CN108467867B

CN108467867B - HD-Zip I transcription factor GmHDL57 gene and application thereof

Info

Publication number: CN108467867B
Application number: CN201810330571.XA
Authority: CN
Inventors: 柯丹霞; 舒勇; 彭昆鹏
Original assignee: Xinyang Normal University
Current assignee: Xinyang Normal University
Priority date: 2018-04-13
Filing date: 2018-04-13
Publication date: 2021-05-07
Anticipated expiration: 2038-04-13
Also published as: CN108467867A

Abstract

The invention discloses an HD-Zip I transcription factor GmHDL57 gene for improving the salt resistance of leguminous plants, wherein the sequence of the gene is a nucleotide sequence shown in SEQ ID NO. 1, and the sequence of the encoded protein is an amino acid sequence shown in SEQ ID NO. 2. The invention also discloses application of the HD-Zip I transcription factor gene GmHDL57 in crowtoe for improving the salt resistance of leguminous plants. The salt resistance of the gene in the Lotus corniculatus of leguminous plants is researched by using an overexpression technology. The result shows that compared with a control plant, the salt resistance of the plant is obviously enhanced after overexpression, and the method has application prospect for expanding the planting range of the leguminous plants in saline-alkali soil.

Description

HD-Zip I transcription factor GmHDL57 gene and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a HD-Zip I transcription factor GmHDL57 gene and application thereof in enhancing salt resistance of crowtoe.

Background

Homeodomain leucine zipper protein HD-Zip (homeodomain leucine-zipper) is a plant-specific transcription factor comprising a homeodomain HD consisting of 60 or 61 amino acids and a leucine zipper domain LZ. The major proteins involved in abiotic stress responses are the HD-Zip class I proteins. There are 17 HD-Zip class I proteins in Arabidopsis, wherein, ATHB5-7 and ATHB12 are induced to express by drought and exogenous ABA, and the expression of ATHB7 can be induced by salt stress and osmotic stress. The tobacco HD-Zip I transcription factor NaHD20 is induced by dehydration stress and positively regulates the accumulation of ABA in leaves. The medicago truncatula MtHB1 is induced by NaCl stress, and the expression level in roots is in an up-regulation trend. The alfalfa MsHB2 is induced by NaCl and ABA, and the response reaction of the plants to salt stress is negatively regulated and controlled. The wild soybean HD-Zip I transcription factor Gshdz4 is highly expressed in leaves and roots under the induction of alkali stress, and the overexpression of Gshdz4 improves the carbonate tolerance of transgenic arabidopsis, so that a new discovery provides clues for disclosing a wild soybean alkali stress tolerance mechanism. In recent years, the participation of plant HD-Zip transcription factors in abiotic stress response has become a hot research. The current research mainly focuses on arabidopsis, rice, corn, poplar, cucumber and other plants, and the functional research on the protein in soybean is reported less and is still in the starting stage.

At present, 35 HD-Zip I transcription factors are identified in soybean, but few research reports about the participation of the transcription factors in stress-resistant physiology are reported. Early studies found that 1 soybean HD-Zip class I transcription factor GmHZ-1 positively regulates infection by Soybean Mosaic Virus (SMV). At present, research on the stress-resistant molecular mechanism of the HD-Zip I transcription factor in arabidopsis thaliana is mature day by day, and the research in soybean is relatively lagged.

The soybean HD-Zip I salt stress response gene GmHAT5 is obtained by early cloning, the gene is up-regulated by salt stress induction, and the expression level is obviously increased by about two times at 2 time points after the salt stress treatment for 6 h and 12 h. However, stable transformation of the crowtoe seeds is not obtained, and deep research is carried out, so that the method is lack of practical application value. The invention separates another HD-Zip I gene GmHDL57 of soybean, and constructs a plant over-expression vector of the gene, obtains a transgenic crowtoe plant through stable transformation and measures various physiological indexes of a transgenic line under salt stress in view of the most obvious change of the gene expression quantity of a soybean transcription factor GmHDL57 in response to high salt stress. T is used in salt stress treatment₀Transgenic crowtoe seed, namely T₁The transgenic crowtoe seeds can be further subjected to passage screening, and finally popularized and applied, the salt resistance quality of the crowtoe is improved, and the planting range of the crowtoe in saline-alkali soil is expanded.

Disclosure of Invention

The invention aims to provide an HD-Zip I transcription factor GmHDL57 gene for improving the salt resistance of leguminous plants, and the sequence of the gene is a nucleotide sequence shown as SEQ ID NO. 1.

The protein coded by the HD-Zip I transcription factor GmHDL57 gene for improving the salt resistance of leguminous plants has an amino acid sequence shown in SEQ ID NO. 2.

The invention further aims to provide the genetic engineering application of the HD-Zip I transcription factor GmHDL57 gene for improving the salt resistance of leguminous plants, in particular to the application in lotus japonicus.

The purpose of the invention is realized by the following technical scheme:

a preparation method of HD-Zip I transcription factor GmHDL57 gene for improving salt resistance of leguminous plants comprises the following steps: taking the root tissue of the cultured soybean Williams82 seedling, quickly freezing by liquid nitrogen, and extracting the total RNA of the root tissue of the seedling according to the operation instruction of an RNA extraction kit of TaKaRa company. First strand cDNA was prepared using a reverse transcription kit from TIANGEN. According to the gene sequence of GmHDL57 (GenBank: XP _ 006574472.1) published by NCBI website, primers are designed to amplify by taking the first strand of cDNA as a template. And recovering and purifying the target fragment, and connecting with a T vector for sequencing verification. The GmHDL57 gene of the present invention and any DNA of interest or a DNA homologous thereto can be amplified from genome, mRNA and cDNA using PCR (polymerase chain reaction) technique.

Stress expression detection of GmHDL57 gene: the seeds of the cultivated soybean Williams82 are sterilized, placed in 1/2 Hoagland culture medium for germination and growth, and subjected to abiotic stress treatment when the seedlings grow to the V1 stage (the first multi-leaf stage). Seedlings from 3 experimental groups were treated with 1/2 Hoagland medium containing 100. mu. mol/L ABA, 100 mmol/L NaCl, and 30% PEG-6000, respectively. Another 1 group of seedlings were transferred to a 4 ℃ incubator for cold treatment. And 3 repeats are set for each group of 5 seedlings, the root tissues of the seedlings are respectively taken at 0 h before stress and 1, 6, 12, 24 and 48 h after stress, and the seedlings are quickly frozen by liquid nitrogen and stored in a refrigerator at the temperature of minus 80 ℃ for later use. Real-time fluorescent quantitative PCR primers were designed 1 using Primer 5.0 software based on the GmHDL57 gene sequence, and the first strand cDNA of each stressed sample was used as a template according to the instruction of PrimeScript RT reagent Kit (Takara). Soybean actin 11 (ACT 11) was used as an internal reference gene. The expression characteristics of the GmHDL57 gene under abiotic stress were analyzed by Excel table mapping according to the results calculated by the formula of relative quantitation (Δ Ct).

A method for improving salt resistance of leguminous plantThe application of the HD-Zip I transcription factor GmHDL57 gene in crowtoe comprises the following steps: is achieved by genetic transformation. The invention constructs a plant over-expression vector of the GmHDL57 gene, and introduces the GmHDL57 gene into the lotus japonicus by adopting an agrobacterium tumefaciens mediated cotyledonary node transformation method. And (3) PCR amplifying Gus genes by using transgenic and wild type Lotus corniculatus plant leaf DNA as templates, extracting partial PCR positive plant leaf RNA, and performing GmHDL57 gene expression analysis. Collecting plant seeds with positive RT-PCR detection, namely T₀Generating transgenic crowtoe seeds, sterilizing the surfaces of the crowtoe seeds and wild seeds, and transferring the crowtoe seeds and the wild seeds into an MS culture medium to wait for the seeds to germinate. Transferring the seedlings into a pot containing the matrix after one week, selecting the seedlings with consistent growth after 3 weeks for salt stress treatment, setting 3 times of repetition, and taking pictures after 14 days and determining related physiological and biochemical indexes.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention separates a homeodomain leucine zipper transcription factor HD-Zip gene GmHDL57 in soybean root tissues by utilizing a reverse transcription PCR technology, and confirms that the gene is an HD-Zip I transcription factor gene through methods of homologous protein multi-sequence comparison, conservative domain prediction and the like; the influence of 4 abiotic stresses (salt, abscisic acid, drought and cold) on the expression of the GmHDL57 gene in the seedling stage of the soybean is analyzed, and the fact that the expression quantity of the gene induced by high-salt stress is remarkably increased and is 8.5 times of that before the stress is confirmed; meanwhile, the salt resistance of the crowtoe can be obviously improved through the over-expression genetic transformation method which proves that the gene over-expression.

2. Lotus japonicus (Lotus japonicus) is a good leguminous forage and is widely planted worldwide. Because of the advantages of high nitrogen fixation efficiency, good cell regeneration performance, high genetic transformation efficiency and the like, the material has become an ideal material for researching symbiotic nitrogen fixation mechanism, gene ectopic expression and the like. Although the lotus corniculatus has the characteristics of drought resistance and salt and alkali tolerance, the lotus corniculatus is only suitable for being planted on slightly arid land and slightly saline-alkali land, and is planted in medium and severe salinization and arid environments, and the growth of plants is severely limited. The invention proves that the salt resistance of the crowtoe can be obviously improved by the overexpression of the gene through an overexpression technology. Therefore, the gene can be overexpressed in leguminous plants by means of transgenosis, so that the adaptability of the leguminous plants to saline-alkali soil can be greatly improved, a new candidate gene is provided for the improvement of soybean salt-tolerant gene engineering, and the gene has important significance for cultivating a new salt-tolerant lotus japonicus variety, enhancing the growth capacity of the new salt-tolerant lotus japonicus variety in saline-alkali areas and fully developing and utilizing saline-alkali soil resources.

Drawings

FIG. 1 is a schematic diagram of PCR amplification of GmHDL57 gene (1A) and restriction enzyme identification of recombinant plasmid p1301U-GmHDL57 (1B).

FIG. 2 is a schematic diagram of conserved sequence alignment analysis of soybean GmHDL57 and other plant homologous proteins by using DNMAN software, wherein the black line part is a homeobox domain sequence, and the dotted line part is a homeobox binding leucine-like zipper domain sequence:

Sequence source: Glycine soja，accession KHN01166.1

Sequence source: Cajanus cajan，accession XP_020226777.1

Sequence source: Vigna angularis，accession KOM56384.1

Sequence source: Phaseolus vulgaris，accession XP_007157405.1

Sequence source: Vigna radiata，accession XP_014521387.1

Sequence source: Cicer arietinum，accession XP_004489987.1

Sequence source: Lupinus angustifolius，accession XP_019461917.1

Sequence source: Medicago truncatula，accession XP_003613578.1

Sequence source: Arachis duranensis accession XP_015965387.1

Sequence source: Arachis ipaensis，Ai: accession XP_016202544.1。

FIG. 3 is a schematic diagram of the expression analysis of the soybean GmHDL57 gene under abiotic stress.

Fig. 4 is a schematic diagram of transgenic Lotus corniculatus positive plant detection and salt-resistant phenotype identification, wherein 4A: detecting Gus in the transgenic plant and the control plant by PCR; 4B: detecting the expression quantity of GmHDL57 in the transgenic plant and the control plant by RT-PCR; 4C: after being treated for 14 days by different salt concentrations, the phenotype of the GmHDL57 crowtoe and a control plant is transferred.

FIG. 5 is a schematic diagram showing the effect of salt stress on transgenic Lotus corniculatus plant height (5A) and root length (5B).

FIG. 6 is a schematic diagram of determination of physiological indexes of transgenic crowtoe under salt stress, wherein 6A: measuring the content of malonaldehyde; 6B: relative plasma membrane permeability analysis; 6C: measuring the content of chlorophyll; 6D: and (5) measuring the activity of the root system.

Fig. 7 is a schematic diagram of determination of cation content of transgenic crowtoe under salt stress, wherein 7A: na in the leaf⁺Measuring the content; 7B: na in root⁺Measuring the content; 7C: blade middle K⁺Measuring the content; 7D: root of Chinese Kabushiki⁺Measuring the content; 7E: ca in leaves²⁺Measuring the content; 7F: ca in the root²⁺And (4) measuring the content.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the following detailed description is given with reference to the accompanying drawings and preferred embodiments of the present invention.

Example 1

A preparation method of HD-Zip I transcription factor gene GmHDL57 for improving salt resistance of leguminous plants comprises the following steps:

the method comprises the steps of disinfecting seeds of cultivated soybeans Williams82, placing the seeds in a 1/2 Hoagland culture medium for germination and growth, setting the parameters of a light incubator to be 25 ℃, relative humidity to be 60%, light for 18 hours and darkness for 6 hours. Taking the seedling root tissue, quickly freezing by liquid nitrogen, and extracting the total RNA of the seedling root tissue according to the operation instruction of an RNA extraction kit of TaKaRa company. First strand cDNA was prepared using a reverse transcription kit from TIANGEN. Primers F-GmHDL57 (5'-ATGGCGAGTGGCAAGCTT TATGC-3') and R-GmHDL57 (5'-TCAATAGGGCCAGAAACAG-3') are designed according to a GmHDL57 gene sequence (GenBank: XP _ 006574472.1) published by NCBI website, and first strand cDNA is used as a template for amplification (shown in FIG. 1A). The target fragment is recovered and purified, then connected with a T vector and sent to Nanjing Kinshire company for sequencing verification.

Example 2

The stress expression detection of the GmHDL57 gene comprises the following steps:

the method comprises the steps of disinfecting seeds of cultivated soybeans Williams82, placing the seeds in a 1/2 Hoagland culture medium for germination and growth, setting the parameters of a light incubator to be 25 ℃, relative humidity to be 60%, light for 18 hours and darkness for 6 hours. The abiotic stress treatment is carried out when the seedlings grow to the V1 stage (the first multiple leaf stage). Seedlings from 3 experimental groups were treated with 1/2 Hoagland medium containing 100. mu. mol/L ABA, 100 mmol/L NaCl, and 30% PEG-6000, respectively. Another 1 group of seedlings were transferred to a 4 ℃ incubator for cold treatment. And 3 repeats are set for each group of 5 seedlings, the root tissues of the seedlings are respectively taken at 0 h before stress and 1, 6, 12, 24 and 48 h after stress, and the seedlings are quickly frozen by liquid nitrogen and stored in a refrigerator at the temperature of minus 80 ℃ for later use.

Real-time fluorescent quantitative PCR primers, F-GmHDL57-RT (5'-GATGAAGAGGATAACCTTAG-3') and R-GmHDL57-RT (5'-TCAATAGG GCCAGAAACAG-3'), were designed 1 using Primer 5.0 software based on the GmHDL57 gene sequence, using the first cDNA strand of each stressed sample as a template, according to the Takara PrimeScript RT reagent Kit protocol. Taking soybean actin 11 (ACT 11) as an internal reference gene, and taking a real-time fluorescent quantitative PCR upstream primer F-ACT11 (5'-ATTTTGACTGAGCGTGGTTATTCC-3'); the downstream primer was R-ACT11 (5'-GCTGGTCCTGGCTGTCTCC-3'). The expression characteristics of the GmHDL57 gene under abiotic stress were analyzed by Excel table mapping according to the formula of relative quantitation (Δ Ct) (shown in FIG. 3).

The response characteristics of the GmHDL57 gene expression to 4 different abiotic stresses are researched by simulating the abiotic stresses through exogenous abscisic acid, high salt, drought, cold and the like. As can be seen from FIG. 3, the expression level of the GmHDL57 gene rapidly increases after NaCl and PEG stress treatment for 1 h, and the expression level continuously increases with the increase of the treatment time until reaching a maximum value at 48 h, which is 8.5 times and 4.2 times of that before stress respectively. Under the stress of ABA, the expression quantity of the GmHDL57 gene is slowly increased at two time points of 1 h and 6 h, obviously rises to 12 h, then slowly rises, and reaches 3.9 times of that before the stress at 48 h. When the cold stress treatment is carried out at 4 ℃, the expression quantity of the GmHDL57 gene is continuously reduced to reach a minimum value when the expression quantity reaches 12 h, and the minimum value is 0.5 time of that before the stress, and then the expression quantity tends to be stable, so that the cold stress adaptability is shown. The change of the expression quantity of the soybean transcription factor GmHDL57 in response to high salt stress is shown to be most obvious.

Example 3

An application of HD-Zip I transcription factor gene GmHDL57 for improving salt resistance of leguminous plants in crowtoe comprises the following specific implementation steps:

stable transformation of Lotus corniculatus:

(1) construction of overexpression fusion vectors

The target gene with correct sequencing is inserted into a plant overexpression vector p1301U, an upstream primer is F-GmHDL57-OX (5'-CGggatccATGGCGAGTGGCAAG-3'), a downstream primer is R-GmHDL57-OX (5'-GGggtaccTCAATAGGGCCAGAAAC-3'), and lowercase letter regions are enzyme cutting site sequences BamH I and Kpn I, so that an overexpression vector p1301U-GmHDL57 (shown in FIG. 1B) is constructed. Extracting plasmid, and electrically shocking to transform Agrobacterium tumefaciens EHA 105.

(2) Preparation of Agrobacterium liquid

After culturing Agrobacterium EHA105 single colony in YEB liquid culture medium supplemented with 40 mg/L rifampicin and 50mg/L kanamycin at 28 ℃ for 42 h by shaking, centrifuging at 5000 r/min for 10 min to collect the thallus, resuspending with MS liquid culture medium, and diluting to OD600=0.6 for use.

(3) Material treatment

Washing the Lotus japonicus MG20 seeds with 75% alcohol once, sterilizing for 6-8 min with 0.1% mercuric chloride, washing with sterile distilled water for more than 10 times, soaking overnight, washing for 3-4 times, transferring onto MS solid culture medium, spreading two cotyledons 3-4 days later, cutting hypocotyl into 2 mm sections, and inoculating onto callus induction culture medium.

(4) Explant preculture

Taking the cut hypocotyl and pre-culturing the hypocotyl on a callus induction culture medium for 3 d.

(5) Infection and Co-cultivation

Immersing the pre-cultured explant into the prepared agrobacterium liquid for infection for 20 min, taking out, sucking the residual liquid on the surface of the explant by using sterile filter paper, transferring the explant into a callus induction culture medium without antibiotics, and carrying out dark co-culture for 3 d.

(6) Transformation screening

The co-cultured callus was transferred to a callus induction medium containing carbenicillin (300 mg/L) and kanamycin (50mg/L) for culture, bacteriostatic and selection. After 2 weeks, the resistant callus was transferred to a shoot differentiation medium for induced differentiation culture. When the induced bud grows to 2-3 cm, cutting off the bud from the base part, and inserting the bud into a rooting culture medium to induce rooting so as to form a regeneration plant.

(7) Culture conditions

The temperature is 24-26 ℃, the light/dark cycle is 16 h/8 h, the illumination intensity is 2000 Lux, and the subculture is carried out once in 14-21 d.

The formula of the culture medium in the steps is as follows:

MS culture medium: 4.3 g of MS basal salt mix (Sigma), 0.103 g of MS vitamin powder, 0.7-0.8% (mass-to-volume ratio) agar powder and ddH2O, wherein the volume is adjusted to 1000 mL, and the pH value is adjusted to 5.8.

Callus induction medium: MS +2, 4-D2.0 mg/L + KT 2.0 mg/L

Bud differentiation medium: MS +6-BA 1.0 mg/L + NAA 0.1 mg/L

Rooting culture medium: MS + NAA 0.1 mg/L

(8) Molecular biological detection of stably transformed plants

Using transgenic and wild lotus petiolus plant leaf DNA as template, PCR amplifying Gus gene, F-Gus (GTCGCGCAAGACTGTAACCA) and R-Gus (CGGCGAAATTCCATACCTG) as primer. Extracting partial PCR positive plant leaf RNA, and carrying out GmHDL57 gene expression analysis, wherein the primers comprise F-GmHDL57-rt (5'-GATGAAGAGGATAACCTTAG-3') and R-GmHDL57-rt (5'-TCAATAGGGCCAGAAACAG-3'), the crowtoe GPDH is used as an internal reference gene, and the primers comprise F-GPDH (GTGGTGCCAAGAGGTTGTTAT) and R-GPDH (CTGGGAATGATG TTGAAGGAAG).

Salt stress treatment:

collecting plant seeds with positive RT-PCR detection, namely T₀Transgenic BaimaiRoot seeds, sterilizing the surfaces of the root seeds and wild seeds, and transferring the root seeds and the wild seeds to an MS culture medium to wait for the seeds to germinate. Transferring the seedlings into a pot containing a substrate after one week, selecting the seedlings with consistent growth after 3 weeks, dividing the transgenic plants and the wild plants into 3 groups, respectively treating 24 plants in each group with 1/8 Hoagland nutrient solution with final concentration of 0, 100 and 200 mmol.L-1 NaCl, setting 3 times of repetition, photographing after 14 days (shown in C in figure 4) and determining related physiological and biochemical indexes including malonaldehyde content determination, leaf plasma membrane permeability (relative conductivity), chlorophyll content determination, root activity determination (shown in figure 6) and cation Na⁺、K⁺And Ca²⁺Assay (shown in fig. 7).

As can be seen from FIG. 4C, under normal conditions, the growth status of the transgenic plants and the control plants are substantially consistent, while the control plants treated with high salt have significantly poorer growth vigor, short plants, and green and wilting leaves. It is further clear from FIG. 5 that both the plant height and root length of the transgenic lines are superior to those of the control group.

As can be seen from FIG. 6, the malondialdehyde content and relative plasma membrane permeability of the transgenic lines were lower than those of the control group, while the chlorophyll content and root activity were higher than those of the control group.

As can be seen from FIG. 7, there was no significant difference in the ion content in transgenic and control plants under normal growth conditions, indicating that the GmHDL57 gene did not affect the uptake of 3 cations by the plants under normal growth conditions. Na in leaves and roots of transgenic and control plants with increasing salt concentration⁺And Ca²⁺The contents all rise with the increase of K⁺The content is reduced, but Na in the transgenic plant is compared with the control⁺Less content, K⁺And Ca²⁺The content is high. GmHDL57 gene reduced Na under high salt stress⁺Or increase Na⁺Reducing Na in leaves and roots of transgenic plants⁺Thereby maintaining the normal physiological function of the plant. Maintenance of higher cytoplasmic K under salt stress⁺And Ca²⁺The concentration can enhance the salt tolerance of the plant. In this study, under salt stress conditions, K in transgenic Baimai root leaves and roots⁺And Ca²⁺The contents of the genes are obviously higher than those of a control plant, which fully shows that the overexpression of the GmHDL57 gene is beneficial to K pair cells⁺And Ca²⁺Thereby regulating the ion homeostasis balance within the cell.

The above description is only for the specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and other modifications or equivalent substitutions made by the technical solution of the present invention by the ordinary skilled in the art should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Sequence listing

<110> Xinyang college of teachers and schools

<120> HD-Zip I transcription factor GmHDL57 gene and application

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ttccaaggtt caaaacccgt ggttgatttt gagaatgtaa gtgggagcag gatgacggat 180

aggcctttct ttcaagcgtt ggagaaggaa gagaactgtg atgaggatta cgaggggtgt 240

ttccaccaac cggggaagaa aaggaggctc acaagcgaac aagttcagtt ccttgaaagg 300

aactttgagg tagagaacaa gcttgaaccc gaaaggaaag tccaacttgc aaaagagctt 360

ggcttgcagc caaggcaagt tgctatatgg ttccaaaacc gaagggcaag gttcaagacc 420

aagcagctag aaaaagacta tggcgtgttg aaagctagtt atgacagact caaaagtgac 480

tatgaaagtc ttgttcaaga gaatgacaag ttaaaagcag aggtgaattc tctggagagc 540

aaattgattc ttagagataa agagaaggag gagaattcgg atgacaagtc atctcctgat 600

gatgctgtca attcttcttc accccacaac aacaaggagc ctatggattt attaattatt 660

tcaaaaaatg caacaacaac aacaacatct gaaaatggga ccaaagtgtt gtcaccactc 720

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1 5 10 15

Gly Ala Gly Ala Leu Pro Cys Ser Ser Gly Val Leu Gly Ser Leu Thr

20 25 30

Ala Gly Thr Ser Ala Pro Ala Ser Pro Gly Gly Ser Leu Pro Val Val

35 40 45

Ala Pro Gly Ala Val Ser Gly Ser Ala Met Thr Ala Ala Pro Pro Pro

50 55 60

Gly Ala Leu Gly Leu Gly Gly Ala Cys Ala Gly Ala Thr Gly Gly Cys

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Pro His Gly Pro Gly Leu Leu Ala Ala Leu Thr Ser Gly Gly Val Gly

85 90 95

Pro Leu Gly Ala Ala Pro Gly Val Gly Ala Leu Leu Gly Pro Gly Ala

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Leu Val Gly Leu Ala Leu Gly Leu Gly Leu Gly Pro Ala Gly Val Ala

115 120 125

Ile Thr Pro Gly Ala Ala Ala Ala Ala Pro Leu Thr Leu Gly Leu Gly

130 135 140

Leu Ala Thr Gly Val Leu Leu Ala Ser Thr Ala Ala Leu Leu Ser Ala

145 150 155 160

Thr Gly Ser Leu Val Gly Gly Ala Ala Leu Leu Leu Ala Gly Val Ala

165 170 175

Ser Leu Gly Ser Leu Leu Ile Leu Ala Ala Leu Gly Leu Gly Gly Ala

180 185 190

Ser Ala Ala Leu Ser Ser Pro Ala Ala Ala Val Ala Ser Ser Ser Pro

195 200 205

His Ala Ala Leu Gly Pro Met Ala Leu Leu Ile Ile Ser Leu Ala Ala

210 215 220

Thr Thr Thr Thr Thr Ser Gly Ala Gly Thr Leu Val Leu Ser Pro Leu

225 230 235 240

Pro Leu Pro Ile Met Val Thr Cys Cys Leu Gly Gly Ala Ala Ala Ser

245 250 255

Ala Leu Ser Ala Val Leu Ala Ser Ala Ser Pro His Cys Thr Ser Pro

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Val Gly Pro Ala Ala Ser Ser His Ala Pro Gly Pro Gly Ala His Ser

275 280 285

Gly Ala Pro Ser Gly Ala Gly Gly Ala Ala Leu Ser Gly Ala Leu Leu

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Met Thr Pro Pro Ser Ser Cys Cys Leu Pro Leu Val Gly Gly His Cys

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Thr Ala Gly Pro Pro Gly Ala Ser Cys Ala Pro Gly Pro Gly Val Gly

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Ala Gly Thr Pro Cys Pro Thr Pro Thr

340 345

Claims

1. An application of an HD-Zip I transcription factor GmHDL57 gene in enhancing the salt resistance of crowtoe is characterized in that the sequence of the GmHDL57 gene is a nucleotide sequence shown in SEQ ID NO. 1.