CN107602679B - TabHLH44 protein and its encoding gene and application - Google Patents

Abstract

The invention discloses TabHLH44 albumen and its encoding gene and applications.The present invention provides albumen, are named as TabHLH44, be it is following 1) or 2): 1) protein shown in sequence 2 in sequence table；2) amino acid sequence shown in sequence 2 in sequence table by the substitution and/or deletion and/or addition of one or several amino acid residues and had into identical function protein as derived from sequence 2.The experiment proves that, the present invention passes through to the processing of wheat environment stress, filter out the transcription factor TabHLH44 gene of response environment stress, it is induced by salt, PEG, cold stress and ABA, it is conducted into arabidopsis, obtains turning TabHLH44 arabidopsis, carried out Function Identification, show that TabHLH44 improves the drought resistance, salt-resistance and frost resistance of transgenic arabidopsis, provides basis to cultivate resistance plant.

Description

TabHLH44 protein and its encoding gene and application

technical field

The invention relates to the field of biotechnology, in particular to a TabHLH44 protein and its encoding gene and application.

Background technique

The harsh growth environment (extreme temperature, salinity, drought, etc.) seriously affects the growth and development of plants and reduces the yield of crops. Plants cannot avoid adversity stress by moving. Therefore, in order to eliminate or reduce the harm caused by adversity stress to themselves, plants have formed a complex regulatory network in the process of evolution, so that plants can pass the cellular level, molecular Adjustment of levels, etc. to adapt to unfavorable growing conditions. In order to reduce the huge loss of crops caused by the unfavorable growth environment and ensure food security, how to improve the stress resistance of crops has attracted the attention of governments around the world. Genetic improvement of crops to improve their resistance to stress is an effective method.

Transcription factor is a regulatory gene and an important molecule in the regulatory network. It participates in almost all life processes of organisms through signal transduction and regulation of stress response genes. Involved in plant response to biotic and abiotic stress, and played a key role in the process of plant response to stress. However, there are relatively few studies on the function of stress-related TabHLH transcription factors in wheat.

SUMMARY OF THE INVENTION

An object of the present invention is to provide TabHLH44 protein and its encoding gene.

The protein provided by the present invention, named as TabHLH44, is as follows 1) or 2):

1) the protein shown in sequence 2 in the sequence listing;

2) A protein derived from Sequence 2 with the amino acid sequence shown in Sequence 2 in the Sequence Listing subjected to substitution and/or deletion and/or addition of one or several amino acid residues and having the same function.

In order to facilitate purification of the protein in (1), a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

Table 1 Sequences of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The protein in the above (2) can be obtained by artificial synthesis, or by first synthesizing its encoding gene and then biologically expressing it. The gene encoding the protein in the above (2) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 1 in the Sequence Listing, and/or making one or several base pairs missense. Mutation, and/or ligation of the coding sequence of the tags shown in Table 1 at its 5' and/or 3' ends.

DNA molecules encoding the above proteins are also within the scope of the present invention.

The above-mentioned DNA molecule is the DNA molecule of any one of the following 1)-4):

1) the coding region is the DNA molecule shown in sequence 1 in the sequence listing;

2) the coding region is the DNA molecule shown in position 271-1458 of sequence 1 in the sequence listing;

3) DNA molecules that hybridize to the DNA sequences defined in 1) or 2) under stringent conditions and encode a protein with the same function;

4) The DNA sequences defined in 1) or 2) have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% %, have at least 98% or at least 99% homology and encode DNA molecules with the same function.

The above stringent conditions can be hybridization in a solution of 6×SSC, 0.5% SDS at 65°C, and then the membrane is washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above DNA molecules are also within the scope of the present invention.

The recombinant vector is a vector obtained by inserting nucleotides 271-1458 of sequence 1 between the two attB sites of the pEarleyGate 100 vector, which is an overexpressing TabHLH44 gene vector, named pEarleyGate 100-TabHLH44.

The primer pair for amplifying the full length of the above DNA molecule or any fragment thereof is also within the scope of protection of the present invention.

The application of the above-mentioned protein, the above-mentioned DNA molecule or the above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in regulating plant stress resistance is also within the scope of protection of the present invention;

Or the application of the above-mentioned protein, the above-mentioned DNA molecule or the above-mentioned recombinant vector, expression cassette, transgenic cell or recombinant bacteria in cultivating transgenic plants with improved stress resistance is also within the scope of protection of the present invention.

In the above application, the stress resistance is salt resistance, drought resistance and/or low temperature resistance;

The plants are monocotyledonous or dicotyledonous.

Another object of the present invention is to provide a method for cultivating transgenic plants with improved stress resistance.

The method provided by the present invention is to introduce the DNA molecule encoding the above-mentioned protein into the target plant to obtain a transgenic plant,

The stress resistance of the transgenic plant is higher than that of the target plant;

In the above method, the stress resistance is salt resistance, drought resistance and/or low temperature resistance;

In the above method, the drought resistance of the transgenic plant is higher than that of the target plant system, and the survival rate of the transgenic plant under drought stress is higher than that of the target plant;

The salt resistance of the above-mentioned transgenic plant is higher than that of the target plant system, and the survival rate or root length of the transgenic plant under salt stress are higher than those of the target plant;

The low temperature resistance of the above-mentioned transgenic plants is higher than that of the target plant system, and the survival rate of the transgenic plants under low temperature stress is higher than that of the target plants.

Drought stress is no watering; salt stress is 150mM NaCl; low temperature stress is -10℃;

In the above method, the plant is a monocotyledonous plant or a dicotyledonous plant.

The low temperature is specifically -10°C;

The dicotyledonous plant is specifically Arabidopsis thaliana.

The experiment of the present invention proves that the transcription factor TabHLH44 gene that responds to the adversity stress is screened out by the treatment of wheat adversity stress, which is induced by salt, PEG, cold stress and ABA, and then introduced into Arabidopsis thaliana to obtain a transgenic TabHLH44 gene. Arabidopsis, its functional identification showed that TabHLH44 improved the drought resistance, salt resistance and freezing resistance of transgenic Arabidopsis, providing a basis for breeding resistant plants.

Description of drawings

Figure 1 shows the expression patterns of TabHLH44 under different stress treatments.

Figure 2 shows the identification of drought resistance in Arabidopsis transfected with TabHLH44.

Figure 3 shows the survival rate of Arabidopsis transfected with TabHLH44 for drought resistance.

Figure 4 is the identification of salt resistance of Arabidopsis thaliana transfected with TabHLH44.

Figure 5 shows the root lengths of transgenic Arabidopsis thaliana under salt-resistant treatment.

Figure 6 is the identification of frost resistance of Arabidopsis transfected with TabHLH44.

Figure 7 shows the survival rate of Arabidopsis transfected with TabHLH44 against freezing treatment.

In each of the above figures, WT: wild type; L1, L2, L3: Arabidopsis plants transduced with TabHLH44.

Detailed ways

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

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Discovery of TabHLH44 gene

1. Discovery of the TabHLH44 gene

In the previous work of the laboratory, several full-length cDNA libraries of wheat were constructed. By sequencing the cDNA plasmids, the bHLH transcription factor was predicted according to the structural characteristics of the transcription factor, and the full-length cDNA sequence of the TabHLH44 gene was obtained.

The nucleotide sequence of the TabHLH44 gene in wheat is sequence 1, and the open reading frame is nucleotides 271-1458 of sequence 1; the encoded protein TabHLH44 has the amino acid sequence 2.

2. Expression of TabHLH44 gene under stress conditions

Chinese spring, drought-resistant wheat Hanxuan No. 10 and salt-resistant wheat Chadianhong, which were used for stress treatment, were cultured for 10 days at 25°C under 16h light/8h dark photoperiod, respectively, and then different stress treatments were performed. Chinese spring was used for cold treatment at 4°C and ABA treatment. Leaf tissue was collected at 0, 3, 7, 12, 24, and 48 h during cold treatment, and root tissue was collected at 0, 1, 3, 7, 12, and 24 h during ABA treatment. . The drought-resistant wheat, Hanxuan No. 10, was treated with 16.1% PEG6000, and the root tissue was taken at 0, 1, 3, 7, 12 and 24 h. Salt-resistant wheat tea starch red, used for 250 mM salt treatment, root tissue was harvested at 0, 1, 3, 7, 12 and 24 h. The RNA of the sample was extracted, and the cDNA was obtained by reverse transcription as a template. The gene-specific primers (F primer, ATGACAGGTTCGCGTTCGTC; R primer, TCTTGTTCCCATTCGTTGGG) were used to amplify, and the Tubulin gene (F primer, ACCGTGGTGATGTTGTGC; R primer, TGGTGGCTGGTAGTTGATA) was used for amplification. Internal reference.

The results are shown in Figure 1. It can be seen that under the condition of NaCl treatment, within 3h of treatment, the expression of TabHLH44 gradually increased with the increase of treatment time, and then the expression decreased, and the expression increased significantly when the treatment was 12h, until 24h. The TabHLH44 gene remained highly expressed. Under PEG treatment, the expression level of TabHLH44 increased slowly, reached the maximum value at 7h, then began to decrease, and began to increase again at 24h. Under the condition of cold treatment, the expression level of TabHLH44 increased rapidly at 3h, began to decrease at 7h, increased again at 24h, and maintained a high expression level until 48h. Under the condition of ABA treatment, the expression level of TabHLH44 increased at 3h, and then decreased. The results showed that the expression of TabHLH44 was induced by NaCl stress, PEG stress, cold stress and ABA stress, and could respond to a variety of stress. Therefore, further functional identification of TabHLH44 was carried out in Arabidopsis.

Example 2. Application of TabHLH44 gene in stress resistance

1. Preparation of Arabidopsis transfected with TabHLH44

1. Use the Gateway method to construct an Arabidopsis-transformed overexpression vector

The BP plasmid is a plasmid obtained by inserting the TabHLH44 gene shown at positions 271-1458 of SEQ ID NO: 1 into the entry vector pDONR ^™ /Zeo (Invitrogen, 12535035).

LR reaction: The above-mentioned BP plasmid and pEarleyGate 100 overexpression vector were subjected to LR reaction by the method of Gateway technology to obtain a recombinant vector. The LR reaction system is shown in Table 2 below:

Table 2 is the LR reaction

The recombinant vector is a vector obtained by inserting nucleotides 271-1458 of sequence 1 between the two attB sites of the pEarleyGate 100 vector, which is an overexpressing TabHLH44 gene vector, named pEarleyGate 100-TabHLH44.

2. Transformation of Agrobacterium

The recombinant vector pEarleyGate 100-TabHLH44 was transferred into Agrobacterium GV3101 (GV3101 Agrobacterium competent cells, Shanghai Chaoyan Biotechnology Co., Ltd., CC3201) to obtain recombinant bacteria GV3101/pEarleyGate 100-TabHLH44 (the plasmid was extracted and sequenced correctly).

3. Agrobacterium infection and transformation of Arabidopsis and screening of transgenic plants

A) Culture of Arabidopsis

1) Under sterile conditions, wild-type Arabidopsis clo-0 (Nitschke S, Cortleven A, Iven T, Feussner I, Havaux M, Riefler M, Schmülling T. 2016.CircadianStress Regimes Affect) was treated with 10℅ of sodium hypochlorite solution. the Circadian Clock and Cause Jasmonic Acid-DependentCell Death in Cytokinin-Deficient Arabidopsis Plants.Plant Cell.[Epub aheadof print]) seeds were sterilized for 15min, washed with sterile water, and then plated on MS medium for culture.

2) Put the MS petri dish at 4°C for vernalization for 2 days, and then transfer it to a 22°C greenhouse for cultivation. After culturing on MS medium for 1 week, transfer the seedlings into the soil to continue cultivation. When transferred to the soil, Moisturize for 2-3 days.

3) When the Arabidopsis plants grow to the point where most of the flower buds are about to bloom, Agrobacterium infection and transformation are started.

B) Agrobacterium infection to transform Arabidopsis thaliana

1) Use a sterile toothpick to pick up the correct Agrobacterium strain identified by GV3101/pEarleyGate 100‐TabHLH44 strain PCR, in YEB solid medium (containing antibiotics Rif: 30 μg/mL, Gen: 30 μg/mL, Kan: 50 μg/mL) ) were streaked, placed upside down in a 28°C incubator, and incubated in the dark for 2-4 days.

2) After growing a single colony, inoculate a single colony into YEB liquid medium (containing antibiotics Rif: 30 μg/mL, Gen: 30 μg/mL, Kan: 50 μg/mL), place it on a shaker at 28°C, and cultivate at 210 rpm overnight.

3) Prepare 200 ml of YEB liquid medium (containing antibiotics Rif: 30 μg/mL, Gen: 30 μg/mL, Kan: 50 μg/mL), and add 1 mL of overnight cultured Agrobacterium solution to it. Incubate overnight at 28°C with a shaker at 210 rpm until the color of the bacterial liquid turns orange.

4) Pour 200 mL of the cultured Agrobacterium liquid into a sterile centrifuge tube, collect the bacteria at room temperature, and centrifuge at 5000 rpm for 15 min.

5) Prepare 100 mL of transformation permeate at the ratio of 1/2MS+5% sucrose+20μl silwet, and suspend the collected Agrobacterium cells in the transformation permeate.

6) A large petri dish was loaded with the transformation osmotic solution of Agrobacterium, and the entire inflorescence of Arabidopsis thaliana was invaded into the osmotic solution for 30 s. Infected Arabidopsis thaliana were cultured under normal culture conditions. According to the growth state of Arabidopsis thaliana, the Arabidopsis thaliana can be re-infected one week later to improve the transformation efficiency, and the seeds of the T1 generation transgenic TabHLH44 Arabidopsis thaliana were harvested.

4. Identification of Arabidopsis transfected with TabHLH44

The DNA of T1 generation transgenic TabHLH44 Arabidopsis thaliana leaves was extracted, and the gene-specific primers: F primer, ATGACAGGTTCGCGTTCGTC; R primer TCTGTTCCCATTCGTTGGG were used for PCR amplification, and the 240bp positive trans-TabHLH44 Arabidopsis was obtained.

The RNA of the above-mentioned positive-transfected TabHLH44 Arabidopsis thaliana leaves was extracted, and the cDNA was obtained by reverse transcription as a template. The above-mentioned gene-specific primers were used for RT-PCR amplification, and the AtActin gene was used as an internal reference (F primer, GTCTGGATTGGAGGGTC; R primer, TGAGAAATGGTCGGAAA), The 240bp transfected Arabidopsis with 240bp is the positive T1 generation.

The positive T1-transformed TabHLH44 Arabidopsis was propagated and subcultured to obtain the T2-transformed TabHLH44 Arabidopsis, which was a homozygous-transformed TabHLH44 Arabidopsis line.

Using the same method, the empty vector pEarleyGate 100 was transformed into wild-type Arabidopsis thaliana to obtain the transgenic pEarleyGate 100 Arabidopsis thaliana.

2. Stress resistance study of Arabidopsis transfected with TabHLH44

Homozygous trans-TabHLH44 Arabidopsis and wild-type Arabidopsis were selected for the following treatments, 10 strains per line, the experiment was repeated 3 times, and the results were averaged:

1) Anti-drought treatment

Wild-type Arabidopsis, homozygous trans-TabHLH44 Arabidopsis lines L1, L2, L3 and trans-pEarleyGate 100 Arabidopsis seedlings were cultured on MS medium for 1 week and then transferred to soil, respectively, at 22 °C for 9 h under short-day conditions continue to cultivate. In the early stage, Arabidopsis plants were grown under normal culture conditions. When the seedlings grew for 3 weeks and were relatively robust, stop watering and observe the growth status of the seedlings at any time. When the plants were severely wilted and dry, the plants were rehydrated and rehydrated. The growth state of Arabidopsis plants was observed after 5 days. The normal culture was used as the control.

The results are shown in Figure 2. It can be seen that after drought treatment, the homozygous trans-TabHLH44 Arabidopsis lines L1, L2, and L3 grow better than wild-type Arabidopsis, indicating that the homozygous trans-TabHLH44 Arabidopsis has improved drought resistance.

After 5 days of rehydration, most wild-type plants basically died, while some transgenic plants could return to normal growth state. The survival rates of homozygous trans-TabHLH44 Arabidopsis lines L1, L2, L3 lines and wild-type Arabidopsis were calculated separately. =(Number of surviving plants/Number of total plants)*100%.

The results are shown in Figure 3. After drought treatment, the survival rate of homozygous trans-TabHLH44 Arabidopsis lines L1, L2, and L3 lines was significantly higher than that of wild-type lines.

There was no significant difference between the results of transfected pEarleyGate 100 Arabidopsis and wild-type Arabidopsis.

2) Anti-salt treatment

In a 16 h light culture greenhouse at 22°C, homozygous transgenic TabHLH44 Arabidopsis lines L1, L2, L3, wild-type Arabidopsis and transgenic pEarleyGate 100 Arabidopsis seedlings were grown in MS medium for 5 days, respectively. Line and wild-type seedlings were transferred to MS medium without NaCl (0 mM NaCl, CK) or with 150 mM NaCl, and the MS medium was placed vertically, and the growth status and root length of Arabidopsis seedlings were observed every day.

The results are shown in Figure 4. It can be seen that in 150mM NaCl medium, the homozygous trans-TabHLH44 Arabidopsis lines L1, L2 and L3 grow better than wild-type Arabidopsis, indicating that the homozygous trans-TabHLH44 Arabidopsis is salt resistant Sexual improvement.

The root lengths of the homozygous trans-TabHLH44 Arabidopsis lines L1, L2, L3 and wild-type lines were measured respectively. The results are shown in Figure 5. In NaCl containing 150 mM, the roots of the homozygous trans-TabHLH44 Arabidopsis lines were significantly longer than wild type.

There was no significant difference between the results of transfected pEarleyGate 100 Arabidopsis and wild-type Arabidopsis.

3) Anti-low temperature treatment

3-week-old positive homozygous trans-TabHLH44 Arabidopsis thaliana lines L1, L2, L3, wild-type Arabidopsis (WT) and pEarleyGate 100 Arabidopsis were grown at -10°C for 3h. The normal culture was used as a control (CK, without -10°C low temperature treatment).

The results are shown in Figure 6. It can be seen that at -10 °C, the homozygous trans-TabHLH44 Arabidopsis lines L1, L2, and L3 grow better than wild-type Arabidopsis, indicating that the homozygous trans-TabHLH44 Arabidopsis is resistant to Low temperature performance is improved.

After freezing treatment, most wild-type plants basically died, while some transgenic plants could recover to normal growth state. The survival rates of homozygous trans-TabHLH44 Arabidopsis lines L1, L2, L3 and wild-type were calculated respectively = (number of surviving plants/ total number of plants)*100%.

The results are shown in Figure 7. The survival rate of homozygous trans-TabHLH44 Arabidopsis lines L1, L2, and L3 was significantly higher than that of wild-type lines.

There was no significant difference between the results of transfected pEarleyGate 100 Arabidopsis and wild-type Arabidopsis.

In the above results, Arabidopsis thaliana overexpressing TabHLH44 is resistant to stress, indicating that TabHLH44 is resistant to stress.

Claims (9)

1. A protein, which is the protein represented by SEQ ID NO: 2 in the sequence listing. 2. A DNA molecule encoding the protein of claim 1. 3. DNA molecule as claimed in claim 2 is characterized in that: described DNA molecule is the DNA molecule of following 1) or 2): 1) The coding region is the DNA molecule shown in Sequence 1 in the sequence listing; 2) The coding region is the DNA molecule shown at positions 271-1458 of sequence 1 in the sequence listing. 4. A recombinant vector, expression cassette or recombinant bacteria containing the DNA molecule of claim 2 or 3. 5. A primer pair for amplifying the full length of the DNA molecule of claim 2 or 3 or any fragment thereof. 6. the application of the described protein of claim 1, the described DNA molecule of claim 2 or 3 or the described recombinant vector of claim 4, expression cassette or recombinant bacteria in regulating plant stress resistance; Or the application of the protein described in claim 1, the DNA molecule described in claim 2 or 3 or the recombinant vector described in claim 4, the expression cassette or the recombinant bacteria in cultivating stress resistance to improve transgenic plants; The stress resistance is salt resistance, drought resistance and/or low temperature resistance. 7. application according to claim 6, is characterized in that: The plants are monocotyledonous or dicotyledonous. 8. a method for cultivating stress resistance to improve transgenic plants, for introducing the DNA molecule encoding the protein of claim 1 into a target plant, to obtain a transgenic plant, The stress resistance of the transgenic plant is higher than that of the target plant; The stress resistance is salt resistance, drought resistance and/or low temperature resistance. 9. The method according to claim 8, wherein: The plants are monocotyledonous or dicotyledonous.