CN108997487B

CN108997487B - Application of stress resistance-related protein Z76 in regulation and control of plant stress resistance

Info

Publication number: CN108997487B
Application number: CN201811024442.4A
Authority: CN
Inventors: 贺琳; 袁红梅; 徐晶宇; 杨克军; 程殿君
Original assignee: Institute Of Industrial Crops Of Heilongjiang Academy Of Agricultural Sciences; Heilongjiang Bayi Agricultural University
Current assignee: Institute Of Industrial Crops Of Heilongjiang Academy Of Agricultural Sciences; Heilongjiang Bayi Agricultural University
Priority date: 2018-09-04
Filing date: 2018-09-04
Publication date: 2021-06-15
Anticipated expiration: 2038-09-04
Also published as: CN108997487A

Abstract

The invention discloses an application of stress-resistance-related protein Z76 in regulation and control of plant stress resistance. The stress-resistant related protein Z76 provided by the invention is a1) or a2) or a 3): a1) the amino acid sequence is protein shown as a sequence 2 in a sequence table; a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table; a3) the protein which is related to the plant stress resistance is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table. Experiments prove that the Z76 gene is overexpressed in wild arabidopsis thaliana to obtain Z76 gene-transferred arabidopsis thaliana; compared with wild arabidopsis thaliana, the Z76 transgenic arabidopsis thaliana has enhanced stress resistance. Therefore, the protein Z76 has important theoretical significance and practical value in cultivating plants with enhanced stress resistance.

Description

Application of stress resistance-related protein Z76 in regulation and control of plant stress resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of stress-resistance-related protein Z76 in regulation and control of plant stress resistance.

Background

Under the stress of adversity, a series of response reactions are generated in plants, and a plurality of physiological, biochemical and developmental changes are accompanied. The reaction mechanism of the plant to the stress is determined, and scientific data is provided for the research and application of the stress-resistant gene engineering. At present, the research on plant stress resistance is gradually deepened to the cellular and molecular level, and combined with the research on genetics and genetic engineering, the plant growth characteristics can be improved by utilizing biotechnology, and the adaptability of plants to stress is further improved.

Under the adverse conditions of environmental stresses such as drought, high salinity and the like, the plants can be correspondingly adjusted on molecular, cellular and overall levels so as to reduce the damage caused by the environment to the maximum extent and survive. Many genes are induced to express by stress, and the products of the genes not only can be directly involved in the stress response of plants, but also can regulate the expression of other related genes or be involved in signal transduction pathways, so that the plants can avoid or reduce damage, and the resistance to the stress environment is enhanced.

Disclosure of Invention

The invention aims to improve the stress resistance of plants.

The invention firstly protects the application of the protein Z76 in regulating and controlling the stress resistance of plants; the protein Z76 may be a1) or a2) or a 3):

a1) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

a3) the protein which is related to the plant stress resistance is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table.

Wherein, the sequence 2 in the sequence table is composed of 267 amino acid residues.

In order to facilitate the purification of the protein in a1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

The protein according to a3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 1 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The application of the nucleic acid molecule for coding the protein Z76 in regulating the stress resistance of plants also belongs to the protection scope of the invention.

In the above application, the nucleic acid molecule encoding the protein Z76 can be a DNA molecule represented by b1) or b2) or b3) or b 4):

b1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

b3) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b1) or the (b2) and codes the protein Z76;

b4) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under strict conditions and codes the protein Z76.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 1 in the sequence table is composed of 804 nucleotides, and the nucleotide of the sequence 1 in the sequence table encodes an amino acid sequence shown as a sequence 2 in the sequence table.

In the above application, the regulation of the plant stress resistance may be an increase in the plant stress resistance.

In the above application, the stress resistance may be salt resistance and/or drought resistance.

In the above application, the plant may be any one of the following c1) to c 8): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a cruciferous plant; c5) corn; c6) maize hybrid 344 inbred line; c7) arabidopsis thaliana; c8) the wild type Arabidopsis thaliana Columbia-0 subtype.

The invention also provides a method for cultivating transgenic plants, which comprises the following steps: improving the expression quantity and/or activity of the protein Z76 in the original plant to obtain a transgenic plant; the stress resistance of the transgenic plants is enhanced compared to the starting plants.

In the above method, the "increasing the expression level and/or activity of the protein Z76 in the starting plant" can be achieved by a method known in the art, such as multiple copies, modification of promoters, regulatory factors, transgenes, etc., to increase the expression level and/or activity of the protein Z76 in the plant.

In the above method, the "improvement in the expression level and/or activity of the protein Z76 in the starting plant" may be achieved by introducing a substance which improves the expression level and/or activity of the protein Z76 into the starting plant.

The "introduction of a substance which increases the expression level and/or activity of the protein Z76" into a starting plant may be specifically carried out by introducing a nucleic acid molecule encoding the protein Z76 into the starting plant.

In the above method, the nucleic acid molecule encoding the protein Z76 may be a DNA molecule represented by b1) or b2) or b3) or b 4):

In the above method, the "introducing into a starting plant a nucleic acid molecule encoding the protein Z76" may be carried out by introducing into a starting plant a recombinant vector; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein Z76 into an expression vector. The recombinant vector can be specifically the recombinant plasmid pROK II-Z76 mentioned in the examples. The recombinant plasmid pROK II-Z76 is obtained by inserting a DNA molecule shown in a sequence 1 in a sequence table into a restriction enzyme cutting recognition site of restriction enzymes KpnI and SacI of a pROK II vector.

The invention also provides a plant breeding method, which comprises the following steps: increasing the content and/or activity of said protein Z76 in the plant, thereby increasing stress resistance.

In any of the above methods, the stress resistance may be salt resistance and/or drought resistance.

In any of the methods above, the plant may be any of the following c1) to c 8): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a cruciferous plant; c5) corn; c6) maize hybrid 344 inbred line; c7) arabidopsis thaliana; c8) the wild type Arabidopsis thaliana Columbia-0 subtype.

Any of the above-described drought can be simulated (under laboratory conditions) by the addition of mannitol. The higher the concentration of added mannitol, the greater the degree of drought in the environment.

Experiments prove that the Z76 gene is overexpressed in wild arabidopsis thaliana to obtain Z76 gene-transferred arabidopsis thaliana; compared with wild arabidopsis thaliana, the Z76 transgenic arabidopsis thaliana has enhanced stress resistance. Therefore, the protein Z76 has important theoretical significance and practical value in cultivating plants with enhanced stress resistance.

Drawings

FIG. 1 is an analysis of the expression pattern of the Z76 gene under drought stress and salt stress.

FIG. 2 is a statistical result of the growth state and germination rate of Arabidopsis thaliana under the salt stress treatment condition.

FIG. 3 is a statistical result of root length of Arabidopsis thaliana under salt stress treatment conditions.

FIG. 4 shows the root growth status of Arabidopsis thaliana under the salt stress treatment.

FIG. 5 is a statistical result of growth status and germination rate of Arabidopsis thaliana under drought stress treatment conditions.

FIG. 6 is a statistical result of root length of Arabidopsis thaliana under drought stress treatment conditions.

FIG. 7 shows the root growth status of Arabidopsis under drought stress treatment.

Fig. 8 shows the results of measurement of physiological indices.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Wild type Arabidopsis thaliana (Arabidopsis thaliana) (Columbia-0 subtype) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana. Nature genetics.2004, 36: 167-. Arabidopsis thaliana (Columbia-0 subtype) is hereinafter referred to as wild type Arabidopsis thaliana for short.

The pROK II vector is described in the following documents: huiyan Guo, Young Wang, Liuqiang Wang, Ping Hu, Yanmin Wang, Yuanyuan Jia, Chunrui Zhang, Yu Zhang, Yiming Zhang, Chao Wang and Chuanying Yang. expression of the MYB transcription factor gene BplMYB46 infectious diseases both deletion strategy and second cell wall displacement in beta plant topology Journal (2017) 15: 107-121.

the TRIzol reagent is a product of Invitrogen corporation. ReverTra Ace qPCR RT Master Mix with gDNA Remover is a product of TOYOBO.

In the following examples, the inbred line of maize alloy 344 is abbreviated as maize.

In the examples described below, statistical analysis was performed using SPSS 16.0(SPSS Inc, Chicago, IL, USA) software. One-way anova compared whether different strains (WT, OE-3, and OE-16) differed significantly under the same treatment conditions (asterisks for P <0.05, and double asterisks for P < 0.01).

In the following examples, mannitol was added at various concentrations to simulate a drought environment. The higher the mannitol concentration, the greater the drought level of the environment.

Example 1 cloning of Gene encoding stress-resistant related protein Z76

1. Extracting total RNA of the corn root system, and then carrying out reverse transcription to obtain cDNA of the corn root system.

2. Taking cDNA of a corn root system as a template, and adopting a primer Z76-KpnI-F: 5'-CGGGGTACCATGGACGACGGGGACCTGGAT-3' and primer Z76-SacI-R: 5'-CCGAGCTCTCACTTCTTTTCAACATTTGG-3' to obtain a PCR amplification product of about 819 bp.

3. And (3) taking the PCR amplification product obtained in the step (2), carrying out agarose gel electrophoresis, and then recovering a fragment of about 819 bp.

4. And (3) connecting the fragment recovered in the step (3) with pGM-T (a component in the pGM-T kit) by adopting a pGM-T kit (a product of Tiangen Biochemical technology (Beijing) Co., Ltd.) to obtain the recombinant T vector. The method comprises the following specific steps:

(1) blunt-ended target fragment plus A

Reaction system 1 was prepared. Reaction system 1 was 20. mu.L, consisting of 15. mu.L of the fragment recovered in

step

3, 4. mu.L of Tailing-A Reaction Buffer (a component in pGM-T kit) and 1. mu.L of Taq DNA Polymerase (a component in pGM-T kit).

Treating the reaction system at 72 ℃ for 30min to obtain the target fragment.

(2) Ligation of the fragment of interest into the T vector

Reaction system 2 was prepared. The reaction system 2 was 10. mu.L composed of 1. mu.L of 10 XT 4DNA Ligation BBuffer (Components of pGM-T kit), 1. mu. L T4DNA Ligase (concentration 3U/. mu.L), 1. mu.L pGM-T (concentration 50 ng/. mu.L), 4. mu.L of the fragment of interest, and 3. mu.L ddH₂And (C) O.

Treating the reaction system for 1h at the temperature of 2 ℃ to obtain a connecting product.

(3) Transformation of

(3-1) uniformly spreading 16. mu.L of IPTG solution (concentration of 50mg/mL) and 40. mu. L X-Gal solution (concentration of 20mg/mL) on LB solid plate containing Amp, and keeping away from light at 37 ℃ for 1 h;

(3-2) adding 10 mu L of the ligation product into a 100 mu L escherichia coli DH5 alpha competent centrifuge tube, flicking and uniformly mixing, and carrying out ice bath for 30 min;

(3-3) after completing the step (3-2), performing heat shock at 42 ℃ for 90s, and immediately carrying out ice bath on the centrifuge tube for 3min without shaking the centrifuge tube;

(3-4) after the step (3-3) is completed, adding 800 μ L of LB liquid medium preheated at 37 ℃ into the centrifuge tube, and performing shaking culture at 37 ℃ and 130rpm for 1 h;

(3-5) after the step (3-4) is finished, taking the centrifuge tube, centrifuging, discarding the supernatant, uniformly mixing the bacterial liquid, sucking 100 mu L of the mixture, uniformly coating the mixture on the LB solid plate manufactured in the step (3-1), inverting the LB solid plate, and culturing at 37 ℃ overnight.

(4) Enzyme digestion and sequencing

(4-1) white single colonies on LB solid plates were picked and colony PCR was performed using the primer Z76-KpnI-F and the primer Z76-SacI-R. If the PCR amplification product of a single colony contains a fragment of about 819bp, the single colony is a positive single colony.

(4-2) picking positive single colony with toothpick, inoculating in LB liquid culture medium containing Amp, culturing overnight at 37 ℃ to obtain culture bacterial liquid.

(4-3) taking the culture bacterial liquid, and extracting plasmids.

(4-4) taking the plasmid extracted in the step (4-3), and performing enzyme digestion by using restriction enzymes KpnI and SacI.

As a result, the cleavage product contained a fragment of about 819 bp.

And (4-5) sending the plasmid extracted in the step (4-3) to Huada gene for sequencing. The sequencing primers were T7F and M13R.

The sequencing result shows that the plasmid (i.e. recombinant T vector) contains a DNA molecule (i.e. Z76 gene) shown as a sequence 1 in a sequence table, and codes a Z76 protein or a protein Z76 shown as a sequence 2 in the sequence table.

Example 2 expression Pattern analysis

Expression mode of Z76 gene under drought stress

1. Obtaining of cDNA

(1) Selecting full corn seeds with consistent size, washing the corn seeds with tap water, adding 10% (v/v) sodium hypochlorite solution for disinfection for 30min, washing the corn seeds with distilled water, soaking the corn seeds for 12h, accelerating germination, placing the corn seeds in an incubator at 27 ℃ for dark culture for 24h, planting the corn seeds on a perforated foam plate until the roots grow to about 1.5cm, and culturing the corn seeds in 1/2Hoagland culture solution. When the corn grows to three leaves and one heart, selecting corn seedlings with consistent growth, and treating the corn seedlings with 1/2Hoagland nutrient solution containing 10% (w/v) PEG for 12 h. Taking experimental materials (roots or leaves), wrapping with tinfoil paper, treating with liquid nitrogen for more than 30s, and storing at-80 deg.C.

(2) After the step (1) is completed, total RNA of the experimental material is extracted by using TRIzol reagent, and RNA treated for 12h by PEG is obtained.

(3) And (3) after the step (2) is completed, taking the RNA treated by PEG for 12h, and carrying out reverse transcription on the first strand cDNA by adopting a ReverTra Ace qPCR RT Master Mix with gDNA Remover to obtain the cDNA treated by PEG for 12 h.

According to the method, 12h in the step (1) is replaced by 24h and 72h respectively, and other steps are not changed, so that the cDNA of 24h treated by PEG and the cDNA of 72h treated by PEG are obtained in sequence.

Control cDNA was obtained by replacing 12h in step (1) with 0h and leaving the rest unchanged, according to the procedure described above.

2. And (2) detecting the relative expression quantity of the Z76 gene in the corn root by using each cDNA obtained in the step (1) as a template and using a Bio-Rad Chromo4real-time PCR system to perform real-time quantitative PCR (using corn Actin1(GRMZM2G126010_ T01) as an internal reference).

The reaction system was 25. mu.L consisting of 12.5. mu.L of 2 XSSYBR Green Realtime PCR Master Mix (product of Toyobo Co.), the forward primer, the reverse primer, and 2. mu.L of cDNA template (about 10. mu.L)0ng) and RNase free ddH₂And (C) O. The concentration of the upstream primer and the concentration of the downstream primer in the reaction system are both 0.5. mu.M.

Reaction procedure: 30s at 94 ℃; 12s at 94 ℃, 30s at 58 ℃, 30s at 72 ℃ and 45 cycles; 80 ℃ for 1 s.

Primers for detecting the Z76 gene are 5'-CGTCCGAGAACAACAGCAA-3' and 5'-ATACGGGAACGCACCAATC-3'.

The primers for detecting the maize Actin1 are as follows: 5'-GATGATGCGCCAAGAGCTG-3', and 5'-GCCTCATCACCTACGTAGGCAT-3'.

The relative expression level of the Z76 gene in the control cDNA was designated as 1, and the relative expression levels of the Z76 gene in the other cDNAs (12 h-PEG-treated cDNA, 24 h-PEG-treated cDNA, or 72 h-PEG-treated cDNA) were shown in FIG. 1 (leaf in the left panel and root in the right panel). The result shows that under drought stress, the relative expression level of the Z76 gene in the leaves is gradually increased along with the prolonging of the treatment time, and the relative expression level of the Z76 gene in the root system is firstly slowly increased and then decreased.

II, expression pattern of Z76 gene under high salt stress

1. Obtaining of cDNA

(1) Selecting full corn seeds with consistent size, washing the corn seeds with tap water, adding 10% (v/v) sodium chlorate solution for disinfection for 30min, washing the corn seeds with distilled water, soaking the corn seeds for 12h, accelerating germination, placing the corn seeds in an incubator at 27 ℃ for dark culture for 24h, planting the corn seeds on a perforated foam plate until the roots grow to about 1.5cm, and culturing the corn seeds in 1/2Hoagland culture solution. When the corn grows to three leaves and one heart, selecting corn seedlings with consistent growth, and treating the corn seedlings with 1/2Hoagland nutrient solution containing 200mM NaCl for 12 hours. Taking experimental materials (roots or leaves), wrapping with tinfoil paper, treating with liquid nitrogen for more than 30s, and storing at-80 deg.C.

(2) After the step (1) is completed, total RNA of the experimental material is extracted by using TRIzol reagent, and RNA which is subjected to salt treatment for 12 hours is obtained.

(3) And (3) after the step (2) is completed, taking RNA subjected to salt treatment for 12h, and carrying out reverse transcription on the RNA by adopting a ReverTra Ace qPCR RT Master Mix with gDNA Remover to obtain first strand cDNA so as to obtain cDNA subjected to salt treatment for 12 h.

According to the method, the 12h in the step (1) is replaced by 24h and 72h respectively, and other steps are not changed, so that the cDNA of salt treatment 24h and the cDNA of salt treatment 72h are obtained in sequence.

2. And (2) detecting the relative expression quantity of the Z76 gene in the corn roots by using each cDNA obtained in the step (1) as a template and using a Bio-Rad Chromo4real-time PCR system to perform real-time quantitative PCR (taking corn Actin1(GRMZM2G126010_ T01) as an internal reference).

The reaction system, the reaction program, the primer for detecting the Z76 gene and the primer for detecting the maize Actin1 are all synchronous in step one 2.

The relative expression level of the Z76 gene in the control cDNA was designated as 1, and the relative expression levels of the Z76 gene in the other cDNAs (cDNA treated with salt for 12h, cDNA treated with salt for 24h, or cDNA treated with salt for 72 h) were shown in FIG. 1 (leaf in the left panel and root in the right panel). The results show that under the salt stress, the relative expression level of the Z76 gene in the leaves and the root system is increased and then decreased along with the prolonging of the treatment time.

The above results indicate that the Z76 gene can be induced to be expressed by salt and drought in maize roots. The Z76 gene is an important regulator in the process of adapting corn roots to abiotic stress.

Example 3 obtaining of Z76 Gene-transferred Arabidopsis thaliana and identification of stress resistance thereof

Construction of recombinant plasmid

1. The recombinant T vector constructed in example 1 was digested with restriction enzymes KpnI and SacI, and an digested fragment of about 819bp was recovered.

2. The pROK II vector was digested with restriction enzymes KpnI and SacI, and an about 11kb vector backbone was recovered.

3. And connecting the enzyme digestion fragment with a vector framework to obtain the recombinant plasmid pROK II-Z76.

II, obtaining recombinant agrobacterium

1. And (3) introducing the recombinant plasmid pROK II-Z76 prepared in the step one into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, and naming the recombinant Agrobacterium as EHA105/pROK II-Z76.

2. The pROK II is introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which is named EHA105/pROK II.

III, obtaining of Z76 transgenic Arabidopsis thaliana

1. Transferring the EHA105/pROK II-Z76 prepared in step two to wild type Arabidopsis thaliana to obtain T.thaliana inflorescence Floral dip transformation method (Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. plant J16: 735-743.)₁Seeds of Z76 transgenic Arabidopsis thaliana were generated.

2. Will T₁Seeds of Arabidopsis thaliana with transferred Z76 gene are planted in MS solid culture medium containing 50mg/L kanamycin, and Arabidopsis thaliana (resistant seedling) capable of growing normally is T₁Transferring Z76 positive seedling. T is₁The seeds received by the positive seedlings of the transfer Z76 gene are T₂Seeds of Z76 transgenic Arabidopsis thaliana.

3. The T of different strains screened in the step 2₂Seeds of Z76 transgenic Arabidopsis thaliana were selected by sowing in MS solid medium containing 50mg/L kanamycin, if the ratio of the number of Arabidopsis thaliana (resistant seedlings) capable of growing normally to the number of Arabidopsis thaliana (non-resistant seedlings) incapable of growing normally in a certain line was 3: 1, the strain is a strain with one copy of the Z76 gene inserted therein, and the seeds received by the resistant seedlings in the strain are T₃Seeds of Z76 transgenic Arabidopsis thaliana.

4. The T screened out in the step 3₃Seeds of Z76 transgenic Arabidopsis thaliana are sown on MS solid culture medium containing 50mg/L kanamycin again for screening, and the seeds which are all resistant seedlings are T₃The transgenic Arabidopsis with Z76 gene was homozygous.

Randomly selecting two T₃Lines of transgenic Z76 transgenic Arabidopsis thaliana were homozygous for the generations (designated OE-3 and OE-16, respectively) and subjected to subsequent experiments.

According to the method, EHA105/pROK II-Z76 is replaced by EHA105/pROK II, and other steps are the same, so that a T3 generation homozygous empty vector transfer Arabidopsis thaliana plant, called empty vector transfer Arabidopsis thaliana for short, is obtained.

Fourth, salt tolerance identification

The conditions for normal culture were: 22 +/-2 ℃. The period of light-dark alternate culture is specifically as follows: 16h light culture/8 h dark culture. The illumination intensity during illumination culture is 80-100 μmol · m^-2·s^-1。

The Arabidopsis seeds to be tested are wild Arabidopsis seeds, empty vector Arabidopsis seeds, T3 generation seeds of OE-3 or T3 generation seeds of OE-16.

1. Germination rate test

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

(1) adding the to-be-detected arabidopsis thaliana seeds into an EP (European patent application) tube, firstly soaking in 75% (v/v) ethanol water solution for 1min, washing with ultrapure water for 3-5 times, then sterilizing with 10% (v/v) NaClO for 10min, washing with ultrapure water for 8-10 times, and finally airing on sterile filter paper to obtain the sterile to-be-detected arabidopsis thaliana seeds.

(2) After the step (1) is completed, 100 sterile seeds of arabidopsis thaliana to be tested are sown on a culture medium to be tested (1/2MS solid culture medium, 1/2MS solid culture medium containing 50mM NaCl, 1/2MS solid culture medium containing 100mM NaCl or 1/2MS solid culture medium containing 150mM NaCl), and are normally cultured for 3d, and the germination rate (the germination rate is the number of germinated seeds of arabidopsis thaliana to be tested/100 grains multiplied by 100%) is counted; the growth state was observed in the normal culture for 5 days.

The growth state of a part of Arabidopsis seeds on solid medium is shown in the left panel of FIG. 2 (WT is wild type Arabidopsis seed). The statistical results of germination rates of part of Arabidopsis seeds cultured normally for 3d are shown in the right picture in FIG. 2 (WT is wild type Arabidopsis seeds): normally culturing for 3d, wherein the growth vigor of the to-be-detected Arabidopsis seeds is basically consistent on 1/2MS solid culture medium, and the germination rate has no significant difference (which indicates that the over-expression of the Z76 gene has no influence on the germination of plants under the non-stress condition); normally culturing for 3d, wherein the germination rates of OE-3 and OE-16 are both obviously higher than those of wild arabidopsis thaliana on 1/2MS solid culture medium containing 50mM NaCl, 1/2MS solid culture medium containing 100mM NaCl or 1/2MS solid culture medium containing 150mM NaCl, and the germination rates of the wild arabidopsis thaliana and the empty vector arabidopsis thaliana are not obviously different; after the culture is normally carried out for 5 days, the growth vigor of OE-3 and OE-16 is obviously superior to that of wild arabidopsis thaliana on 1/2MS solid culture medium containing 50mM NaCl, 1/2MS solid culture medium containing 100mM NaCl or 1/2MS solid culture medium containing 150mM NaCl, and the germination rates of the wild arabidopsis thaliana and the empty vector arabidopsis thaliana are not obviously different.

The results show that overexpression of the Z76 gene improves the salt tolerance of Arabidopsis thaliana at the germination stage.

2. Root length test

(2) And (3) after the step (1) is finished, sowing the sterile arabidopsis thaliana seeds to be detected in 1/2MS solid culture medium, and normally culturing for 7d to obtain the arabidopsis thaliana seedlings to be detected.

(3) After completion of step (2), 50 test Arabidopsis seedlings with uniform growth vigor were transferred to a test medium (1/2MS solid medium, 1/2MS solid medium containing 50mM NaCl, or 1/2MS solid medium containing 100mM NaCl), cultured normally (vertically) for 6d to observe growth state and measure root length.

The growth state of a part of Arabidopsis seeds on solid medium is shown in FIG. 4(WT is wild type Arabidopsis seeds). The root length statistics are shown in FIG. 3(WT is wild type Arabidopsis seed): on an 1/2MS solid culture medium, the growth vigor of the seeds of the arabidopsis to be detected is basically consistent, and the root length has no obvious difference (which indicates that the over-expression of the Z76 gene has no influence on the root length of the plant under the non-stress condition); on 1/2MS solid medium containing 50mM NaCl or 1/2MS solid medium containing 100mM NaCl, the root lengths of OE-3 and OE-16 are both significantly higher than that of wild type Arabidopsis, and there is no significant difference between the root lengths of wild type Arabidopsis and empty vector Arabidopsis.

The results show that the Z76 gene is involved in regulating the growth of plant roots under salt stress. Overexpression of the Z76 gene increased salt tolerance in Arabidopsis.

The above results indicate that salt resistance of OE-3 and OE-16 is significantly enhanced compared to wild type Arabidopsis thaliana.

Fifth, identification of drought tolerance

1. Germination rate test

(2) After completion of step (1), 100 sterile seeds of arabidopsis thaliana to be tested were sown on a test medium (1/2MS solid medium, 1/2MS solid medium containing 100mM mannitol, 1/2MS solid medium containing 200mM mannitol, or 1/2MS solid medium containing 300mM mannitol), cultured normally for 4d, observed for growth state and counted for germination rate (germination rate ═ number of germinated seeds of arabidopsis thaliana to be tested/100 × 100%).

The growth state of a part of Arabidopsis seeds on solid medium is shown in the left panel of FIG. 5 (WT is wild type Arabidopsis seed). The germination rate statistics of some Arabidopsis seeds are shown in the right diagram of FIG. 5 (WT is wild type Arabidopsis seed): on an 1/2MS solid culture medium, the growth vigor of the arabidopsis seeds to be detected is basically consistent, and the germination rate has no obvious difference (which indicates that the over-expression of the Z76 gene has no influence on the germination of plants under the non-stress condition); on an 1/2MS solid culture medium containing 100mM mannitol, the germination rate of OE-16 is obviously higher than that of wild type Arabidopsis, the difference between the germination rates of OE-3 and wild type Arabidopsis is not obvious, and the germination rates of the wild type Arabidopsis and an empty vector Arabidopsis are not obviously different; on 1/2MS solid medium containing 200mM mannitol or 1/2MS solid medium containing 300mM mannitol, the germination rates of OE-3 and OE-16 are both significantly higher than that of wild type Arabidopsis, and there is no significant difference between the germination rates of wild type Arabidopsis and empty vector Arabidopsis.

The result shows that the over-expression of the Z76 gene improves the drought tolerance of arabidopsis thaliana in the germination stage.

2. Root length test

(1) adding the to-be-detected arabidopsis thaliana seeds into an EP (European patent application) tube, firstly soaking in 75% (v/v) ethanol water solution for 1min, washing with ultrapure water for 3-5 times, then sterilizing with 10% (m/v) NaClO for 10min, washing with ultrapure water for 8-10 times, and finally airing on sterile filter paper to obtain the sterile to-be-detected arabidopsis thaliana seeds.

(3) After completion of step (2), 50 test Arabidopsis seedlings with uniform growth vigor were transferred onto a test medium (1/2MS solid medium, 1/2MS solid medium containing 200mM mannitol, or 1/2MS solid medium containing 300mM mannitol), cultured normally (vertically) for 6d to observe growth state and measure root length.

The growth state of a part of Arabidopsis seeds on solid medium is shown in FIG. 7(WT is wild type Arabidopsis seeds). Partial root length statistics are shown in fig. 6(WT is wild type arabidopsis seeds): on an 1/2MS solid culture medium, the growth vigor of the seeds of the arabidopsis to be detected is basically consistent, and the root length has no obvious difference (which indicates that the over-expression of the Z76 gene has no influence on the root length of the plant under the non-stress condition); on 1/2MS solid medium containing 200mM mannitol or 1/2MS solid medium containing 300mM mannitol, the root length of OE-3 and OE-16 is significantly higher than that of wild Arabidopsis, and there is no significant difference between the root length of wild Arabidopsis and empty vector Arabidopsis.

The result shows that the Z76 gene is involved in regulating the growth of plant roots under drought stress. Overexpression of the Z76 gene improves drought tolerance of Arabidopsis thaliana.

Sixth, measurement of physiological index

And (3) disinfecting the arabidopsis seeds to be detected (wild arabidopsis seeds, OE-3T 3 generation seeds or OE-16T 3 generation seeds), sowing the seeds on a 1/2MS culture medium, and transplanting the seeds into nutrient soil to continue to grow for 3-4 weeks after the seeds germinate for 8-10 days to obtain the arabidopsis seedlings to be detected.

At a temperature of 22 +/-2 ℃ and an illumination intensity of 400 mu mol.m^-2·s^-1And in an artificial climate chamber with the photoperiod of 16h illumination/8 h darkness and the relative humidity of 65-75%, respectively adding an MS liquid culture medium containing 200mM NaCl, an MS liquid culture medium containing 400mM Manniol or water (contrast) into nutrient soil to treat the arabidopsis seedlings to be detected for 72h, then taking leaves, and carrying out determination and analysis on various physiological and biochemical indexes.

1. Determination of Water loss

The water loss of the leaves of Arabidopsis thaliana seedlings to be tested was determined according to the method described in the literature (Huiyan Guo, Young Wang, Liuqiang Wang, Ping Hu, Yanmin Wang, Yuanyuuan Jia, Chunrui Zhang, Yu Zhang, Yiming Zhang, Chao Wang and Chuanying Yang. expression of the MYB transfer factor gene BplMYB46 infection strain tolerance and second cell wall displacement in beta plant project (2017) 15: 107-. The method comprises the following specific steps: taking 100-200mg of arabidopsis seedling leaves (water treatment control) to be tested, and immediately weighing the fresh weight FW; then placing the leaves into a culture dish, standing for 0.5h, 1h, 1.5h, 2h, 3h, 4h or 5h at room temperature, and weighing the fresh weight FW (h) of the leaves of the arabidopsis thaliana seedlings to be measured. And finally, placing the leaves of the arabidopsis thaliana seedlings to be detected in an oven at 80 ℃ to dry to constant weight, and weighing the DW. Calculating the water loss rate of the blade in each time period:

total water content is FW-DW;

water content fw (h) -DW at each time point;

the water loss rate at each time point was 1- (FW (h) -DW)/(FW-DW).

The results are shown in A in FIG. 8 (WT is a wild type Arabidopsis seed). The results show that the water loss rate of OE-3 and OE-16 is significantly reduced compared to wild type Arabidopsis.

2. Determination of proline content

The proline content of the leaves of the Arabidopsis seedlings to be tested was determined according to the method described in the literature (Bates, L.S., Waldren, R.P., and Teare, I.D. (1973), Rapid determination of free proline for water-stress students. plant Soil 39, 205-. The method comprises the following specific steps:

(1) accurately weighing 0.1g of leaf of arabidopsis thaliana seedling to be detected, adding 1mL of 3% (w/v) sulfosalicylic acid solution, grinding, homogenizing, transferring into a centrifuge tube, extracting in boiling water bath for 10min, cooling, centrifuging at 3000r/min for 10min, and obtaining the supernatant which is the proline extracting solution.

(2) Standard Curve preparation

(2-1) reagents were added according to the following Table

(2-2) after the step (2-1) is finished, shaking up, developing in a boiling water bath for 30min (the time is strictly controlled, otherwise, precipitation is caused), cooling to room temperature, adding 2mL of toluene, fully shaking, extracting a red product, and standing for a period of time (10-30min) after extraction to separate the red product (the red substance enters the toluene layer). The upper layer of red material was gently pipetted into the cuvette using a syringe or dropper, and the absorbance was measured at 520mn in the spectrophotometer.

And (3) drawing a standard curve by taking the proline content as an abscissa and the corresponding light absorption value as an ordinate.

(3) Sample assay

And (3) replacing the standard proline in the step (2) with 2mL of the proline extracting solution obtained in the step (1), and obtaining the absorbance value without changing other steps. And (3) obtaining the content of proline in the proline extracting solution according to the standard curve drawn in the step (2). Further, obtaining the proline content in the leaves of the arabidopsis seedlings to be detected according to the following formula;

c is the content (mu g) of proline in the proline extracting solution; v: the total volume of the extracting solution is 5 mL; vt: the liquid volume when measuring, namely 2 mL; w: sample mass.

The results of the partial detection are shown in B of FIG. 8 (WT is a wild type Arabidopsis seed). The results show that under the conditions of drought stress or salt stress, the proline content of OE-3 and OE-16 is significantly increased compared with that of wild type Arabidopsis.

3. Determination of relative conductivity

The relative conductivity of the leaves of the Arabidopsis seedlings to be tested was determined according to the method described in the literature (Lutts S, Kinet J M, Bouhormont J.1996.NaCl-induced sensecence in leaves of rice (Oryza sativa L.) culture differentiation in saline resistance. Annals of Botany, 78, 389. 398.). The method comprises the following specific steps:

(1) a small beaker with the specification of 50mL is taken, washed with deionized water repeatedly for three times, and then balanced with the deionized water for 24 hours.

(2) Taking 100mg of leaves of arabidopsis seedlings to be detected, washing the leaves for three times by using deionized water, shearing the leaves, putting the leaves into a small beaker completing the step (1), and adding 50mL of ddH₂O。

(3) And (3) after the step (2) is finished, putting the small beaker into a vacuum pump, and carrying out vacuum infiltration for 15min until the leaves are semitransparent.

(4) And (4) after the step (3) is finished, taking the beaker, measuring the electric conductivity value of each solution by using a conductivity meter to stretch into the beaker, and measuring the electric conductivity value S1 of each solution.

(5) After the completion of the step (4), the beaker is placed in a water bath kettle at 90 ℃, water bath is carried out for 20min, and after the beaker is cooled to room temperature, the conductivity value S2 at this time is measured again.

(6) The relative conductivity was calculated. Relative conductivity was (S1/S2) × 100%.

The results of the partial detection are shown in FIG. 8C (WT is a wild type Arabidopsis seed). The results indicate that under conditions of drought stress or salt stress, the relative conductivities of OE-3 and OE-16 are reduced compared to wild type Arabidopsis.

Fourth, determination of superoxide dismutase (SOD) Activity

The SOD activity of the leaves of arabidopsis seedlings to be tested was measured according to the method described in the literature (Wang, y.c., Gao, c.q., Liang, y.n., Wang, c.c., Yang, c.p., and Liu, g.f. (2010).

The results of the partial detection are shown in FIG. 8D (WT is a wild type Arabidopsis seed). The results show that the SOD activities of OE-3 and OE-16 are significantly improved compared with wild type Arabidopsis under drought stress or salt stress conditions.

Fifthly, measuring the chlorophyll content

The total chlorophyll content in the leaves of the arabidopsis seedlings to be detected is detected by referring to a recording method in a monograph (Liheng. plant physiological and biochemical experiment principle and technology [ M ]. Beijing: higher education Press).

The results of the partial detection are shown in FIG. 8, E (WT is a wild type Arabidopsis seed). Results show that the total in vivo chlorophyll content of OE-3 and OE-16 is significantly increased compared with that of wild type Arabidopsis under drought stress or salt stress conditions.

Sixth, H₂O₂Determination of content

The H of the leaf of an Arabidopsis seedling to be tested was examined according to the method described in the literature (Velikova, V., Yordanov, I., and Edreva, A. (2000), Oxidative stress and sodium antioxidant system in acid chain-linear plants: protective roll of exogenous polyamines. plant Science 151, 59-66.)₂O₂And (4) content.

The results of the partial detection are shown in FIG. 8, panel F (WT is a wild type Arabidopsis seed). The results indicate that H for OE-3 and OE-16 under drought stress or salt stress conditions, as compared to wild type Arabidopsis thaliana₂O₂The content is obviously reduced.

Determination of content of hepta-superoxide anion

The superoxide anion content of the leaves of Arabidopsis thaliana seedlings to be tested was determined according to the method described in the literature (Able A J, Guest D I, Sutherland M W.1998.use of a new tetrazolium-based assay to study the production of superoxide radals by t bacco cell cultures from cultured cells having been transformed with an effective azopotes of photophtora parasitica var. nickelata. journal of Plant Physiology, 117, 491, 499).

The results of the partial detection are shown in FIG. 8, G (WT is a wild type Arabidopsis seed). The results show that under drought stress or salt stress conditions, the superoxide anion content of OE-3 and OE-16 is significantly reduced compared to wild type Arabidopsis.

The results show that the stress resistance of OE-3 and OE-16 is significantly enhanced compared with that of wild type Arabidopsis.

<110> institute of economic crops of department of agriculture, academy of sciences, Heilongjiang province, university of agricultural reclamation, Heilongjiang

Application of stress resistance-associated protein Z76 in regulation and control of plant stress resistance

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 804

<212> DNA

<213> Zea mays L.

<400> 1

atggacgacg gggacctgga tttctccaac ccggacacgt acctgtgtcc ggccctcggc 60

accgacccgc ccagcagctg ctccatggac agctacttcg acgagatcct caaggacacg 120

gagcacctcg cctgcacgca cacccacacc tgcaacccgc cggtgcacga cctctcgcac 180

acccacacct gcgtccacgt ccataccaag atcgtcgcgg cctcgccggg cgacgccgag 240

tcgccgtccg agaacaacag caacggcaac gccgcctcta agaagcgccc gtcggggaac 300

cgcgccgccg tcaggaagta ccgggagaag aagaaggccc acaccgcatc gctcgaggag 360

gaggtggtgc acctccgggc gctcaaccag cagctcatga aaaagctcca gagccacgcc 420

gcgctcgagg cggaggcggc caggctccgc tgcctgctcg tcgacatcag gggcaggatc 480

gagggggaga ttggtgcgtt cccgtatcag aggccgccgg cggtcaagaa cgtcgacctc 540

ctgtctagtg ttgaccagga aagcctcctt ggcagcgctg ctgcccaggt tgccaactcc 600

tgtgatttta gatgcaacca tcagatgtac tgcaaccctg ggatgcaggg cgctatcagc 660

ggtcaggtgt tgggacaagg tgcctgtgat gtcgccagta tgcaatgcat tggaagcacc 720

aagtctggat ccactaagct acctgtttgt gggggtctgg atacactacc tgctgtctgc 780

ttaccaaatg ttgaaaagaa gtga 804

<210> 2

<211> 267

<212> PRT

<213> Zea mays L.

<400> 2

Met Asp Asp Gly Asp Leu Asp Phe Ser Asn Pro Asp Thr Tyr Leu Cys

1 5 10 15

Pro Ala Leu Gly Thr Asp Pro Pro Ser Ser Cys Ser Met Asp Ser Tyr

20 25 30

Phe Asp Glu Ile Leu Lys Asp Thr Glu His Leu Ala Cys Thr His Thr

35 40 45

His Thr Cys Asn Pro Pro Val His Asp Leu Ser His Thr His Thr Cys

50 55 60

Val His Val His Thr Lys Ile Val Ala Ala Ser Pro Gly Asp Ala Glu

65 70 75 80

Ser Pro Ser Glu Asn Asn Ser Asn Gly Asn Ala Ala Ser Lys Lys Arg

85 90 95

Pro Ser Gly Asn Arg Ala Ala Val Arg Lys Tyr Arg Glu Lys Lys Lys

100 105 110

Ala His Thr Ala Ser Leu Glu Glu Glu Val Val His Leu Arg Ala Leu

115 120 125

Asn Gln Gln Leu Met Lys Lys Leu Gln Ser His Ala Ala Leu Glu Ala

130 135 140

Glu Ala Ala Arg Leu Arg Cys Leu Leu Val Asp Ile Arg Gly Arg Ile

145 150 155 160

Glu Gly Glu Ile Gly Ala Phe Pro Tyr Gln Arg Pro Pro Ala Val Lys

165 170 175

Asn Val Asp Leu Leu Ser Ser Val Asp Gln Glu Ser Leu Leu Gly Ser

180 185 190

Ala Ala Ala Gln Val Ala Asn Ser Cys Asp Phe Arg Cys Asn His Gln

195 200 205

Met Tyr Cys Asn Pro Gly Met Gln Gly Ala Ile Ser Gly Gln Val Leu

210 215 220

Gly Gln Gly Ala Cys Asp Val Ala Ser Met Gln Cys Ile Gly Ser Thr

225 230 235 240

Lys Ser Gly Ser Thr Lys Leu Pro Val Cys Gly Gly Leu Asp Thr Leu

245 250 255

Pro Ala Val Cys Leu Pro Asn Val Glu Lys Lys

260 265

Claims

1. The application of the protein Z76 in regulating and controlling the stress resistance of plants; the protein Z76 is a protein with an amino acid sequence shown in a sequence 2 in a sequence table;

the stress resistance is salt resistance or drought resistance;

the plant is maize or arabidopsis thaliana.

2. The use of claim 1, wherein: the regulation and control of the plant stress resistance is to increase the plant stress resistance.

3. Use of a nucleic acid molecule encoding the protein Z76 of claim 1 for modulating stress resistance in a plant;

the stress resistance is salt resistance or drought resistance;

the plant is maize or arabidopsis thaliana.

4. Use according to claim 3, characterized in that: the nucleic acid molecule encoding the protein Z76 is a DNA molecule shown as b1) or b 2):

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table.

5. Use according to claim 3, characterized in that: the regulation and control of the plant stress resistance is to increase the plant stress resistance.

6. A method of plant breeding comprising the steps of: increasing the content or activity of the protein Z76 as defined in claim 1 in plants, thereby increasing stress tolerance;

the stress resistance is salt resistance or drought resistance;

the plant is maize or arabidopsis thaliana.