CN110669121B - Method for improving salt tolerance of cotton by using LeDNAJ gene and application - Google Patents

Abstract

The invention discloses a method for improving salt tolerance of cotton by utilizing LeDNAJ genes and application thereof. The invention provides an application of LeDNAJ protein or related biological materials thereof in regulating and controlling the salt tolerance of plants; the related biological material is a nucleic acid molecule capable of expressing the LeDNAJ protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule; the LeDNAJ protein is shown as SEQ ID No. 1. The LeDnaJ transgenic positive cotton has significantly enhanced salt tolerance compared to the recipient cotton. The invention has important significance for breeding new species of salt-tolerant cotton.

Description

Method for improving salt tolerance of cotton by using LeDNAJ gene and application

Technical Field

The invention relates to the field of biotechnology, in particular to a method for improving salt tolerance of cotton by utilizing LeDNAJ genes and application thereof.

Background

Cotton is a pioneer crop of saline-alkali soil, and the saline-alkali soil accounts for 666 million hectares in about 1 hundred million hectares of cultivated land in China; in addition, a large amount of saline-alkali wasteland exists. The salt has double functions on the growth of cotton. The salt with lower concentration (less than 0.2%) is beneficial to the emergence and growth of cotton and can improve the yield and quality.

The coastal saline-alkali soil is wide in China, long in coastline, and not only has a large amount of coastal saline-alkali soil, but also has inland saline-alkali soil. In the face of the trend of population increase, industrial development and gradual reduction of the cultivated land area, the improvement and utilization of saline-alkali soil are greatly paid attention in various aspects. The cotton is one of crops with strong salt tolerance and is a pioneer crop of saline-alkali soil. Generally, the salt content of the plough layer is below 0.3%, but when the salt concentration of the soil is more than 0.3%, the cotton can be damaged. Screening and utilizing salt-tolerant cotton varieties is one of the most economical and effective methods for saline-alkali soil improvement. But the salt tolerance level of cotton germplasm resources is lower, and the production requirement cannot be met. The introduction of resistance target genes into upland cotton by genetic engineering means, using recombinant DNA and transgenic technology, to improve the saline tolerance (resistance) of cotton has become increasingly urgent.

In 1980, Georgopoulos et al first isolated a 41KD heat shock protein from E.coli, named DnaJ, and later discovered its homologous sequence in many eukaryotes including humans, animals, plants, yeast, etc., and recent studies showed that the homologous sequence of the protein exists in almost all organisms and contains a highly conserved 70 amino acid J-domain. The DnaJ-like protein (HSP40) is an accessory protein of a molecular chaperone HSP70 protein, and HSP70 plays an important role in folding, assembly and transportation of intracellular nascent proteins, and can prevent the denaturation of unfolded proteins and mediate the solubility and the renaturation of aggregated proteins after adversity digestion under adversity conditions. Numerous studies have found that the DnaJ protein and the heat shock protein HSP70 form an ordered molecular complex that functions as a chaperone machine in cells.

In plants, Chaihuaiao et al (2000) separate a heavy metal stress response gene DnaJ-1ike protein by differentially screening HgC 12-stressed kidney bean seedling leaf cDNA library, the expression of the gene in leaves can be strongly induced by heavy metals (Hg, CA, As, Zn, Cu and the like), and the DnaJ-1ike protein is presumed to play an important role in protecting cell membranes and the structure and function of enzyme protein and improving the stress resistance of plants. Zhangda et al (2006) cloned a 609bp DNA sequence of a DnaJ-like gene from a suppression subtractive hybridization library of halophyte salicornia, and coded 1 202aa open reading frame, and Northern Blatting shows that the expression of the gene in a detector (200mmol/L NaCl) is obviously enhanced. Plum blossom, Guangdong, et al (2007) clone a full-length cDNA of a molecular chaperone gene of potato by using a leaf of a bacterial wilt-resistant variety of potato induced by a microspecies of ralstonia solanacearum as a material. The molecular chaperone gene has 84% of consistency with a partial nucleotide sequence of Arabidopsis DnaJ-like 20 and 59% of consistency with an encoded amino acid sequence thereof. The semi-quantitative RT-PCR result shows that the gene is induced by ralstonia solanacearum and regulated by jasmonic acid at the same time, and has the function of promoting the recovery activity of stress damaged cells under the stress of the ralstonia solanacearum and the jasmonic acid. At present, there are reports related to DnaJ gene transformation research in plants and cultivation of novel transgenic plant varieties with salt stress resistance.

Disclosure of Invention

The invention aims to provide a method for improving the salt tolerance of cotton by using LeDNAJ genes and application thereof.

In a first aspect, the invention claims the use of LeDNAJ proteins or their related biomaterials for modulating salt tolerance in plants.

The related biological material is a nucleic acid molecule capable of expressing the LeDNAJ protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the LeDNAJ protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In the application, the activity and/or expression quantity of the LeDNAJ protein or the coding gene thereof in the plant is improved, and the salt tolerance of the plant is improved. The LeDNAJ protein or the coding gene thereof has reduced activity and/or expression in the plant, and the plant salt tolerance is reduced.

In a second aspect, the invention claims a method of breeding a plant variety with improved salt tolerance.

The method for breeding a plant variety with improved salt tolerance as claimed in the present invention may comprise the step of increasing the expression level and/or activity of the LeDNAJ protein in the recipient plant. The LeDNAJ protein is any one of the proteins shown in the (A1) - (A4) above.

In a third aspect, the invention claims a method of breeding a plant variety with reduced salt tolerance.

The method for breeding a plant variety with reduced salt tolerance as claimed in the present invention may comprise the step of reducing the expression level and/or activity of the LeDNAJ protein in the recipient plant. The LeDNAJ protein is any one of the proteins shown in the preceding paragraphs (A1) - (A4).

In a fourth aspect, the invention claims a method of breeding transgenic plants with improved salt tolerance.

The method for cultivating transgenic plants with improved salt tolerance, which is claimed by the invention, can comprise the following steps: introducing nucleic acid molecules capable of expressing LeDNAJ protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased salt tolerance as compared to the recipient plant. The LeDNAJ protein is any one of the proteins shown in the preceding paragraphs (A1) - (A4).

In a fifth aspect, the invention claims a method of breeding transgenic plants with reduced salt tolerance.

The method for cultivating transgenic plants with reduced salt tolerance, which is claimed by the invention, can comprise the following steps: carrying out suppression expression on coding genes of LeDNAJ protein in a receptor plant to obtain a transgenic plant; the transgenic plant has reduced salt tolerance as compared to the recipient plant. The LeDNAJ protein is any one of the proteins shown in the preceding paragraphs (A1) - (A4).

In the fourth aspect, the "introducing into a recipient plant a nucleic acid molecule capable of expressing the LeDNAJ protein" may be achieved by introducing into the recipient plant a recombinant expression vector comprising a gene encoding the LeDNAJ protein.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor.

When the LeDNAJ gene is used to construct a recombinant plant expression vector, any one of an enhanced promoter or a constitutive promoter (e.g., cauliflower mosaic virus (CAMV)35S promoter, Ubiquitin promoter from maize (Ubiquitin)) or a tissue-specific expression promoter (e.g., seed-specific expression promoter) may be added before the transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters. In addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or initiation codons of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are wide ranging from natural to synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.), or chemical resistance marker genes (e.g., herbicide resistance gene), etc., which are expressed in plants.

In the present invention, the promoter for promoting transcription of the encoding gene in the recombinant expression vector is a 35S promoter. And a terminator for terminating the transcription of the coding gene in the recombinant expression vector is an Nos terminator.

In the above method, the introduction of the recombinant expression vector into the recipient plant may specifically be: plant cells or tissues are transformed by using conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

Transformed cells, tissues or plants are understood to comprise not only the end product of the transformation process, but also the transgenic progeny thereof.

In the above aspects, the "nucleic acid molecule capable of expressing the LeDNAJ protein" is a gene encoding the LeDNAJ protein.

Further, the encoding gene of the LeDNAJ protein can be any one of the following DNA molecules:

(B1) a DNA molecule represented by positions 12-479 of SEQ ID No. 2;

(B2) DNA molecule shown in SEQ ID No. 2;

(B3) a DNA molecule which hybridizes with the DNA molecule defined in (B1) or (B2) under stringent conditions and encodes the LeDNAJ protein;

(B4) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence defined by (B1) or (B2) or (B3) and encodes the LeDNAJ protein.

In the above genes, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Mixing with 1mM EDTAHybridization in hybridization solution, rinsing at 65 ℃ in 0.1 XSSC, 0.1% SDS; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In each of the above aspects, the plant may be a dicot.

Further, the dicotyledonous plant may be a plant of the family Malvaceae.

Further, the malvaceae plant may be cotton (e.g., gossypium hirsutum).

In one embodiment of the invention, the plant is specifically the gossypium hirsutum variety R15.

In a particular embodiment of the invention, the salt tolerance is embodied as salt tolerance during the germination and/or seedling stage. The salt tolerance is more specifically embodied in that the relative plant height, the relative fresh weight of the overground part and/or the relative dry weight of the overground part of the transgenic positive plant in the 7-day seedling stage is greater than that of the non-transgenic receptor plant after being treated under the condition of 250mM NaCl treatment; and/or the relative elongation of the root length of transgenic positive plants treated for 3 days germination under 250mM NaCl treatment is greater than that of non-transgenic recipients. The relative plant height is the ratio of the plant height of the plant under the stress of 250mM NaCl salt to the plant height under the normal condition; the relative fresh weight of the overground part is the ratio of the fresh weight of the overground part of the plant under the stress of 250mM NaCl salt to the fresh weight of the overground part under the normal condition; the relative dry weight of the aerial parts is the ratio of the dry weight of the aerial parts of the plants under 250mM NaCl salt stress to the dry weight of the aerial parts under normal conditions. The relative elongation of the root length refers to the ratio of the root length under 250mM NaCl salt stress to the root length under normal conditions.

The invention constructs the full length of the LeDnaJ gene of tomato into a plant expression vector containing a 35s promoter and an NOS terminator, transforms the plant expression vector into a upland cotton variety R15 by an agrobacterium-mediated method, and obtains pure cotton progeny of the LeDnaJ gene through PCR detection and Southern detection of a target gene LeDnaJ and preliminary salt tolerance identification of transgenic progeny. And the experiment proves that the salt tolerance of the cotton with the positive LeDnaJ transgenosis is obviously enhanced compared with that of receptor cotton. The invention has important significance for breeding new species of salt-tolerant cotton.

Drawings

FIG. 1 shows a construction map of pCAMBIA2301-35s-LeDnaJ vector. LB: a left border sequence; NPTII: kanamycin marker gene; 35S P: a 35S promoter; LeDnaJ: a gene of interest; and (4) GUS: a reporter gene; and (3) Nos: a terminator; RB: a right border sequence; kanamycin, Kanamycin marker gene.

FIG. 2 is a sterile shoot cultured for 5 days.

FIG. 3 is co-culture of hypocotyl segments after infection.

FIG. 4 shows the first inoculation into resistant callus induction medium for 20 days.

FIG. 5 shows inoculation into resistant callus induction medium for 40 days.

FIG. 6 is embryogenic callus development.

FIG. 7 shows the process of regenerating plants from embryogenic callus. a is embryogenic callus; b is torpedo-shaped embryo; c is primary regeneration seedling; d is mature regeneration seedling; e is a regeneration plant.

FIG. 8 shows the PCR amplification of target gene of T1 transgenic plant. Marker: DL2000DNA marker. The negative control in the figure refers to receptor material, water is used as blank control, DNAJ-and L-DNAJ-are different plants, the amplified band of about 480bp is positive in the figure, and the band without the band is negative.

FIG. 9 shows RT-PCR detection of transgenic cotton. A is actin background for detecting cotton total RNA; b is a target fragment in the cDNA of the transgenic cotton amplified by RT-PCR. M: DL2000 DNAmarker; 1-38: the genome DNA is detected as positive transgenic plant cDNA by PCR. Lane 4: represents ST-1; lane 5: represents ST-2; lane 6: representation ST-3, lane 10: represents ST-4; lane 11: represents ST-5; lane 13: represents ST-6; lane 15: represents ST-7; lane 16: represents ST-8; lane 17: represents ST-9; lane 21: represents ST-10; lane 22: represents ST-11; lane 25: represents ST-12; lane 28: represents ST-13; lane 29, representing ST-14; lane 30: represents ST-15; lane 31: representation ST-16, lane 32: represents ST-17; lane 33: represents ST-18; lane 38: represents ST-19.

FIG. 10 shows the detection of target genes by Sourther hybridization. Lane 1 represents DNAJ-1, single copy; lane 2 represents DNAJ-2, double copy; lane 3 represents DNAJ-3, double copy.

FIG. 11 shows PCR (top) and RT-PCR (bottom) validation of LeDnaJ transgenic cotton lines. M is marker; p: a positive plasmid; CK: r15; d1, D2 and D3 represent three transgenic lines DNAJ-1, DNAJ-2 and DNAJ-3 respectively.

FIG. 12 is a comparison of germination of LeDnaJ transgenic cotton lines and control germination period salt-treated for 3 days. CK denotes transgenic acceptor cotton material R15.

FIG. 13 is a comparison of the growth of LeDnaJ transgenic cotton lines and control seedling stage salt treatment for 7 days. CK denotes transgenic acceptor cotton material R15.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The nucleotide sequence of tomato LeDnaJ gene related in the following examples is specifically shown in SEQ ID No.2, and the 12 th to 479 th sites are ORF, and encode protein shown in SEQ ID No. 1.

Example 1 construction of recombinant expression vector pCAMBIA2301-35s-LeDnaJ

1 pair of specific primers are designed respectively at the upstream and downstream of LeDnaJ gene:

LeDnaJ-F-BamH I：5’-CGCGGATCCATGGCTTCTTCTTCTTTTCTTCTCTC-3’；

LeDnaJ-R-Kpn I：5’-GGGGTACCCTACCAACACTGATCGGTTTCCCATC-3’。

And adding BamH I and Kpn I restriction enzyme sites (underlined parts in the primer) at both ends, cloning the target cDNA sequence to pCAMBIA2301 vector, and constructing expression vector pCAMBIA2301-CaMV 35S-LeDnaJ. All effector plasmids were identified correctly by PCR, restriction and sequencing.

The structure of the recombinant expression vector pCAMBIA2301-35s-LeDnaJ is described as follows: a recombinant plasmid which is formed by inserting a DNA fragment shown in 12 th to 479 th positions of SEQ ID No.2 between enzyme cutting sites BamH I and Kpn I of a pCAMBIA2301 vector.

The construction map of the recombinant expression vector pCAMBIA2301-35s-LeDnaJ is shown in figure 1.

Example 2 transformation and characterization of Cotton

The recombinant expression vector pCAMBIA2301-35s-LeDnaJ constructed in example 1 was transformed into upland cotton variety R15 by Agrobacterium mediated method. The method comprises the following specific steps:

culture medium for genetic transformation of upland cotton

Agrobacterium culture medium:

streaking plate medium: LB solid culture Medium +50 mg. L^-1Kanamycin +20 mg. L^-1Rifampicin.

Liquid culture medium: LB liquid culture Medium +50 mg. L^-1Kanamycin +20 mg. L^-1Rifampicin.

Plant culture medium:

minimal Medium (MSB): inorganic salts of MS medium + organics of B5 medium.

Sterile seedling culture medium: 1/2MS inorganic salt + agar 6.5 g.L ^-1。

Co-culture medium: MSB +0.1 mg. L^-1 2.4-D+0.1mg·L^-1KT+30g·L^-1Glucose +6.5 g.L^-1Agar.

Kanamycin (Km) resistant callus induction medium: MSB +0.1 mg. L^-1 2.4-D+0.1mg·L^-1KT+0.91g·L^-1MgCl₂+2.0g·L^-1Gelrite+50mg·L^-1Km+500mg·L^-1Cef+30g·L^-1And (3) glucose.

Embryogenic callus induction medium: MSB +1.9 g.L^-1KNO₃+0.91g·L^-1MgCl₂+2.5g·L^{- 1}Gelrite+30g·L^-1And (3) glucose.

Embryo maturation and germination medium: MSB + NH₄NO₃+1.9g·L^-1KNO₃+0.5g·L^-1Radonamide +1.0 g.L^-1Glutamine +0.91 g.L^-1MgCl₂+2.5g·L^-1Gelrite+30g·L^-1And (3) glucose.

Regenerated plantlet growth medium: inducing culture medium for homoembryogenic callus.

All media were adjusted to pH 6.5 with KOH and pH between 5.6-5.9 after sterilization; all cultures were incubated at 25 ℃ and 2 ℃ under light at 3000. sup. th and 5000. sup. lx, at 16h/8h D/N.

Genetic transformation of upland cotton

1. Sterile seedling preparation

Sulfuric acid (H) for cotton seeds₂SO₄) Removing short fibers, washing sulfuric acid on the surfaces of the seeds by tap water, and airing; sterilizing the seeds with 70% ethanol surface for 1min, and discarding ethanol. 10% -15% hydrogen peroxide (H)₂O₂) Sterilizing the seeds for 3-4 h. Washing the seeds with sterile water for 3 times, adding sterile water, and placing in a culture room for 18-24h until the seeds are exposed; peeling seed coats under aseptic conditions, inoculating on a seedling culture medium, and culturing at 25-28 deg.C for 5d (figure 2).

2. Agrobacterium transformation and culture

(1) The recombinant expression vector pCAMBIA2301-35s-LeDnaJ constructed in example 1 was transferred into competent cells of Agrobacterium strain EHA105 by heat shock method to obtain an engineered Agrobacterium for plant transformation experiments, named EHA105(pCAMBIA2301-CaMV 35S-LeDnaJ).

(2) Activating agrobacterium: the agrobacterium carrying the target gene is inoculated in LB liquid culture medium, and is cultured overnight under shaking at 28 ℃, and is reserved when OD600 value is 0.3-0.7.

(3) And (4) using LB solid culture medium of overnight cultured agrobacterium culture solution to draw a single colony, and culturing for 48h at the temperature of 28 ℃.

(4) 3-5 single colonies were picked and cultured overnight in LB liquid medium containing 50mg kanamycin and 25mg rifampicin, and when the strain entered logarithmic phase (OD600 value is around 2.0), the bacterial liquid was diluted to OD600 of 0.3-0.5 with the liquid co-culture medium for further use.

3. Infection and Co-culture

Cutting hypocotyl of aseptic seedling into 0.5-0.7cm sections with scalpel blade in sterilized culture dish of super clean bench, infecting (soaking) the cut hypocotyl sections with diluted bacterial liquid for 7-8min, adsorbing excess bacterial liquid on the hypocotyl sections with sterilized filter paper, placing on co-culture medium with a layer of sterilized filter paper, sealing with sealing film, and co-culturing at 22-25 deg.C for 2d (FIG. 3).

4. Induction, proliferation and subculture of resistant callus

And inoculating the hypocotyl section after co-culture into a resistant callus induction culture medium, and carrying out subculture once for 30 d. The expansion of both sides of the hypocotyl cut can be observed after the first inoculation for about 10 days, and a small amount of callus grows out of the section from 20 days to 30 days (figure 4). When soft callus blocks with the diameter of about 0.5cm appear at the two ends of the hypocotyl (figure 5), the calluses at the two ends are peeled off by a scalpel and a pair of tweezers and transferred into the same culture medium for separate culture, so that the agrobacterium tumefaciens pollution can be effectively removed and the browning phenomenon of the calluses can be prevented.

5. Induction of embryogenic callus

The diameter of the callus on the resistant callus induction medium reached 1-3cm, which was subcultured to a sexual callus induction medium with the hormone removed and potassium nitrate added, and cultured under the conventional conditions in the culture room, the same medium was subcultured every one month or so, 2-4 times, and yellow-green or gray-green rice-grain-shaped embryogenic callus began to appear at the bottom and around the callus pieces (FIG. 6).

6. Regeneration of

Picking out embryogenic callus, transferring the embryogenic callus into an embryo maturation and germination culture medium paved with filter paper, wherein the embryo maturation and germination culture medium is 0.1-0.2 g/bottle (a in figure 7) to further form an embryoid (b in figure 7), the germinated embryoid grows to about 1cm of a hypocotyl (c in figure 7), picking out and inserting the embryoid into a regenerated plantlet growth culture medium to continue growing, a part of the embryoid can quickly grow into a plantlet with normal root, stem and leaf (d in figure 7), the embryoid grows to 3-5 true leaves or 3-5cm above the epicotyl (e in figure 7), taking 2-3 leaves of the plantlet under aseptic condition, extracting DNA by a small-amount method, carrying out PCR amplification of a marker gene and a target gene, and cutting off 3-5cm of the upper part of the positive plantlet to be used as a scion for grafting.

Identification of transformed plants

After transformation, PCR, RT-PCR and Southern detection were performed on the transformed plants.

1. PCR detection

The first step is as follows: extraction of Cotton genome (Using modified CTAB method)

DNA extraction A CTAB method (Paterson, 1993) is adopted for DNA extraction, and the specific steps are as follows:

firstly, taking about 1g of tender cotton leaves, immediately putting the tender cotton leaves into a precooled mortar, adding liquid nitrogen, quickly grinding the tender cotton leaves into powder, immediately putting the powder into a 2ml centrifuge tube, adding 1ml of extracting solution, uniformly shaking the mixture, carrying out ice bath for 10 minutes, carrying out 8000rpm for 20min, and discarding the supernatant.

② adding 0.7ml of lysis buffer preheated at 65 ℃ into the precipitate, mixing evenly, and carrying out water bath at 65 ℃ for 30 min. Uniformly mixing once every 10min to fully disperse the sample.

③ after the water bath is finished, adding the chloroform with the same volume, slowly inverting the centrifuge tube for 30-50 times, and then centrifuging for 15min at 12000 r/s.

And fourthly, adding chloroform-isoamylol (24: l, volume ratio) with the same volume into the supernatant to re-extract once.

Fifthly, taking the supernatant, adding ice-cooled isopropanol with the volume 0.6 times that of the supernatant, slowly inverting the centrifuge tube until flocculent precipitates are integrated.

Sixthly, standing for 30min (or putting the precipitate into a refrigerator with the temperature of minus 20 ℃ for 20min to 40min), picking out the precipitate, transferring the precipitate into a l.5ml centrifuge tube, washing the precipitate for 2 to 3 times by using 70 percent alcohol, air-drying the precipitate, adding 200ml TE for dissolving, and preserving the precipitate at the temperature of minus 20 ℃.

The second step is that: reaction conditions for PCR amplification of the target fragment:

(1) PCR (polymerase chain reaction) primer for detecting target gene LeDNAJ

DNAJ-F：5’-GCTCTAGACCAAATCTATGGCTTCT-3’；

DNAJ-R：5’-TCCCCCGGG-CTACCAACACTGATCGGTT-3’。

(2) PCR amplification of fragments of interest

10 μ l reaction: template 1.0 μ L; 10 × PCR buffer 1.0 μ L; MgCl₂(25mmol/L) 0.6. mu.L; dNTP (10mmol/L) 0.3. mu.L; primer F (10. mu. mol/L) 1.0. mu.L; primer R (10. mu. mol/L) 1.0. mu.L; rTaq 0.1. mu.L; sterile dd H₂O 5.0μL。

PCR amplification procedure: 5min at 94 ℃; 30s at 94 ℃, 45s at 55 ℃, 45s at 72 ℃ and 36 cycles; 10min at 72 ℃; keeping the temperature at 4 ℃.

The third step: and (5) agarose electrophoresis detection.

1.0% agarose gel, run under 1 XTAE buffer system.

2. RT-PRC detection

The first step is as follows: RNA extraction

(1) Taking 0.5g fresh cotton tissue, adding 0.1g cross-linked polyvinylpyrrolidone (PVPP), fully grinding in liquid nitrogen to powder, quickly transferring the frozen powder into a 10ml centrifuge tube, adding 5ml CTAB extract and 500 μ L0.1M Tris-HCl with pH of 8.0, and carrying out water bath at 65 ℃ for 20 min; turning and mixing in the midway.

(2) Adding equal volume of chloroform, mixing, and standing in ice bath for 10 min.

(3) Centrifuging at 10000rpm for 20min at 4 deg.C. Subpackaging in 4 1.5ml centrifuge tubes.

(4) The supernatant was mixed with 1/3 volumes of 8M LiCl and mixed at-70 ℃ for 30min or-20 ℃ overnight.

(5) Centrifuging at 10000rpm for 20min at 4 deg.C. The supernatant was discarded, washed twice with 70% ethanol and dissolved in 30. mu.L of DEPC water.

(6) Adding 10U of DNase without RNase activity and 25U of RNase inhibitor, digesting by 10 Xbuffer for 30min, adding equal volume of chloroform, and extracting once.

(7) The supernatant was transferred to a new tube and 1/10 volumes of 3M pH 5.2NaAc and an equal volume of isopropanol or 2.5 volumes of absolute ethanol were added and ice-cooled at-70 ℃ for 3 h.

(8) Centrifuging at 4 deg.C and 10000rpm for 20min, discarding supernatant, washing with 70% ethanol twice, and dissolving in 30 μ L DEPC water. Thus obtaining the cotton RNA.

The second step is that: first Strand cDNA Synthesis

42℃(2h)→70℃(10min)→4℃

And (3) detecting by taking the cDNA as a template, wherein the PCR system and the reaction procedure are the same as the step 1.

In addition, the primers for detecting actin using actin as a control were:

Actin-F：5’-CAGGAAACCAAGAAGGGCAAGAAAA-3’；

Actin-R：5’-TGTCCGTCAGGCAACTCAT-3’。

3. southern hybridization analysis of copy number

Reagent and its preparation

(1) And (3) denatured liquid: 0.5NaOH, 1.5M NaCl

(2) Neutralizing liquid: 1.5M NaCl, 0.1M Tris-HCl (pH7.4)

(3)20 XSSC: 175.3g NaCl, 88.2g sodium citrate, adjusted to pH7.0 with 10M NaOH, added with water to a volume of 1000ml, autoclaved.

(4) Maleic acid buffer: 0.1M maleic acid, 0.15M NaCl, pH adjusted to 7.5(20 ℃ C.) with NaOH (solid).

(5) Washing buffer solution: maleic acid buffer, 0.3% Tween 20 (v/v).

(6) Detection buffer solution: 0.1M Tris-HCl, 0.1M NaCl, pH 9.5(20 ℃).

(7) TE buffer solution: 10mM Tris-HCl, 1mM EDTA, pH 8.0.

(8) Blocking solution: using maleic acid buffer according to 1: 10 ratio 10 × blocking solution (vial No. 6) was diluted to 1 × working solution.

(9) Antibody solution: anti-digoxigenin-AP (tube 4) in the original tube was centrifuged at 10000rpm for 5 minutes before each use, then carefully pipetted the desired amount from the surface and blocked with blocking solution as 1: anti-digoxigenin-fluoroscein was diluted at a ratio of 5000.

(10) Color development liquid: to 10ml of assay buffer was added 200. mu.L of NBT/BCIP stock (kit tube 5) (ready for use, protected from light).

(11) And (3) washing solution I: 2 XSSC, 0.1% SDS.

(12) Washing the membrane liquid: 1 XSSC, 0.1% SDS.

The above reagents are from kit companies: roche, name: DIG-High Prime DNA Labeling and Detection Starter Kit II; the goods number is: 11585614910.

the first step is as follows: enzyme digestion of genomic DNA

Enzyme digestion system of genomic DNA: 30 μ g of genomic DNA; endonuclease 15. mu.L (20U/. mu.L); 10 × buffer40 μ L; ddH₂Make up to 400. mu.L of O.

In the experiment, EcoRI restriction enzyme is selected to carry out enzyme digestion on genome DNA.

The method comprises the following specific steps:

(1) after the addition of the above system, the enzyme digestion is carried out for 16 hours at 37 ℃, and a 2 mu L agarose gel electrophoresis detector is taken to judge whether the enzyme digestion is complete.

(2) After the enzyme digestion is completed, adding equal volume of chloroform for extraction, adding isopropanol for precipitation for one hour, drying the precipitate, adding ddH ₂And O is completely dissolved.

The second step: digoxin labeled probe

(1) The DNA fragment (about 1. mu.g) recovered from the PCR product was treated with sterilized ddH₂Diluting O to 16 μ L, sealing with parafilm, heating in boiling water for 10min, and rapidly placing in crushed ice for more than 2 min.

(2) After brief centrifugation at 4 ℃ 4. mu.L of DIG-High Prime (tube 1 of Kit; company of Kit: Roche, name: DIG-High Prime DNA Labeling and Detection Kit II; cat # 11585614910) was added, the mixture was repeatedly aspirated by a pipette tip, and the mixture was incubated at 37 ℃ for 20 hours

(3) The reaction was stopped by adding 2. mu.l of 0.5mol/L EDTA (pH8.0)

(4) The resulting extract was purified with QIAquick Nucleotide Removal Kit from QIAGEN, and stored at-20 ℃ for further use

The third step: rotary film

The transfer operation method is used for printing and recording the capillary hybridization, and the specific operation is as follows:

and sequentially adding the enzyme-cut DNA into 0.8% agarose gel, performing low-pressure electrophoresis at 30V, and stopping electrophoresis when bromophenol blue runs to 3/4 of the gel. Excess was cut off, the length and width of the gel were recorded, and the right corner was cut as a mark.

Placing the gel in a denaturing solution with a volume of several times for denaturation for 45min, and continuously oscillating during denaturation;

rinsing the gel with ultrapure water, neutralizing the neutralization solution for 15min, and repeating the steps once;

The water absorption paper with the thickness of 5-7cm, the filter paper with the thickness of 1cm and the soaked nylon membrane are stacked in the porcelain plate from bottom to top, and the nylon membrane and the filter paper cannot have air bubbles.

The gel was placed on the nylon membrane with the back side down, and several filter papers wetted in advance with 10 XSSC were placed on the membrane, taking care to avoid air bubbles between the gel, the nylon membrane and the filter papers.

Transfer was initiated by placing a strip of filter paper of appropriate width and dipping both ends in 10 XSSC. Note: the filter paper can not contact with the absorbent paper and the filter paper below the nylon membrane, so that the short circuit caused by capillary action is avoided.

18-24h later, the nylon membrane is taken down, washed by 2 XSSC and fixed for 2 hours at 80 DEG C

The fourth step: hybridization and membrane washing

Hybridization was carried out according to the instructions of the digoxin Kit (DIG High Prime DNA labelling and Detection Starter Kit I) from Roche. The hybridization temperature is 37-42 ℃, and the operation steps are as follows:

(1) soaking the fixed nylon membrane in 2 XSSC, placing into a clean hybridization tube, adding a proper volume of DIG Easy Hyb preheated at 37 ℃, paying attention to remove bubbles between the nylon membrane and the tube wall, and prehybridizing for 30min-2 h.

(2) The purified probe was placed in a boiling water bath for 5min and rapidly cooled on ice for 5 min.

(3) The probe was added to pre-heated DIG Easy Hyb and mixed well, carefully added to the bottom of the hybridization tube.

(4) The hybridization tubes were placed in a hybridization oven for 20 hours.

(5) And rinsing the nylon membrane for 5min at room temperature by using the membrane-washing solution I, and repeating the rinsing once.

(6) And rinsing the nylon membrane twice with a membrane washing solution II preheated to 55 ℃ for 7min each time.

(7) The nylon membrane is rinsed with washing buffer for 5min and soaked in 100ml blocking solution for 30 min.

(8) The membrane was immersed in 20ml of antibody solution for 30 min.

(9) Washing twice with large-volume membrane washing solution for 15min each time.

(10)20mL of detection buffer was equilibrated for 2-5min, and then developed in the freshly prepared developing solution (10mL) for 6-12 hours in the absence of light, taking care to avoid shaking after the development is started.

(11) After the development, the film was rinsed with TE for 5min to stop the reaction, and photographed or scanned for recording.

The PCR detection result is shown in FIG. 8, the amplified target band is consistent with the positive control (plasmid pCAMBIA2301-35s-LeDnaJ) and has the size of about 480bp, and the target gene LeDnaJ is preliminarily proved to be integrated into the cotton genome. While the control (receptor material R15) did not develop the band of interest, excluding possible gene interference of the receptor itself.

The RT-PCR detection result of the transgenic cotton positive plant detected by PCR is shown in figure 9, in figure 9A, after the PCR positive individual plant reversal product is amplified by the internal reference primer Actin, the reversal product is successful because the target band of about 200bp is existed. In FIG. 9B, the transcription level was detected by semi-quantitative RT-PCR, and the strain whose amplification product was 480bp or so was positive in expression level.

To further determine whether the foreign gene LeDnaJ has been transferred into the cotton genome and analyzed for copy number in the cotton genome. Selecting several transgenic plant materials with positive PCR and RT-PCR detection, extracting genome DNA, and performing Southern hybridization. FIG. 10 shows the hybrid bands with arrows, and the single copy in lane 1 (plant No. DNAJ-1), and the double copy in lane 2 (plant No. DNAJ-2) and lane 3 (plant No. DNAJ-3).

As the exogenous gene transformation process needs a stabilization process and the character needs to be stabilized, in the tissue culture stage, a culture medium added with kanamycin is adopted, 3 pure cotton line descendants (numbered as DNAJ-1, DNAJ-2 and DNAJ-3) of the LeDnaJ gene transferred by T5 generations are obtained through multi-generation selfing screening, and are proved to be positive through PCR and RT-PCR detection (figure 11), Southern hybridization proves that the Lane 1 is that the DNAJ-1 is single copy, the Lane 2 is that the DNAJ-2 is double copy, and the Lane 3 is that the DNAJ-3 is double copy, and thus the LeDnaJ exogenous genes in the three transgenic lines are integrated into a receptor cotton material by different copy numbers.

Fourth, the salt tolerance identification of the positive transformed plant

The invention carries out salt tolerance experiments on three T5 generation strains (DNAJ-1, DNAJ-2 and DNAJ-3) obtained in the third step in the germination stage and the seedling stage.

1. Identification of salt tolerance of LeDNAJ gene-transferred cotton strain in germination period

Disinfecting the seeds to be tested, and then placing the seeds in 30% H₂O₂Shaking at room temperature for 2-3h, and then ddH₂O wash 3-5 times, and incubate sterilized seeds on solid MS medium containing 20% NaCl, 3 replicates per material, each replicate containing 50 seeds. And when the seeds grow for 7 days, counting the germination rate of the seeds. Seeds with an exposure of more than 5mm are considered as germinated seeds. The germination rate of the seeds, i.e., the number of seeds with a white exposure of more than 5 mm/total number of seeds, is measured for the length of the root system of the germinated seeds, and the measurement tool is a ruler (cm).

TABLE 1 salt stress 3 transgenic pure lines and control germination statistics

Note: D-1D-2D-3 is 3 transgenic pure lines respectively

*: significant difference at 0.05 level

We performed statistics on the root length of 3 transgenic materials and their control in germination phase under salt stress. Germination was performed on MS medium with 250mM NaCl, and the root elongation of 3 transgenic pure lines was significantly higher than that of the control three days after germination (FIG. 12). The ratio of the root length under the salt stress to the root length under the normal condition is taken as the relative elongation value of the root length, and the results show that the relative root lengths of three transgenic pure lines of DNAJ-1, DNAJ-2 and DNAJ-3 are all remarkably higher than that of a control R15 (table 2), which indicates that the LeDnaJ gene improves the salt tolerance of upland cotton.

TABLE 2 comparison of root length of 3 transgenic inbred lines and control germinated under salt stress

Material	Root length under 0mM NaCl(cm)	Root growth root (cm) under 200mM NaCl	Relative root length
				D-1	4.4±0.41	1.9±0.22	0.4318**
D-2	3.9±0.36	1.7±0.18	0.4358**
				D-3	4.1±0.38	1.7±0.23	0.4146**
R15	4.3±0.36	0.6±0.11	0.1395

Note: D-1D-2D-3 is 3 transgenic pure lines DNAJ-1, DNAJ-2 and DNAJ-3 respectively. *: significantly different at the 0.05 level compared to R15;^**the difference was significant compared to R15 at the 0.01 level.

2. Salt tolerance identification of LeDNAJ gene-transferred cotton strain in seedling stage

Placing the cotton seedlings in a room with the temperature controlled at 28 ℃, and carrying out light and dark treatment for 12 hours respectively; salt treatment was started when the plant had grown to 2 leaves and 1 heart. Keeping seedlings with consistent growth for treatment, wherein the salt treatment concentration is 250mM, 50ml of saline water is poured into each cup, 50ml of clear water is poured into each cup at the same time, and 10 seedlings are treated by comparing the salt with the clear water; meanwhile, 10 seedlings are taken as a control on the same day of treatment, and relevant indexes including plant height, overground part and underground part growth amount and the like are measured. And 7 days after treatment, measuring the plant related indexes including the relative height of the plant, the relative growth amount of the overground part and other salt-tolerance related characters under the salt stress and the clear water contrast.

The salt tolerance of 3 pure cotton lines of the LeDNAJ gene at the seedling stage is subjected to phenotype identification. When the plants grow to 2 leaves and 1 heart, 250mM NaCl and clear water are respectively used for treating the plants in the seedling stage, meanwhile, the seedlings on the treatment day are used as a control, the plant related indexes under salt stress and clear water control are measured 7 days later, and the plant height of the transgenic line is obviously higher than that of the control R15 (figure 13) after 7 days later. The plants on the day of treatment are used as a control, the growth amount of the plants under salt stress relative to the growth amount of the plants in clear water treatment, namely the relative growth amount is used as a salt tolerance index and comprises relative plant height, relative fresh weight of the overground part and relative dry weight of the overground part, and the result shows that the relative plant height, relative fresh weight of the overground part and relative dry weight of the overground part of 3 transgenic lines are remarkably or extremely remarkably higher than the control (Table 3), which indicates that the LeDnaJ gene improves the salt tolerance of the upland cotton.

TABLE 3 phenotypic statistics of 3 transgenic inbred lines and control seedling stages under salt stress

Material	Relative plant height	Relatively fresh and heavy	Relative dry weight
				D-1	0.514±0.009**	0.431±0.007*	0.554±0.023*
D-2	0.486±0.016*	0.406±0.014*	0.528±0.035*
				D-3	0.454±0.014*	0.381±0.012*	0.496±0.011*
R15	0.379±0.015	0.305±0.015	0.406±0.014

Note: D-1D-2D-3 is 3 transgenic pure lines DNAJ-1, DNAJ-2 and DNAJ-3 respectively. *: significantly different at the 0.05 level compared to R15;^**the difference was significant compared to R15 at the 0.01 level.

Sequence listing

<110> Jiusheng Setaria species industries Ltd

<120> method for improving salt tolerance of cotton by utilizing LeDNAJ gene and application thereof

<130> GNCLN192399

<141> 2019-11-14

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Pro Phe Ser Val Ser Ala Ala Tyr Ser Thr Ala Glu Arg Thr Ser Thr

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Tyr Arg Lys Leu Ala Arg Ile Leu His Pro Asp Val Arg Asn Ser Ser

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Pro Glu Lys Arg Ala Asn Tyr Asp Arg Asn Leu Phe Gly Asn Arg Ile

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Ala Arg Pro Val Asp Phe Ser Thr Ala Gly Ala Arg Ser His Tyr Thr

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cttccaaatc tatggcttct tcttcttttc ttctctccac ttcaattacc ggctctaaac 60

tctccgccgc tgcaccaccg cggagttctg ttagcttcaa gcagcggccg ttctctgttt 120

ccgccgcgta ctccactgcg gagaggactt ctactgctac tactagtagc tctacaatcg 180

cctcacatac atcgttatac gaagttttag ggattcgatt tggagctaat tctcatgaaa 240

ttaagtctgc ttaccggaaa ttagccagaa ttttgcatcc agatgttcgt aattcgtcgg 300

cggaggactt tataagagtc caatcagcgt atgctactct ttccgatccg gaaaaacgtg 360

ctaattatga tcggaaccta tttggaaata gaattgcaag gcctgttgat ttctcaacgg 420

cgggagctcg cagccattat actgttcgcc gaggatggga aaccgatcag tgttggtaga 480

att 483

Claims (9)

1. The application of LeDNAJ protein or related biological materials thereof in regulating and controlling the salt tolerance of plants;

   the related biological material is a nucleic acid molecule capable of expressing the LeDNAJ protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

   the LeDNAJ protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No. 1;

   (A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

   the plant is cotton or tomato.
2. 2. Use according to claim 1, characterized in that: the activity and/or expression level of the LeDNAJ protein or the coding gene thereof in the plant is improved, and the salt tolerance of the plant is improved;

   The LeDNAJ protein or the coding gene thereof has reduced activity and/or expression in the plant, and the plant salt tolerance is reduced.
3. 3. Use according to claim 1 or 2, characterized in that: the nucleic acid molecule capable of expressing the LeDNAJ protein is a coding gene of the LeDNAJ protein; the coding gene of the LeDNAJ protein is any one of the following DNA molecules:

   (B1) a DNA molecule represented by positions 12-479 of SEQ ID No. 2;

   (B2) DNA molecule shown in SEQ ID No. 2;

   (B3) a DNA molecule which hybridizes with the DNA molecule defined in (B1) or (B2) under stringent conditions and encodes the LeDNAJ protein;

   (B4) a DNA molecule which has more than 80% of homology with the DNA sequence defined in (B1) or (B2) or (B3) and encodes the LeDNAJ protein.
4. 4. A method for breeding a plant variety having improved salt tolerance, comprising the step of increasing the expression level and/or activity of a LeDNAJ protein in a recipient plant;

   the LeDNAJ protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No. 1;

   (A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

   the plant is cotton or tomato.
5. 5. A method for breeding a plant variety with reduced salt tolerance, comprising the step of reducing the expression level and/or activity of a LeDNAJ protein in a recipient plant;

   the LeDNAJ protein is any one of the following proteins:

   (A1) a protein having an amino acid sequence of SEQ ID No. 1;

   (A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

   the plant is cotton or tomato.
6. 6. A method of breeding transgenic plants with improved salt tolerance comprising the steps of: introducing nucleic acid molecules capable of expressing LeDNAJ protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased salt tolerance as compared to the recipient plant;

   the LeDNAJ protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No. 1;

   (A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

   the plant is cotton or tomato.
7. 7. A method of breeding a transgenic plant with reduced salt tolerance comprising the steps of: carrying out suppression expression on the coding gene of the LeDNAJ protein in the receptor plant to obtain a transgenic plant; the transgenic plant has reduced salt tolerance as compared to the recipient plant;

   The LeDNAJ protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No. 1;

   (A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

   the plant is cotton or tomato.
8. 8. The method of claim 6, wherein: introducing into the recipient plant a nucleic acid molecule capable of expressing the LeDNAJ protein is achieved by introducing into the recipient plant a recombinant expression vector comprising a gene encoding the LeDNAJ protein.
9. 9. The method of claim 6, wherein: the nucleic acid molecule capable of expressing the LeDNAJ protein is a coding gene of the LeDNAJ protein; the coding gene of the LeDNAJ protein is any one of the following DNA molecules:

   (B1) a DNA molecule represented by positions 12-479 of SEQ ID No. 2;

   (B2) DNA molecule shown in SEQ ID No. 2;

   (B3) a DNA molecule which hybridizes with the DNA molecule defined in (B1) or (B2) under stringent conditions and encodes the LeDNAJ protein;

   (B4) a DNA molecule which has more than 80% of homology with the DNA sequence defined in (B1) or (B2) or (B3) and encodes the LeDNAJ protein.

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