CN117903272A

CN117903272A - Application of soybean gene GsPP C-51 in improving drought tolerance of plants

Info

Publication number: CN117903272A
Application number: CN202410245819.8A
Authority: CN
Inventors: 盖钧镒; 刘成; 王吴彬
Original assignee: Sanya Research Institute Of Nanjing Agricultural University
Current assignee: Sanya Research Institute Of Nanjing Agricultural University
Priority date: 2024-03-05
Filing date: 2024-03-05
Publication date: 2024-04-19

Abstract

The invention discloses an application of soybean gene GsPP C-51 in improving drought tolerance of plants, wherein soybean gene GsPP C-51 is a gene encoding protein of an amino acid sequence shown in SEQ ID No. 1. Drought, PEG and exogenous ABA treatment significantly induced GsPP C-51 expression; gsPP2C-51 overexpression improves drought tolerance of transgenic hairy root complex soybean plants and transgenic Arabidopsis thaliana, and enhances SOD, POD and CAT activity under drought stress, but does not improve salt and cold tolerance. These results provide a basis for resolving the role of GsPP C-51-a1 in soybean abiotic stress responses.

Description

Application of soybean gene GsPP C-51 in improving drought tolerance of plants

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to application of soybean gene GsPP C-51 in improving drought tolerance of plants.

Background

Sustainable production of soybeans is threatened by unpredictable climate change, especially sustained drought in many parts of the world. Over the last decades, global crop production losses amount to about 300 million dollars (Gupta et al, 2020) due to frequent drought, which reduces soybean yield by about 40% (Specht et al, 1999). Annual wild soybeans (g.soja) are considered as ancestors of cultivated soybeans (Broich and Palmer, 1980) and have higher drought resistance than cultivated soybeans. Although the overall economic trait of wild soybeans is inferior to that of cultivated soybeans, the potential of utilizing wild soybeans to improve cultivated soybeans, particularly in widening the genetic basis of cultivated soybeans, has been recognized and demonstrated one by one (La et al, 2019; prince et al, 2015). Therefore, it is important to search for drought tolerance genes of wild soybeans and transfer them into cultivated soybeans.

Many morphological and physiological traits have been used as indicators for detecting drought tolerant QTLs (quantitative trait loci), such as canopy wilting, water use efficiency, root system-related traits, and seed-related traits. Hwang et al (2015) used canopy wilting as an indicator to evaluate the phenotype of R2 to R5 and identified eight QTL clusters in five Recombinant Inbred (RIL) populations. Steketee et al (2020) detected 45 SNP (single nucleotide polymorphism sites) associated with the canopy wilting index based on 162 soybean lines of Maturity Group (MG) VI-VIII. WUE (water use efficiency) is one of important physiological traits related to drought resistance. Plant WUE is generally defined as biomass accumulated per unit of water usage. In recent years, genetic variation of WUE has been reported for many field crops, including soybean (Liu et al, 2005a; mian et al, 1998). Assessment of canopy wilting and WUE is often affected by subjective awareness of the person and growth phase of the soybean, and typically requires a period of time to elapse. In this team, growth-related traits such as plant height, stem dry weight, root length, etc. are considered suitable indicators of drought resistance, as all individual biological processes will ultimately be reflected in plant growth and biomass. Liu et al (2005 b) identified 5, 3 and 5 QTLs, respectively, using relative values of dry root weight, total root length and root volume. Khan et al (2018) identified 111 drought-tolerant QTLs with a high proportion of QTL x environmental interactions for 262 alleles using root and aerial length as an indicator; the genetic variation accounts for 88.55-95.92% of the phenotypic variation (Wang et al 2020). Using 24694 multiallelic markers, 75 and 64 QTLs were detected using relative strain and strain height as indicators.

Many transcription factors have been reported to be involved in the regulation of drought response, such as MYB (Du et al 2012), ERF (Zhai et al 2017), NAC (Yang et al 2020), DREB (Chen et al 2007; kidokoro et al 2015) and PP2C (Moli et al 2020). Here, PP2Cs (protein phosphatase 2C gene) is one of the largest transcription factor families in soybean, but little research has been conducted. The plant hormone ABA (abscisic acid) plays an important role in the regulation of the response under abiotic stress, in particular drought stress. ABA has been shown to be involved in the regulation of abiotic stress signaling by ABA-dependent and ABA-independent gene expression. The key regulator of the ABA signaling network is a subset of serine/threonine phosphatases, represented by type 2C protein phosphatases (PP 2 Cs). PP2Cs in Arabidopsis are divided into ten groups (A-J) (Schweighofer et al, 2004). In soybean, PP2C is divided into 10 subfamilies (A, B, C, D, E, F, F2, G, H, and I) (Chen et al, 2018 a). Group A of Arabidopsis PP2C consists of ABI1, ABI2, HAB1, HAB2, AHG1 and AtPP2CA, and has been shown to be a negative regulator of ABA signaling and response. Some members of branch B have been shown to be negative regulators of MAPK activity (Li et al 2020; zhang et al 2020). Members of the C-arm, including POLTERGEIST and POLTERGEIS-LIKE, are involved in stem cell identity and stem and root meristems (Cober et al, 2010; fang et al, 2017). In branch E, EGR1, EGR2 and EGR3 are negative growth regulators that inhibit growth during drought stress (Sun et al, 2018), and AtPP2C6-6 can interact with histone acetyltransferase GCN5 to regulate stress response genes (Cen et al, 2020). A branched I protein phosphatase 2C, taPP2C158, interacts with TaSnRK1.1 and dephosphorylates it, is a negative regulator of wheat drought tolerance. However, little is known about PP2C branch F1 in soybean or other plants, especially in stress response to drought.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: the gene capable of improving the drought tolerance of soybeans and the application thereof are provided against the problems of insufficient application of wild soybean resources, limited drought tolerance gene excavation, little research on F1 type PP2C genes and the like.

The technical scheme of the invention is as follows: the application of soybean gene GsPP C-51 in improving plant drought tolerance, wherein soybean gene GsPP C-51 is a gene encoding protein of an amino acid sequence shown in SEQ ID No. 1.

Because of the degeneracy of the codons, the gene encoding the protein of the amino acid sequence shown in SEQ ID No.1 may have a number of different sequences, preferably the nucleotide sequence of the soybean gene GsPP C-51 is shown in SEQ ID No. 2.

Further, plant drought tolerance is improved by overexpressing soybean gene GsPP C-51 in plants.

Further, the plant is soybean or arabidopsis thaliana.

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

Drought, PEG and exogenous ABA treatment induced expression of GsPP C-51-a1 significantly; over-expression of GsPP C-51-a1 increased drought tolerance of transgenic hairy root complex soybean plants and transgenic Arabidopsis thaliana, and enhanced SOD, POD and CAT activity under drought stress, but did not increase salt and cold tolerance. These results provide a basis for resolving the role of GsPP C-51-a1 in soybean abiotic stress responses.

Drawings

Fig. 1 soybean parental drought resistance evaluation, (a) two parental growth states in greenhouse under Drought Treatment (DT) and Control (CK), bar=10 cm; (B) Under drought stress, the relative aerial part dry weight of N24852 (RSDW) is higher than NN1138-2; (C) Parental growth status under hydroponic (CK) and 15% PEG6000 (PEG) treatments, bar=10 cm;

(D) N24852 has a higher RSDW under PEG stress than NN 1138-2; (E-G) frequency distribution of SojaCSSLP population RSDW in the E1, E2 and E3 environments, respectively; data are mean ± SD (standard deviation); statistical significance was determined by a two-tailed t-test: * P <0.05 and P <0.001.

FIG. 2 expression and characterization of Glyma.14G162100, (A) relative expression of Glyma.14G162100 in two parental soybean leaves under 15% PEG6000 stress with soybean UBI3 gene as internal reference, data analysis using one-way analysis of variance followed by Fisher protected minimal significant differences (P=0.05); (B) Homology relationship of Glyma14g162100 between soybean and different species, glyma14g162100 belongs to F1 group of PP2C based on homology with Arabidopsis; (C) GsPP2 subcellular localization of 2C-51 in tobacco epidermal cells, green Fluorescent Protein (GFP) is a control containing only GFP coding region. Scale bar, 20 μm; (D) Sequence differences of the Glyma14g162100 gene in the 247 soybean germplasm populations, with the black boxes representing exons; the horizontal lines between the black boxes are introns; the gray box is the UTR region, a1-a4 representing the four haplotypes of Glyma14g 162100; the data in the wild (wild) and cultivated (cultivated) columns represent the amount of soybean material per haplotype/allele.

FIG. 3 variation of expression of transgenic hairy root complex soybean plants and GsPP C-51-a1 under PEG treatment, (A) hairy root complex plants containing 15% PEG6000, GFP representing empty vector pBinGFP4 as control; gsPP2C-51-a1 represents over-expression GsPP C-51-a1;0h, 2h, 6h, 12h and 24h represent the time of 15% PEG6000 treatment of plants, respectively; RW stands for plant rehydration after 24 hours of treatment; (B) Treating the hairy root composite soybean plant with PEG6000 for different time leaf forms; (C) Fresh weight of 4cm ² leaves; (D) 35S GsPP2C-51-a1 was overexpressed in hairy root complex plants before treatment (0 h), with soybean UBI3 gene as an internal reference; (E) GmPP2 relative expression of 2C-51-a1 in hairy roots, with soybean UBI3 gene as internal control; data are mean ± SD, statistical significance determined by a two-tailed t-test: * P <0.05, P <0.01 and P <0.001, ns, are not significant.

FIG. 4 shows the growth state of Arabidopsis thaliana under water-deficient stress, (A) the morphology of Arabidopsis thaliana after three weeks of drought stress; (B and C) fresh and dry weight of Arabidopsis thaliana; (D) morphology of arabidopsis after drought treatment and rehydration; (E) fresh weight of arabidopsis thaliana, wild type arabidopsis thaliana; GFP represents the Arabidopsis empty vector transformants #11 and #12 are two independent overexpressing GsPP C-51-a1 lines; data are mean ± SD, statistical significance determined by a two-tailed t-test: * P <0.01.

FIG. 5 germination and growth of Arabidopsis under ABA treatment, (A and B) germination of Arabidopsis grown on 1/2MS medium (Mock) and 2. Mu.M ABA, respectively; (C) GsPP2 germination and overexpression investigation analysis of 2C-51-a1 Arabidopsis after 3 weeks under 2. Mu.M ABA stress, wild type Arabidopsis; GFP represents the Arabidopsis empty vector transformants #11 and #12 are two independent overexpressing GsPP C-51-a1 lines; (D) Morphology of Arabidopsis grown on 1/2MS medium (Moc k); (E) Arabidopsis thaliana adults were grown on 4.0. Mu.M ABA for two weeks; (F) Chlorophyll levels of WT and both transgenic lines under Mock and ABA treatment, white streaks of 1 cm; data are mean ± standard deviation, statistical significance determined by double-sided t-test: * P <0.001; ns, is not significant.

FIG. 6GsPP stomatal movement of 2C-51-a1 overexpressing lines under ABA and PEG treatment, (A) stomatal in mock treatment (stomatal open solution; SOS) (upper panel), 80. Mu.M ABA (middle panel) and 20% PEG6000 (lower panel) of 4 week old WT and GsPP2C-51-a1 overexpressing transgenic lines #11 and #12, 100 stomata per genotype, photographs taken at 60 x magnification under the bright field of an Olympus BX53 fluorescence microscope, scale bar, 2 μm; (B) Graph of percentage of different types of stomata (closed and open) in WT and GsPP C-51-a1 overexpressing transgenic lines in control, ABA and PEG treatments.

FIG. 7 hormone and enzyme activity of GsPP C-51-a1 transgenic lines under drought stress, abscisic acid (ABA), jasmonic Acid (JA), salicylic Acid (SA), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), malondialdehyde (MDA) and hydrogen peroxide (H ₂O₂) #11 and #12, gsPP C-51-a1 overexpressing transgenic lines, numerical data were collected in fresh leaves and analyzed using one-way variance analysis, then Fisher protected minimal significance difference (P=0.05), data as mean.+ -. SD.

Detailed Description

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.

1. Experimental materials and methods

1. Experimental materials

An improved wild soybean chromosome fragment substitution line population SojaCSSLP5 (Liu et al, 2021) consisting of 177 CSSL and 1366 SNPLDB markers was used for drought studies. Its parents are NN1138-2 (G.max) which is a elite variety used as recurrent parent and N24852 (G.soja); the latter is an annual wild soybean with a strong drought tolerance and is used as a donor parent. The CSSL of ,BC₃F₈、BC₃F₇、BC₃F₆、(BC₂F₃)BC₁F₆、(BC₂F₂)BC₂F₆、BC₄F₇、BC₅F₆、BC₄F₆ and BC ₅F₆ generations in 177 CSSL are 23, 12, 3, 62, 3, 25, 14, 21 and 14, respectively.

A chinese germplasm population consisting of 247 parts of material, including 85 parts wild soybean and 162 parts cultivated soybean, was used, these materials were sequenced at 30 x depth for source analysis of identified genes/alleles.

2. Parent and SojaCSSLP population drought resistance identification

First, drought resistance of both parents under water stress and 15% PEG6000 conditions, respectively, was evaluated. In the greenhouse of the south Beijing jiangpu test station (N31 DEG 02', E118 DEG 04'), the drought tolerance of SojaCSSLP and the parents was evaluated in 3 environments of 2016 spring (E1), summer (E2) and 2017 spring (E3). The design of a split area is adopted, saturated water is used as a control, water shortage is used as drought stress, and three biological processes are repeated. Soybean seeds were sown in a pot (diameter 10.0cm, depth 12.5cm, weight 60.0 g) containing about 1800 g of dry sand and 400mL of Hoagland solution. Sowing 5 seeds, and keeping two robust seedlings after emergence of seedlings for subsequent experiments. The border pots around the test material were considered as guard bars. The water in the flowerpot is saturated until drought stress begins after the recurrent parent NN1138-2 grows true leaves and fully expands. For the drought stress group, the moisture content was measured every other day by a weighing method and recorded as W _WC, and W _WC was maintained at 10%, 5% and 1% when NN1138-2 of the control group was in stages V0-V1, V1-V2 and V2-V3, respectively. For the control group, the pots always maintained sufficient water. After one month, plants from the aerial parts (above cotyledonary nodes) were harvested and placed in separate paper bags. And then dried in an oven at 90 c for 2 days. From the water deficit treated and control aerial dry weight data, sojaCSSLP and parental RSDW values were calculated as: RSDW = W _SDT/W_SCK, where W _SDT is the dry weight of the drought treated aerial parts and W _SCK is the dry weight of the control aerial parts.

3. Determination of QTL/fragment and candidate Gene

QTL analysis the RSTEP-LRT-ADD model in ICIMAPPING V4.1 software (additive QTL likelihood ratio test based on stepwise regression) was used and the LOD threshold was set to 2.5. The additive effects (Add) and phenotypic variances of QTL interpretation are estimated from the markers at the highest peaks. Based on the detected QTL/fragment, candidate gene predictions are based on the GO (gene ontology) annotation in SoyBase (http:// soyb ase. Org) of genes related to water, salt stress or hormonal stress. Sequence analysis of candidate genes was based on resequencing data of the two parents (depths of N24852 and NN1138-2 were 11X and 9X, respectively). To analyze the expression level of candidate genes under drought conditions, parents 0h, 6h, 12h and 24h were treated with 15% PEG6000, their leaves and root systems were collected and immediately frozen in liquid nitrogen and then stored at-80 ℃ for subsequent qRT-PCR.

4. RNA extraction and qRT-PCR

Total RNA was extracted from 100mg of tissue per sample using RNA extraction kit (TIANGEN). cDNA was synthesized by using PRIME SCRIPT RT MASTER Mix kit (TAKARA, japan). Fluorescent quantitative PC R (qRT-PCR) was performed using LIGHTCYCLE R and 480 systems (Roche) and SYBR Premix Ex Taq kit (TAKARA, japan). The PCR mixture contained 10ng cDNA, 0.75. Mu.L of 10. Mu.M forward and reverse primer, 12.5. Mu. LSYBR Premix Ex Taq (Takara), and water was added to give a final volume of 25. Mu.L. PCR conditions were 95℃for 30 seconds followed by 95℃for 5 seconds and 54℃for 30 seconds, 40 cycles. GmUBI3 was used as an internal control. Three biological replicates were performed in all assays and the relative expression of GsPP C-51 in soybean was calculated using the 2 ^-ΔΔCt method.

5. Construction of transgenic vectors

For GsPP C-51-a1 (allele a1 of GsPP C-51 in N24852) over-expression, full-length cDNA was amplified from wild-type soybean N24852 and cloned into plant binary vector pBinGFP to yield over-expression vector pBinGFP S:: gsPP2C51-a1. The vector was then introduced into Arabidopsis col-0 by Agrobacterium-mediated transformation. pBinGFP35S GsPP C-51-a1 was transformed into A.rhizogenes strain K599, and the hypocotyl of soybean (williams) was infected to obtain transgenic soybean hairy root complex plants.

6. Subcellular localization

For subcellular localization, an expression vector pBinGFP S carrying Green Fluorescent Protein (GFP) GsPP C-51-a1 was introduced into leaf epidermal cells of 3 to 4 week old Nicotiana benthamiana plants by Agrobacterium EHA105 (Batistic et al, 2010). The expression position of GsPP C-51-a1 protein was observed in tobacco leaves 2-3 days after incubation using a laser scanning confocal microscope (Zeiss LSM 780).

7. Preparation of GsPP C-51-a1 transgenic soybean hairy root composite plant

Williams82 seeds were grown on nutrient-loaded soil: vermiculite is in a flowerpot with a volume ratio of 1:1. When the seedling true leaves are fully developed, the hypocotyl below the cotyledonary node is created with a surgical knife to create a 1cm long wound. The wound was then infected with K599 prepared containing pBinGFP S::: gsPP2C-51-a1 plasmid. As a negative control, empty vector was used for the same treatment. The hypocotyl and A.rhizogenes were co-cultured in an illumination incubator at a temperature of 25℃and an illumination/darkness cycle of 16h/8h for 5 days with a humidity of about 80% -90%. When the hairy root is about 1cm long, the root of the plant itself is removed and replaced with the hairy root. Then the plants were hydroponic in a light incubator in Hoagland solution at 25℃with a humidity of 60% and a light/dark cycle of 16h/8h. After two weeks, all plants were transferred to a new Hoagland solution containing 15% PEG 6000. Roots were collected after treatment for 0 hours, 2 hours, 6 hours, 12 hours, 24 hours and rehydration for 24 hours, respectively, and subjected to quantitative qRT-PCR analysis of GsPP C-51-a1, and fresh leaves of 4cm ² (2 cm. Times.2 cm) were weighed to evaluate drought resistance.

8. Germination and growth of GsPP C-51-a1 transgenic Arabidopsis under ABA stress

The identified T ₁ transgenic line was selected with GFP and grown for production of T ₂ and T ₃ seeds. Seedlings (T ₂ or T ₃) with single copy GsPP C-51-a1 inserts were then identified and used for further analysis. About 50 seeds of two over-expressed GsPP C-51-a1 strains (# 11 and # 12) were grown with WT on 1/2MS medium plates containing 2. Mu.M ABA, incubated for 3 days at 4℃for vernalization, and then transferred to an illumination incubator at 22℃under long day (16 h illumination/8 h darkening) conditions. For the vertical growth assay, the medium plates were placed vertically on a rack. After two weeks of growth, the germination rate (appearance of radicle) of the seeds was identified. All experiments were repeated three times and the mean and standard error calculated.

9. Response of transgenic arabidopsis to drought, low temperature, salt and ABA stresses

Two experiments were performed on arabidopsis drought treatment as described by Sakamoto et al (2004), with slight modifications. Drought treatment 1: arabidopsis thaliana seeds of the wild type Arabidopsis thaliana (WT), empty vector (GFP) and overexpressing GsPP C-51-a1 (GsPP C-51-a 1-OE) lines germinated and grown on 1/2MS medium for 10 days. The plants were then transferred to 9 cm pots filled with 1:3 (v: v) nutrient soil: vermiculite. They were grown at a relative humidity of 45% + -5% with 16h light at 22℃and 8h dark at 20 ℃. After 7 days of growth under sufficient water, watering was stopped and drought stress was initiated for three weeks. Drought treatment 2: and drought-treating the arabidopsis plants for four weeks without watering, and then recovering watering. On the eighth day after re-watering, the growth status of the plants was evaluated. All plants in both experiments were then harvested to measure fresh and dry weight.

Abscisic acid (ABA), salt and cold treatment. Wild-type and over-expressed GsPP C-51-a1 Arabidopsis seedlings were transferred to 1/2MS medium containing 4. Mu.M ABA. After two weeks, all leaves were collected to measure chlorophyll content. For salt tolerance and cold resistance analysis, arabidopsis plants were grown on 1/2MS medium at 100mM NaCl and 4℃for three weeks. The growth of wild type and transgenic plants under ABA, salt and cold stress was then evaluated.

10. Measurement of chlorophyll content

Total chlorophyll content was determined according to Zhang et al (2015) and chlorophyll was extracted from fresh leaf tissue of Arabidopsis with 95% pre-chilled ethanol. The extract was centrifuged at 12000rpm for 10min at 4℃and absorbance at 649nm and 664nm was measured using a UV-1800 spectrophotometer. Chlorophyll content was calculated according to LICHTENTHALER (1987):

Ca＝13.36A₆₆₄-5.19A₆₄₉

Cb＝27.43A₆₄₉-8.12A₆₆₄

Ca+b＝5.24A₆₆₄+22.24A₆₄₉，

Wherein Ca, cb and Ca+b are the concentrations of chlorophyll a, chlorophyll b and total chlorophyll, respectively; a ₆₆₄ and A ₆₄₉ are absorbance values at 664nm and 649nm, respectively.

11. Air hole movement observation

The air hole movement was observed as described by Singh et al (2015), but with slight modifications. Briefly, from 4-week-old Arabidopsis thaliana WT and GsPP C-51-a1-OE plants (grown at 22 ℃, 60% relative humidity, 16h light/8 h darkness) grown in an open-pore solution (SOS: 50mmol L ^-1KCl、0.2mmol L^-1CaCl₂ and 10mmol L ^-1 MES-KOH, pH 6.15) under white light (150. Mu. Mol m ^-2s^-1) for 2h, an epidermis of Arabidopsis thaliana was obtained. For ABA and PEG treatments, epidermal cells were analyzed under bright field under white light (under fluorescent microscope (Olym pus, BX 53) in 150 μmol L ^-1 ABA and 15% PEG6000 SOS buffer and examined for pore sizes under random field.

12. Determination of hormone and enzyme Activity

Leaf samples were removed and ground to a powder. The ground sample was accurately weighed in a glass test tube. Then 10 volumes of acetonitrile solution (volume ratio 10:1) and 4 μl of the internal standard mixture at a concentration of 100ng mL ^-1 were added, respectively. The mixture was mixed, extracted overnight at 4 ℃, centrifuged at 12000g for 5min, and the supernatant transferred to a new centrifuge tube for concentration. After concentration, redissolved with 400. Mu.L of 80% methanol/water solution and passed through a 0.22 μm filter. The plant hormones abscisic acid (ABA), jasmonic Acid (JA) and Salicylic Acid (SA) were measured by ESI-HPLC-MS/MS method at a flow rate of 0.3mL min ^-1 and by agilent 1290 high performance liquid chromatograms and AB Qtrap6500 mass spectrometer (Zhang et al 2022). The column temperature was 30℃and the amount of sample introduced was 2. Mu.L.

Fresh leaves were placed in a phosphate liquid (1:9 w/v) (PBS) buffer to 50mM, pH 7.8, 1% (w/v) insoluble polyvinylpyrrolidone (PVP) in an ice bath. The homogenate was centrifuged at 8000g, 10min, 4℃and the supernatant of the crude product was used for the enzyme extract. Malondialdehyde (MDA) content, H ₂O₂ content, peroxidase (POD), superoxide dismutase (SOD) and Catalase (CAT) activity were measured using commercial kits (BC 0025, BC3595, BC0090, BC0175, BC0205, solarbio, china). The absorbance values were 532nm, 415nm, 470nm, 560nm and 240nm, respectively.

13. Statistical analysis of phenotypes

RSDW descriptive statistics, including frequency distribution, mean, range, coefficient of Variation (CV), genetic power (h ²), and analysis of variance (ANOVA), were calculated based on a random model using PROC-GLM procedure of SAS procedure. For transgenic arabidopsis plants and composite soybean plants, the effect of drought, ABA and PEG treatments on fresh or dry weight was analyzed using Student t test. The data for gene expression, hormone content and enzyme activity were analyzed using analysis of variance, followed by minimal significant differences using Fisher's protection.

2. Experimental results

1. Revealing wild QTL/fragment and candidate gene related to drought tolerance based on SojaCSSLP population

Under drought stress and 15% PEG6000 treatment, the relative aerial dry weight (RSDW) values of the two soybean parents under both stresses were 0.58vs.0.44 and 0.68vs.0.37, respectively (a-D in fig. 1), indicating that the relative aerial dry weight (RSDW) of the wild soybean parent N24852 during the young stage was higher than that of the cultivated soybean parent NN1138-2. To reveal the drought resistance QTL of wild soybean N24852, drought tolerance studies were performed on soybean shoots under three environments (E1, E2 and E3) using SojaCSSLP populations derived from N24852 and NN1138-2. RSDW was observed to have a wide range of phenotypic variations and significant differences (ranging from 0.32 to 0.77) in SojaCSSLP (E-G in fig. 1).

Using ICIMAPPING software, 6 QTLs associated with drought tolerance were detected, accounting for 22.08%, 31.43% and 19.20% phenotypic variation in three environments, respectively (table 1). Of all QTLs, only gm14_ldb_21 could be detected in three environments, which explained the 7.79%, 13.89% and 12.99% phenotypic variation, respectively. This suggests that Gm14_LDB_21 is a stable drought-resistant QTL/fragment, which is located on chromosome 14, similar to the position of MPW14.1 previously reported by Wang et al. Therefore, gm14_ldb_21 is an important QTL/fragment related to drought resistance.

Table 1 QTL identification of RSDW in SojaCSSLP5

RSDW, relative value of dry weight of aerial parts. LOD, logarithm of ratio; PVE, percentage of phenotypic variation explained by single QTL; additive effects of the ADD, N24852 alleles. E represents an Environment (Environment).

To determine drought tolerance genes from wild soybean N24852, annotation analysis was performed on all genes of the gm14_ldb_21 region. Four candidate genes, glyma14G102900, glyma114G103100, glyma14G105900 and Glyma14G162100 (http:// soybase org) were predicted based on GO information (Table 2). They are associated with water, ABA and jasmonic acid stress and belong to WRKY, ZINC file and PP2C transcription factors, respectively (table 2). Sequence analysis showed that there were SNP differences in the gene regions of Glyma114G103100, glyma14G105900 and Glyma14G162100 between the wild (N24852) and cultivated (NN 1138-2) parents (Table 2).

Table 2Gm14_LDB_21 major QTL/fragment major candidate genes

I. s and NS represent introns, synonymous mutation and non-synonymous mutation, respectively, of the gene.

To compare the differences in expression of 4 candidate genes under drought stress, two parents NN1138-2 and N24852 were treated with 15% PEG6000 during soybean seedling stage. qRT-PCR analysis showed that only Glym a g162100 showed a trend of increasing and decreasing with time after 0h, 6h, 12h and 24h treatments and reached a peak at 12h (a in fig. 2), and that the expression level of Glyma14g162100 in wild soybean N24852 was significantly higher at 12h than cultivar NN1138-2. The above results indicate that Glyma14g162100 may be a drought-resistant candidate gene for Gm14_LDB_21.

Phylogenetic analysis was performed based on the full-length protein sequence of Glyma14G162100, and the results showed that the gene was homologous to Phvul.001G075400, medtr G028300, os05G49730 and AT2G29380 in kidney bean, alfalfa, rice and Arabidopsis, respectively (FIG. 2B). Meanwhile, there are 66 genes homologous to Glyma14g162100 in soybean. Based on previous studies by Chen et al, glyma14g162100 belongs to PP2C-51 of group F1 of PP2C family. Thus, it was designated Gs PP2C-51. To determine the expression position of GsPP C-51 in plant cells, its coding sequence was fused to a Green Fluorescent Protein (GFP) driven by the 2X CaMV35S promoter. GsPP2C-51-GFP was infiltrated into the E.benthamiana epidermal cells by Agrobacterium using a transient expression system. As shown in FIG. 2C, gsPP C-51-GFP fusion proteins were located in the nucleus.

2. Allelic variation of GsPP C-51 allele in Chinese germplasm population and SojaCSSLP5

A chinese germplasm population consisting of 247 parts of material, wherein 85 parts of wild soybean and 162 parts of cultivated soybean. All materials were sequenced to a depth of about 30×. In this population, glyma.14g162100 contains 3 exons and 2 introns, encoding 386 amino acids (D in FIG. 2), with 4 SNPs detected in the gene region. Based on the sequence data, four allele types (a 1-a 4) were identified in wild soybeans, whereas only a1 and a2 were found in cultivated soybeans, which should be inherited by wild soybeans (D in FIG. 2). Thus, in this germplasm population, all alleles of Glyma14g162100 in cultivated soybeans were inherited from wild soybeans, without any new alleles, 32% of a1 in wild soybeans, 71% of a1 in cultivated soybeans, and 24% of a2 in wild soybeans, 29% of a2 in cultivated soybeans. It follows that evolution of Glyma14g162100 from wild to cultivated was mainly the exclusion of the a3 and a4 alleles, except for the change in allele frequency. In SojaCS SLP, the additive effect of a1 and a2 is calculated, the a1 effect being +0.70 and the a2 effect being-0.01 (D in FIG. 2). Thus, the present case speculates that the a1 allele has the function of positively regulating soybean drought tolerance, and that the a1 allele has inherited from wild soybeans to most cultivars (115 of 162 varieties) in the chinese soybean germplasm population.

3. Verification of drought tolerance of GsPP C-51-a1 Using Soy hairy root composite plants

To verify the effect of GsPP C-51-a1 (or Glyma.14g162100-a 1) on drought stress, transgenic soybean hairy root complex plants were evaluated for response to 15% PEG 6000. There was no significant difference in fresh leaf weight between control and GsPP C-51-a1 transgenic composite plants prior to PEG treatment (A-C in FIG. 3). After 2 hours of treatment with PEG, the leaf weight of the control (GFP) was reduced and significantly lower than the weight of the transgenic composite plants. Control leaves began to wilt 6h after PEG stress, and rehydration treatment (RW) after PEG stress was relieved failed to recover. However, for transgenic composite plants, leaves began to wilt 24 hours after PEG stress and remained viable after 24 hours of re-watering. The relative expression data showed that GsPP C-51-a1 was expressed higher in roots of transgenic complex plants at 0h than in empty vector transformed plants (GFP) (FIG. 3D), meaning 35S: gsPP2C-51-a1 was overexpressed in complex plants prior to treatment. The time expression of GsPP C-51-a1 in the hairy roots of transgenic composite plants was then analyzed. The expression level of GsPP C-51-a1 was induced by PEG treatment and accumulated to the highest level 6 hours after PEG stress (E in FIG. 3). Furthermore, the expression level of GsPP C-51-a1 in the complex plants after 24 hours of rehydration was significantly lower than before the onset of PEG stress. The result shows that GsPP C-51-a1 is induced by osmotic stress, and the drought resistance of the soybean composite plant can be obviously improved.

4. Over-expression of GsPP C-51-a1 improves drought tolerance in Arabidopsis thaliana

In order to further verify the function of GsPP C-51-a1, the present case also performed stable genetic transformation in Arabidopsis. After screening with GFP, two independent transgenic arabidopsis lines (# 11 and # 12) were obtained. The effect of GsPP C-51-a1 was tested under drought stress by two experiments (drought stress 1 and drought stress 2) using #11, #12, WT and empty vector transformed plants (GFP) (FIG. 4). In drought treatment 1, arabidopsis was subjected to drought treatment for three weeks after one week of growth in the incubator, whereby it was found that the fresh and dry weights of transgenic Arabidopsis lines (# 11 and # 12) were higher than those of WT and GFP plants (A-C in FIG. 4). In drought treatment 2, one week old arabidopsis plants were dried by suppressing moisture until the leaves of the plants withered, and then re-watered. After two days, the overexpressing lines (# 11 and # 12) survived and maintained higher fresh weights, but the WT and GFP plants failed to recover (D and E in fig. 4). These results indicate that GsPP C-51-a1 transgenic plants have higher drought tolerance compared to wild type plants, indicating that GsPP C-51-a1 is a positive regulator of drought response.

5. GsPP2C-51-a1 improves physiological characteristics of drought resistance of arabidopsis thaliana

GsPP2C-51-a1 can improve drought tolerance of plants, but the mechanism of action is not clear. The physiological properties of the GsPP C-51-a1-OE strain from germinated to adult Arabidopsis were then analyzed. GO notes show GsPP C-51 might respond to regulation of ABA (Table 2). Therefore, the scheme firstly researches the response mechanism of GsPP C-51-a1 genes to ABA. To examine the sensitivity of GsPP C-51-a1 to exogenous ABA, arabidopsis seeds were germinated on 1/2MS medium with two concentrations of ABA (0 and 2.0. Mu.M) (FIG. 5). Under normal conditions (0. Mu.M ABA), there was no significant difference in seed germination rates for WT, GFP and GsPP C-51-a1-OE Arabidopsis plants (FIGS. 5A and C). Under 2.0. Mu.M exogenous ABA treatment, the seed germination rates of WT, GFP and two GsPP C-51-a1-OE lines were 0, 50% and 50%, respectively (B and C in FIG. 5). These results indicate that GsPP C-51-a1 overexpression mitigates ABA-mediated inhibition of seed germination.

Chlorophyll content and pore movement of adult arabidopsis thaliana are important physiological regulatory factors for drought resistance. In the absence of ABA treatment, there was no significant difference in chlorophyll content between WT and GsPP C-51-a1-OE arabidopsis plants (D and F in fig. 5). However, under 4.0 μm ABA treatment, the leaves of WT senesced (yellow), and the chlorophyll content of seedlings of #11 and #12 was higher than WT (E and F in fig. 5). In addition, arabidopsis plants were also subjected to cold (4 ℃) and salt (100 mM) stress treatments. The results showed that GsPP C-51-OE Arabidopsis plants exhibited the same growth state as WT, which means that GsP P C-51-a1 did not have the ability to withstand cold and salt.

GsPP2C-51-a1 to increase drought resistance may regulate stomatal movement. Thus, this document discusses whether GsPP C-51-a1 affects the sensitivity of protective cells to ABA and PEG in Arabidopsis. At2 hours of 80.0 μM ABA treatment, most of the stomata in WT (93.3%) were closed, whereas the stomata in the transgenic arabidopsis lines showed insensitivity to ABA, with about 85.0% of the stomata remaining open (fig. 6). However, at2 hours of 15% PEG6000 treatment, 94.17%, 84.7% and 86.6% of the pores in WT, #11 and #12, respectively, were closed (fig. 6). These results indicate that GsPP C-51-a1 is sensitive to PEG stress, but it is independent of the ABA pathway, resulting in insensitivity to ABA.

The results show that GsPP C-51-a1 is insensitive to ABA and cannot induce the stomatal closure and the chlorophyll content reduction of transgenic Arabidopsis thaliana. Then, gsPP C-51-a1 is how does it improve drought tolerance? Some plant hormones and physiological substances are involved in plant response to drought stress, so the present case measured the hormone content and physiological parameters of GsPP C-51-a1-OE lines and wild-type Arabidopsis thaliana with or without drought stress treatment. After the onset of drought stress, the levels of the hormones ABA (abscisic acid), JA (jasmonic acid) and SA (salicylic acid) increased in both GsPP C-51-a1-OE lines (# 11 and # 12), whereas the levels of ABA and SA in GsPP C-51-a1-OE lines were higher than that of WT (FIG. 7). SOD (superoxide), POD (peroxidase), CAT (catalase) and MDA (malondialdehyde) levels were comparable between GsPP C-51-a1-OE lines and WT without drought stress. Under drought stress, the SOD, POD and CAT activities of GsPP C-51-a1-OE plants were all increased. Furthermore, the SOD and CAT increase significantly more than the WT in GsPP C-51-a1-OE systems. Interestingly, the H ₂O₂ (hydrogen peroxide) content did not change significantly between normal and drought stress and between the transgenic line and WT. These results indicate that GsPP C-51-a1-OE Arabidopsis plants may have enhanced stress resistance due to increased hormone levels of ABA and SA and increased enzyme activities of SOD, POD and CAT.

Claims

1. The application of soybean gene GsPP C-51 in improving plant drought tolerance, wherein soybean gene GsPP C-51 is a gene encoding protein of an amino acid sequence shown in SEQ ID No. 1.

2. The use according to claim 1, wherein the nucleotide sequence of soybean gene GsPP C-51 is shown as SEQ ID No. 2.

3. The use according to claim 1 or 2, characterized in that the drought tolerance of the plant is improved by over-expressing the soybean gene GsPP C-51 in the plant.

4. The use according to claim 3, wherein the plant is soybean or arabidopsis thaliana.