSCL14 gene overexpression on plant growth phenotype under salt stress

[0047] Seeds of the wild-type strain B104 and the overexpression homozygous strain OE1 obtained in Example 1 were used as treatment materials. Experimental seeds of the same size and full grains were selected at the same time, sterilized with sodium hypochlorite for 15 minutes, and washed with distilled water. The seeds were evenly placed in a sterilized glass culture dish, and the same volume of sterilized distilled water was added to each dish and sealed with paraffin film. They were placed in an incubator and cultured until germination. After whitening, seeds with basically the same germination state were selected and moved into a germination box filled with sterilized sand. Each box was a repeat. After transplanting, the same volume of solution was poured. The experimental group was treated with 100mM NaCl and 150mM NaCl solutions, and the control group was treated with the same volume of water. The experiment was repeated three times and treated for five days. After the treatment was completed, the phenotypes of the plants under different treatments were observed, and the data of the taproot length, the longest seed root length, the chlorophyll content, the seedling length, the number of adventitious roots, and the lateral root density of the wild-type plant B104 and the overexpression plant in the treatment group and the control group were statistically analyzed.

[0048] The results are as follows Figure 4 (Bar = 10 cm) and Figure 5 As shown in the figure, the growth of plants in the salt stress group was significantly lower than that in the control group. Under normal conditions, there was no significant difference between wild-type B104 and OE1 plants. However, under the salt treatment conditions of 100mM NaCl and 150mM NaCl solutions, the main root length and the longest seminal root length of OE1 plants were longer than those of wild-type B104; the number of adventitious roots and the density of lateral roots of OE1 plants were greater than those of wild-type B104; in terms of seedling height and chlorophyll content, OE1 plants were taller than wild-type B104.

[0049] The above results show that the root development of ZmSCL14 gene overexpressing plants under salt stress is significantly better than that of wild-type plant B104. This shows that salt stress can significantly hinder the normal growth and development of plants, while ZmSCL14 gene can significantly improve the salt stress resistance of corn, increase the survival rate of corn, and increase the survival ability of corn seedlings.

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. ZmSCL14 The use of a gene in improving plant stress resistance is characterized in that: Said ZmSCL14 The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1; the stress resistance includes salt stress resistance and drought stress resistance; and the plant is corn.

2. The use according to claim 1, characterized in that: Under salt stress, ZmSCL14 The seed germination rate of the gene overexpression strain was significantly higher than that of the wild-type strain.

3. The use according to claim 1, characterized in that: Under salt stress, ZmSCL14 The plant growth of the gene overexpression strain was significantly better than that of the wild-type strain.

4. The use according to claim 3, characterized in that: The plant growth conditions include main root length, longest seminal root length, adventitious root quantity, lateral root density, chlorophyll content, and seedling length.

5. The use according to claim 1, characterized in that: Under drought stress, ZmSCL14 The seed germination rate of the gene overexpression strain was significantly higher than that of the wild-type strain.

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