Application of soybean ABC transporter GmABCC3 encoding gene

Abstract

The application discloses application of a soybean ABC full-transporter GmABCC3 coding gene. The application of the coding gene of the soybean ABC full-transporter GmABCC3 shown in SEQ ID NO. 1 in genetic engineering modification of soybean aluminum toxicity resistance. The gene is significantly up-regulated by aluminum induction in an aluminum toxicity resistant soybean variety, and the aluminum toxicity resistant soybean variety contains an excellent aluminum induction type promoter GmABCC Pro2 . An excellent haplotype GmABCC3 CDS2 overexpressing the gene can significantly improve the aluminum toxicity resistance of soybean and Arabidopsis. An excellent haplotype GmABCC3 CDS2 overexpressing the gene in a yeast strain can also significantly improve the aluminum toxicity resistance of the yeast. The soybean ABC full-transporter GmABCC3 can be genetically engineered into soybean hairy roots and Arabidopsis, and finally improve the aluminum toxicity resistance of the soybean and Arabidopsis.

Description

Technical Field

[0001] This invention relates to the application of the gene encoding the soybean ABC total transporter protein GmABCC3, and belongs to the field of genetic engineering. Background Technology

[0002] Soybean [Glycine max (L.) Merr.] originated in China and is an important legume crop, widely cultivated in China and worldwide as a significant source of protein and oil. Mature soybean seeds contain approximately 40% protein and 20% oil, providing humans with all eight essential amino acids, as well as various vitamins and essential minerals such as calcium, phosphorus, iron, and zinc. Therefore, soybeans play a vital role in meeting human needs for protein, vegetable oil, and animal feed. They are also widely used as a raw material for industrial products, leading to a continuously increasing global demand. However, abiotic stresses such as aluminum toxicity in acidic soils can severely reduce soybean yield and quality. Statistics show that 37.9% of arable land in Southeast Asia, 30.9% in Latin America and the Caribbean, 21.1% in East Asia, 20.5% in sub-Saharan Africa, 18.1% in North America, and 11.7% in Europe suffer from aluminum toxicity, impacting the yield of soybeans and other crops. Aluminum toxicity in acidic soils is a significant abiotic stress. Globally, approximately 35% of soybean-growing regions are located in acidic soils, and large areas of southern and southeastern coastal my country also suffer from this problem. Therefore, developing aluminum-tolerant soybean varieties is crucial for improving soybean yield and quality in acidic soil regions.

[0003] GmABCC3 is a soybean ABC (ATP-Binding Cassette) holotransporter. The ABC family, to which it belongs, is one of the largest and most functionally diverse protein families known, widely found in all organisms in nature. Based on their motif structure, ABC transporters can be broadly classified into holotransporters and semitranstransporters. Generally, holotransporters have two transmembrane domains (TMD) and two nucleotide-binding domains (NBD / NBF). ABC semitranstransporters typically contain only one TMD and one NBD; some bacterial types of ABC semitranstransporters may contain only one TMD or one NBD. The transport activity of ABC transporters primarily depends on the energy generated from the hydrolysis of adenosine triphosphate (ATP), thereby enabling the transmembrane transport of substrates between the cell and the interior / exterior. Studies have found that ABC transporters participate in many important physiological processes in plants, such as hormone transport, epidermal lipid secretion, seed germination regulation, and the transport of defensive molecules, enabling plants to adapt to changing external environments. Different members of the ABC transporter family are generally responsible for transporting different substrates, but some ABC transporters can even mediate the transport of multiple substances. To date, the functions and substrates of most ABC transporters remain unclear, and many unknown functions await discovery and verification. There are currently no reports of ABC transporters participating in aluminum tolerance in soybean. Using molecular biology techniques, we constructed an overexpression vector for GmABCC3. Transforming soybean rooted strains, Arabidopsis thaliana, and yeast strains revealed that overexpression of the GmABCC3 gene improved aluminum tolerance and reduced aluminum ion concentration in plant roots in all three strains. This discovery not only reveals a new pathway in the mechanism of aluminum tolerance in soybean but also provides genetic resources for breeding aluminum-tolerant soybean varieties. Summary of the Invention

[0004] The purpose of this invention is to disclose the stress resistance genetic engineering application of the soybean ABC holotransfer protein GmABCC3 (Glyma.18G080900), and to discover that GmABCC3 has a natural mutation, with its coding sequence (CDS) haplotype GmABCC3. CDS2 and promoter (Pro) haplotype GmABCC Pro2 To regulate the superior allelic variation of aluminum tolerance in soybean, the superior allelic variation of this gene can be introduced into soybean and Arabidopsis thaliana as the target gene. The superior allelic gene encodes GmABCC3, which alleviates aluminum toxicity by reducing the concentration of aluminum ions in roots and cells, thereby improving the aluminum tolerance of soybean.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] Application of the gene encoding soybean ABC total transporter GmABCC3 in improving soybean aluminum toxicity resistance, with the superior haplotype GmABCC3 of the soybean ABC total transporter GmABCC3 gene described. CDS2 As shown in SEQ ID NO.1.

[0007] As a preferred embodiment of the present invention, overexpression of the superior haplotype GmABCC3 encoding gene GmABCC3 is preferred. CDS2 It can significantly improve the aluminum tolerance of soybeans or Arabidopsis thaliana and reduce the aluminum ion concentration in soybean roots.

[0008] Superior haplotype GmABCC3 encoding gene overexpressing soybean ABC holotransfer protein GmABCC3 CDS2 Application of expression vectors in improving the aluminum toxicity tolerance of soybeans, and the superior haplotype GmABCC3 encoding the soybean ABC holotransfer protein GmABCC3. CDS2 As shown in SEQ ID NO.1.

[0009] As a preferred embodiment of the present invention, the expression vector uses the sequence shown in SEQ ID NO.11 as the superior aluminum-inducible promoter GmABCC. Pro2 .

[0010] Beneficial effects:

[0011] The soybean ABC transporter gene GmABCC3 is induced by aluminum toxicity stress, and its expression level differs between aluminum-sensitive and aluminum-tolerant soybean materials. Promoter and CDS sequencing of the GmABCC3 gene in 46 soybean varieties revealed nine haplotypes (Hap1-9). The most common haplotypes were Hap1 (Pro1+CDS1) or Hap2 (Pro2+CDS2). Comparison of aluminum tolerance among these varieties showed that Pro2 and CDS2 are superior variants for controlling aluminum tolerance in soybeans. CDS1 and GmABCC3 CDS2 The sequence was inserted into an overexpression vector and further transformed into Arabidopsis thaliana and soybean root development to overexpress GmABCC3. It was found that overexpression of GmABCC3 significantly improved the aluminum toxicity tolerance of the transformed plants, reduced the aluminum ion concentration in the plant roots, and GmABCC3... CDS2 The effect is even better. Overexpression of GmABCC3 was also found in yeast strain aluminum toxicity resistance experiments. CDS2 It can significantly improve its resistance to aluminum toxicity. Different promoter haplotypes of GmABCC3... Pro1 and GmABCC3 Pro2Drives GUS gene and GmABCC3 respectively CDS2 Transforming soybean root growth and discovering GmABCC3 in aluminum toxicity stress experiments Pro2 It is a superior haplotype of the aluminum poisoning-inducible promoter. Attached Figure Description

[0012] Figure 1 .Agarose gel electrophoresis image of GmABCC3 after PCR cloning. Marker: DL10000

[0013] Figure 2 . Soybean root phenotypes under aluminum stress, relative expression levels of GmABCC3 after aluminum toxicity stress, and tissue expression patterns under normal conditions. (A) Soybean root phenotypes after 24 h of treatment with 0 μM and 25 μM AlCl3. 84 is an aluminum-sensitive soybean variety, and 644 is an aluminum-tolerant soybean variety. (B) Comparison of relative root growth (RRG) of soybean varieties 644 and 84 after 24 h of treatment with 0 μM and 25 μM AlCl3. (C) Changes and comparisons of GmABCC3 expression levels after 6 h, 12 h, and 24 h of treatment with 25 μM AlCl3 on soybean seeds. (D) Expression of GmABCC3 in various tissues of soybean variety 644. Data represent mean ± standard deviation of three biological replicates. *, * and ***: indicate significant differences at the 0.05, 0.01, and 0.001 levels, respectively (t-test). The same letter above the column indicates that there is no significant difference at the P=0.05 level according to Duncan's multiple comparison analysis.

[0014] Figure 3 Genotyping results of GmABCC3 in 46 soybean varieties and comparison of aluminum tolerance among major haplotypes. (A) Polymorphic site genotyping of GmABCC3 in promoter and CDS sequencing results in 46 soybean varieties. Pro-type is the promoter haplotype of GmABCC3, and CDS-type is the CDS haplotype of GmABCC3. (B) Comparison of aluminum tolerance among major haplotypes of GmABCC3. RRG is relative root growth. The significance test P-value was obtained from the t-test.

[0015] Figure 4 GmABCC3 was overexpressed in Arabidopsis thaliana using the CaMV35S(35S) promoter. CDS1 and GmABCC3 CDS2 Test its resistance to aluminum toxicity. (A)35S:GmABCC3 CDS1 / 2 Semi-quantitative results of GmABCC3 in transgenic Arabidopsis thaliana; AtActin1 was used as an internal control. (B)35S:GmABCC3 CDS1 / 2Quantitative PCR results of GmABCC3 in transgenic Arabidopsis thaliana; AtActin1 was used as an internal control for qRT-PCR. Data represent mean ± standard deviation of three biological replicates. 35S:GmABCC3 CDS1 / 2 Root length phenotype (C) and RRG (D), total root length phenotype (E), and relative total root length (F) of transgenic Arabidopsis and wild-type plants treated with aluminum for 7 days. Aluminum treatment concentrations were 50 μM and 100 μM. Data represent mean ± standard deviation of three biological replicates (n = 10 × 3). (G)35S:GmABCC3 CDS1 / 2 Morin staining of roots from transgenic Arabidopsis thaliana and wild-type plants under aluminum treatment (50 μM, 6 h) and control. (H)35S:GmABCC3 CDS1 / 2 Root aluminum ion concentrations in transgenic Arabidopsis and wild-type Arabidopsis under aluminum treatment (10 μM, 48 h). * and **: indicate significant differences at the 0.05 and 0.01 levels, respectively (t-test). The 35S promoter is used to drive GmABCC3. CDS1 and GmABCC3 CDS2 It is expressed in wild-type Arabidopsis thaliana.

[0016] Figure 5 GmABCC3 was overexpressed in soybean germinal roots using the 35S promoter. CDS1 and GmABCC3 CDS2 Test its resistance to aluminum toxicity. (A)35S:GmABCC3 CDS1 / 2 And the root development of transgenic soybeans without vectors, 24 h of aluminum treatment, and hematoxylin staining under control; (B) 35S: GmABCC3 CDS1 / 2 GmABCC3 was expressed relative to empty-vectored transgenic soybean hair roots; GmEF-1 was used as an internal reference gene for qRT-PCR, and its expression level was normalized to that in empty-vectored transgenic hair roots. (C)35S:GmABCC3 CDS1 / 2 The relative aluminum ion concentration in the roots of transgenic soybeans without a vector was measured by absorbance (A 490) at a wavelength of 490 nm. Data represent the mean ± standard deviation of three biological replicates. (D) GmABCC3 after 4 weeks of aluminum toxicity treatment CDS1 / 2 Root phenotypes of unloaded transgenic soybeans, with a scale bar of 4 cm. GmABCC3 CDS1 / 2 The relative primary root length (E) and relative total root length (F) of transgenic soybeans without a root vector were measured. Data represent mean ± standard deviation of three biological replicates (n = 7 × 3). * and **: indicate significance at the 0.05 and 0.01 levels, respectively (t-test). The 35S promoter is used to drive GmABCC3. CDS1 and GmABCC3 CDS2 The expression.

[0017] Figure 6 Differences in the effects of different promoter haplotypes of GmABCC3 on aluminum tolerance in soybean root development. GmABCC3 Pro1 / 2 Comparison of GUS staining (A) and GUS enzyme activity (B) in the roots of GUS-transgenic soybeans; (C) GmABCC3 Pro1 / 2 :GmABCC3 CDS2 Relative expression of GmABCC3 in soybean rooting control and aluminum treatment; data represent mean ± standard deviation of three biological replicates. (D)GmABCC3 Pro1 :GmABCC3 CDS2 and GmABCC3 Pro2 :GmABCC3 CDS2 The root phenotypes of unloaded transgenic soybeans after 4 weeks of root development in the control and aluminum-treated groups were also analyzed; specifically, the relative taproot length (E), relative total root length (F), relative total root tip number (G), relative root fresh weight (H), and root aluminum ion concentration (I) were measured. Data represent mean ± standard deviation of three biological replicates (n = 12 × 3). * and **: indicate significance at the 0.05 and 0.01 levels, respectively (t-test).

[0018] Figure 7 Expression of GmABCC3 in yeast enhances its aluminum tolerance. (A) Comparison of aluminum tolerance between yeast cells transfected with GmABCC3 and those transfected with the empty pYES2 vector. conc. refers to concentration. (B) Comparison of intracellular aluminum ion concentration in yeast cells transfected with GmABCC3 and those transfected with the empty pYES2 vector under aluminum toxicity treatment. The aluminum ion concentration in yeast cells was measured at 24 h and 36 h of yeast growth after treatment with 400 μM AlCl3. pYES2 served as the empty vector control. GmABCC3 was incorporated into the pYES2 vector and transformed into yeast strain BY4741 to test its aluminum tolerance. * and **: indicate significant differences at the 0.05 and 0.01 levels, respectively (t-test). Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0021] Example 1: Cloning of the soybean ABC total transporter protein GmABCC3 gene

[0022] The primary roots of aluminum-tolerant soybean variety 644 during its seedling stage were used as the sampling object. RNA was extracted using the Tiangen RNA Extraction Kit (DP452, TIANGEN) according to the manufacturer's instructions. The purity and integrity of the RNA were detected by 0.8% agarose gel electrophoresis, and its concentration was determined using a Nanodrop-2000 spectrophotometer. Then, reverse transcription was performed using a PrimerScript™ RT reagent kit (TaKaRa) according to the manufacturer's instructions to obtain cDNA of soybean variety 644. The gene ID (Glyma.18G080900) corresponding to GmABCC3 was found in the NCBI and Phytozome v12 soybean databases. Specific primers were designed based on the nucleotide sequences in the databases. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The PCR amplification procedure was as follows: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 15 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 120 seconds, for a total of 35 cycles, followed by incubation at 72℃ for 5 minutes, and finally isothermal treatment at 12℃. The coding sequence (CDS) of GmABCC3 was cloned from the 644 cDNA and ligated into a T vector for sequencing. The resulting soybean GmABCC3 gene sequence, with a complete coding region of 3906 bp, was obtained. The coding region CDS sequence is shown in SEQ ID NO.1, and its size is 3906 bp. Figure 1 The amino acid sequence of GmABCC3 is shown in SEQ ID NO.2.

[0023] Example 2: Analysis of soybean aluminum toxicity tolerance and the induced expression of the GmABCC3 gene under aluminum toxicity stress

[0024] Aluminum-sensitive soybean variety 84 and aluminum-tolerant variety 644 were cultured in a 0.5 mM CaCl2 solution (pH 4.3) for 24 h after 3-4 days of germination. Then, they were treated with a 0.5 mM CaCl2 solution containing 25 μM AlCl3, while the control group received a 0.5 mM CaCl2 solution containing 0 μM AlCl3, with the pH remaining at 4.3. Root tip samples were taken at 6 h, 12 h, and 24 h after treatment, flash-frozen in liquid nitrogen, and stored at -80 °C for total RNA extraction and cDNA acquisition (using the same method as above). The relative root growth (RRG) trait of aluminum tolerance was obtained by comparing the primary root growth after 24 h of aluminum treatment with the primary root growth of the control group. Sampling for GmABCC3 expression patterns in soybean tissues was conducted at the following time points: root tissues were sampled approximately 7 days after germination in soybean variety 644; stem and leaf tissues were sampled during the trifoliate leaf stage; floral organs were sampled during full bloom; and pods were sampled during pod formation. The primer sequences for GmABCC3 quantitative real-time PCR are shown in SEQ ID NO. 5 and SEQ ID NO. 6. The soybean constitutive gene GmEF-1α (Glyma.17G186600) was used as an internal reference for detecting changes in GmABCC3 gene expression under aluminum toxicity stress; its primer sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8. Quantitative real-time PCR (qRT-PCR) was performed using a Roche 480 instrument from Roche Ltd., Switzerland, and the operation and results analysis were conducted according to the instruction manual. Root phenotypes showed that the root system of aluminum-tolerant variety 644 exhibited better elongation growth under aluminum toxicity stress and higher RRG (residual root growth factor) than that of aluminum-sensitive variety 84. Figure 2 A, 2B). qRT-PCR results showed that GmABCC3 expression was not induced by aluminum toxicity in aluminum-sensitive variety 84, but was upregulated in aluminum-resistant variety 644, indicating a significant difference in GmABCC3 expression between the two varieties. Figure 2 C). In terms of tissue expression patterns, GmABCC3 was expressed at the highest levels in roots and leaves, and at the lowest levels in pods. Figure 2 D).

[0025] Example 3: Detection of allelic variations in the GmABCC3 gene and analysis of superior haplotypes with aluminum toxicity resistance

[0026] Forty-six samples were used, including 24 aluminum-tolerant soybean varieties with RRG ≥ 0.74 and 22 aluminum-sensitive soybean varieties with RRG ≤ 0.42. Using their DNA and cDNA as templates, PCR and sequencing were performed on the promoter (primer pairs see SEQ ID NO. 9 and SEQ ID NO. 10) and CDS (primer pairs see SEQ ID NO. 3 and SEQ ID NO. 4) of the GmABCC3 gene. Association analysis was used to identify variant sites significantly associated with RRG for haplotype analysis of the GmABCC3 gene. Ultimately, seven variant sites on the GmABCC3 promoter classified the 46 samples into seven promoter haplotypes (Pro1-7), two variant sites on the GmABCC3 CDS classified the 46 samples into three CDS haplotypes (CDS1-3), and nine variant sites on the GmABCC3 gene classified the 46 samples into nine haplotypes (Hap1-9). Figure 3 A). The typing results for promoter haplotype (Pro-type), CDS haplotype (CDS-type), and GmABCC3 overall haplotype (Haplotype) all show that the first two haplotypes (Pro1-2 / CDS1-2 / Hap1-2) contain the largest number of varieties. Figure 3 A). Therefore, the differences in the RRG of aluminum toxicity trait among Pro1-2, CDS1-2, and Hap1-2 were mainly analyzed and compared. The results showed that soybean varieties containing the Pro2, CDS2, and Hap2 haplotypes of GmABCC3 had significantly higher RRGs of aluminum toxicity trait than varieties containing the Pro1, CDS1, and Hap1 haplotypes, respectively. Figure 3 B). This indicates that Pro2 (see SEQ ID NO.11) and CDS2 (see SEQ ID NO.1) are the aluminum-resistant promoter haplotypes and CDS haplotypes of GmABCC3, respectively.

[0027] Example 4: Application of superior aluminum-resistant allelic variants of the GmABCC3 gene in root development in Arabidopsis and soybean.

[0028] Based on the information of the overexpression vector pCAMBIA3301, the NcoⅠ and BstEⅡ restriction sites were selected. Using cDNA from varieties 84 and 644 as templates, PCR was performed using primer pairs (SEQ ID NO.12 and SEQ ID NO.13) to obtain GmABCC3. CDS1 and GmABCC3 CDS2 The sequence was obtained by cloning GmABCC3 using a one-step cloning method (Novizan, China). CDS1 and GmABCC3 CDS2The vector was constructed and positioned after the cauliflower mosaic virus promoter CaMV35S(35S). Two different CDS haplotypes (GmABCC3) were overexpressed in wild-type Arabidopsis thaliana (Col-0). CDS1 and GmABCC3 CDS2 ( ), with Col-0 as a control. Transcriptional analysis showed that GmABCC3 was indeed detectable in Arabidopsis lines with overexpression. CDS1 and GmABCC3 CDS2 The expression is present, but it cannot be detected in Col-0. Figure 4 A, B). Arabidopsis thaliana plants with uniform root lengths were vertically germinated and grown on 1 / 2 MS solid medium for approximately 5 days. They were then transferred to 1 / 2 MS solid medium (pH = 4.5) containing 0, 50, and 100 μM AlCl3, respectively, and vertically cultured for 7 days. Root elongation and total root length were measured using a root scanner. The relative root elongation / growth (RRG) and relative total root length indices were used to assess aluminum tolerance. The results showed that overexpression of GmABCC3... CDS1 and GmABCC3 CDS2 The Arabidopsis lines all showed higher relative root elongation / growth (RRG) and relative total root length (P<0.05) than the Col-0 lines, and overexpressed GmABCC3. CDS2 Arabidopsis strains (OE-4 and OE-5) showed better aluminum toxicity phenotype. Figure 4 C, D, E, F). Overexpression of GmABCC3 was also found in Morin staining and root aluminum ion assays. CDS2 Compared to the Col-0 line, the Arabidopsis thaliana strain showed lighter Morin staining in its root tips and lower aluminum ion concentration in its roots (P<0.05). Figure 4 G,H).

[0029] Rooting in transgenic soybeans is a rapid method for detecting candidate gene functions in soybean plants. Using the vector pCAMGFP-CvMV:GWox carrying the green fluorescent protein reporter gene GFP, soybean rooting transformation can be performed, enabling rapid identification of transgenic positive roots under a fluorescence microscope, facilitating subsequent gene function studies. Following the Gateway system's entry and expression vector construction methods, cDNA from 84 and 644 varieties were used as templates, and PCR was performed using primer pairs (SEQ ID NO. 14 and SEQ ID NO. 15) to transform GmABCC3... CDS1 and GmABCC3 CDS2 The gateway system's introductory carrier pENTR is then incorporated, and GmABCC3 is converted via the LR reaction. CDS1 and GmABCC3 CDS2The expression vector pCAMGFP-CvMV:GWox was constructed, and the target gene was overexpressed, driven by the 35S promoter. Empty-vector transgenic soybean rooting served as a control. qRT-PCR results showed that the 35S:GmABCC3 transgenic... CDS1 and 35S:GmABCC3 CDS2 In soybean rootlets transformed with the empty vector, the relative expression level of GmABCC3 was significantly higher than that in soybean rootlets transformed with the empty vector (P<0.01). Hematoxylin staining and relative aluminum ion concentration experiments in soybean rootlets also showed that the expression level of GmABCC3 in the rootlets transformed with the empty vector was significantly higher. CDS1 and 35S:GmABCC3 CDS2 Compared to soybean roots transformed with no vector, the root tips of the transformed soybeans showed lighter hematoxylin staining and lower relative aluminum ion concentration (P<0.01). Furthermore, the roots transformed with 35S:GmABCC3 showed significantly lower hematoxylin staining and relative aluminum ion concentration (P<0.01). CDS2 Soybeans showed better root development ( Figure 5 A, B, C). After 4 weeks of control and stress treatment with gradually increasing aluminum concentration (from 25 μM to 1000 μM), the results also showed that under long-term aluminum toxicity stress, the 35S:GmABCC3... CDS1 and 35S:GmABCC3 CDS2 Soybean roots grown from 35S:GmABCC3 are longer and have more lateral roots compared to those grown from unloaded soybeans. Therefore, in terms of aluminum tolerance relative to root length and relative to total root length, 35S:GmABCC3... CDS1 and 35S:GmABCC3 CDS2 The root development of soybeans converted to 35S:GmABCC3 was significantly higher than that of soybeans converted to no-load, and the root development of soybeans converted to 35S:GmABCC3 was significantly higher. CDS2 Soybeans showed better root development ( Figure 5 (D, E, F). All the above results indicate that overexpression of GmABCC3 can improve aluminum tolerance in Arabidopsis and soybean, reduce root aluminum ion concentration, and GmABCC3... CDS2 It is an excellent CDS haplotype resistant to aluminum toxicity.

[0030] To verify and compare different promoter haplotypes of GmABCC3 Pro1 and GmABCC3 Pro2 The aluminum toxicity resistance effect. First, through promoter activity experiments, using the EcoRI and NcoRI restriction sites of pCAMBIA3301, and using DNA from varieties 84 and 644 as templates, PCR was performed using primer pairs (SEQ ID NO.16 and SEQ ID NO.17, and SEQ ID NO.18 and SEQ ID NO.19). GmABCC3 was cloned using a one-step cloning method. Pro1 and GmABCC3 Pro2The pCAMBIA3301 gene was inserted to replace the 35S variant to drive GUS gene expression in soybean rooting. Results showed that, regardless of control conditions or aluminum toxicity treatment, the expression of the GUS gene was significantly reduced by the insertion of pCAMBIA3301 into the rooting mechanism. Pro2 GUS soybeans have better root development than GmABCC3. Pro1 GUS staining of soybean root hairs is deeper, and GUS enzyme activity is also higher. Figure 6 A, B).

[0031] Similarly, based on the multiple cloning site information of the pBinGFP4 vector and the one-step cloning method (Novizan, China), the KpnⅠ and BamHI restriction sites were selected. Using cDNA from variety 644 as a template, PCR was performed using primer pairs (SEQ ID NO.20 and SEQ ID NO.21) to extract GmABCC3. CDS2 The vector was then constructed. Using the SacⅠ and XhoⅠ restriction sites, and with DNA from varieties 84 and 644 as templates, PCR was performed using primer pairs (SEQ ID NO.22 and SEQ ID NO.23, and SEQ ID NO.24 and SEQ ID NO.25) to extract GmABCC3. Pro1 and GmABCC3 Pro2 To compare GmABCC3, pBinGFP4 was inserted to replace 35S. Pro1 and GmABCC3 Pro2 Driver GmABCC3 CDS2 Differences in expression and aluminum toxicity tolerance were observed in soybean rooting experiments. The expression of GmABCC3 was found to be different. Pro2 :GmABCC3 CDS2 In soybean root germinated plants under aluminum toxicity stress, the relative expression of GmABCC3 was significantly higher than that of transgenic GmABCC3. Pro1 :GmABCC3 CDS2 Soybean rooting with pBinGFP4 empty vector ( Figure 6 C). After 4 weeks of control and stress treatment with gradually increasing aluminum concentration (from 25 μM to 1000 μM), the results also showed that under long-term aluminum toxicity stress, the GmABCC3 variant was converted. Pro2 :GmABCC3 CDS2 Soybean root development relative to GmABCC3 Pro1 :GmABCC3 CDS2 Soybeans transplanted with pBinGFP4 (unloaded) develop longer roots and more lateral roots. Therefore, in terms of aluminum tolerance indices, relative root length and relative total root length, the GmABCC3 variant shows superior growth. Pro2 :GmABCC3 CDS2 Soybean root development was also significantly higher than that of GmABCC3. Pro1 :GmABCC3 CDS2Soybean rooting was compared with that of soybeans without pBinGFP4 (P<0.01). Figure 6 D, E, F). Simultaneously, transfer to GmABCC3. Pro2 :GmABCC3 CDS2 Soybean root development also showed a higher relative number of root tips and a higher relative fresh root weight, as well as a lower root aluminum ion concentration (P<0.05). Figure 6 G,H,I). The above results indicate that GmABCC3 CDS2 and GmABCC3 Pro2 It is an excellent variant type for regulating aluminum toxicity tolerance in soybeans.

[0032] Example 5: GmABCC3 gene enhances the aluminum resistance of yeast strains.

[0033] To further verify the aluminum resistance of GmABCC3, a one-step cloning method was used, along with the Kpn I and BamHI restriction sites of the vector pYES2. Using cDNA from strain 644 as a template, PCR was performed using primer pairs (SEQ ID NO.26 and SEQ ID NO.27) to construct GmABCC3 (CDS2) into the vector pYES2, which was then transformed into yeast strain BY4741. The empty pYES2 vector served as a control group. Yeast strains containing different plasmids were first cultured to OD200. 600 = 0.4 and corrected to be consistent, then diluted layer by layer to 1 / 10. 6 Finally, appropriate amounts of these yeast cultures were spotted onto solid culture media (pH = 4.2) containing 0, 400, and 600 μM AlCl3, respectively. After standing for 30 minutes, the plates were inverted and incubated at 30°C for 3-4 days. From the yeast growth on the culture media, there was no significant difference between yeast containing empty pYES2 and yeast expressing GmABCC3 under aluminum-free stress; however, under aluminum-free stress, yeast expressing GmABCC3 showed better growth than yeast containing empty pYES2, especially under 600 μM AlCl3 treatment at a 1 / 10 dilution. 4 Yeast containing the empty vector pYES2 could only be observed as tiny single colonies, while yeast expressing GmABCC3 could still grow relatively quickly to form larger single colonies. Figure 7 A). To compare the concentration of aluminum ions in yeast cells, yeast containing different plasmids were first cultured to OD200. 600 = 0.4 and corrected to be consistent, then inoculated into yeast liquid medium (pH = 4.2) containing 400 μM AlCl3 at a ratio of 1:100 and cultured by shaking. The OD of the yeast culture was measured every 12 hours. 600Yeast cells were collected, dried, and their aluminum concentration was measured at 24 h and 36 h, respectively. The results showed that under aluminum toxicity treatment for 24 h and 36 h, the aluminum ion concentration in yeast cells expressing GmABCC3 was significantly lower than that in yeast cells containing the empty pYES2 vector (P<0.01). Figure 7 B). These results indicate that the GmABCC3 gene participates in the regulation of aluminum toxicity by reducing the concentration of aluminum ions in cells. sequence list <110> Nanjing Agricultural University <120> Application of the soybean ABC total transporter protein GmABCC3 encoding gene <160> 27 <170> SIPOSequenceListing 1.0 <210> 1 <211> 3906 <212> DNA <213> Soybeans (Glycine max) <400> 1 atggctaagc ttgcttctct tgaagagccc cttttgaatg gtgactctaa agtacagaac 60 aattctgatc ccagtaagac taaaggaaat aactattcaa ttgctggagt tttcagcatt 120 cttactttct catggatcag tccaataata accctaggca atgagaagac attagagcat 180 gaggacctcc cacttcttgc tactgatgac agtgcctatg gggtttttcc aacttttaga 240 aacaaactag agtccgagtg cggtagtgtt aggaatgtga ccactcttaa actcgtgaag 300 gtgttatattct tgtcaacatg gcaaggaatt cttttgtcag gtttatttgc attactatac 360 acgtgtgctt cttatgttgg accctttctt attgaaatct ttgttcaata cctcaatggg 420 gaacaaaagt ttaaaaatga aggctatgtt ttggctatgg catttgttgc tgcaaagctt 480 gtggaatgtc tttcccagag gcactggatg tttaggttcc agcaagttgg ggttaggatg 540 caatcaaagt tggtcgcaat gatctatgct aaaggtttga ctctttcatg tcaatccaag 600 gaggttcgca gcactgggga aatcatcaac ttaatgactg ttgatgcaga aaggattggt 660 gaattttgtt ggtacatgca tgatccatgg atgtgtgttt tgcaagttgc tttggctttg 720 ttaattctat atagaagtgt aggggttgct tcaatagctg ctcttgctgc cactgtaatt 780 gtgatgttgt taaactttcc cgtgtcatca ttgcaagaaa agttccaagg caaggtaatg 840 gagttcaaag ataaaagaat gaaggctacg tctgagattc tgaagaatat caggattcta 900 aaactgcaag cgtgggagat gaagttcttg tcaaagatta ttcagcttag gaagactgag 960 gagatatggc taaagaaatt tctagctagt acagcaatta ttaaatttct cttccataac 1020 gccccaactt ttattgctgt ggttacattt ggtgcttgtg ctcttattgg aatcccactt 1080 gaatctggga aggtcttatc tgcacttgca acattcagaa ttcttcaaat gcccatttac 1140 ggtcttcctg acacaatttc aatgattgca caaacttaaag tttcccttga aaggattgca tcatttcttc gtctagaaga gttgcagacc gtgtagtag aaaagcttcc atggggtagt tctgataagg ctattgaatt agtagtag tatttctctt gggatttatc ttcccctaat acaacactga aaaacataaa tctcacaatt tttcatggta tgagggttgc tgtatgtggt actgttgggt caggcaagtc tagcttactt tcttgtata taggggagt accaaagata tcgggaacgc tgaagatatg tggaaccag gcttatgttt ctcaatcgcc atggatacag ggtggcaaga tagaagataa catattattt ggtaaagaga tggacagggg aaagtataag aaggtgctag aagcatgttc cttgacaaaa gaccttgaga ttctaccatt tggtgatcag actattattg gagagaaggg aatcaatttg agtggtggac agaagcaaag agtgcaaata gcccgtgctc tataccagga tgctgatgta tatctgtttg atgatccctt cagtgctgtg 1740. gatgcccata caggatccca tctctttaag gagtgcttgc ttggtctttt aaaatcaaaa actgtgatat acattactca tcaagtagaa ttcttacctg atgctgatct aatattggtc agagagaag gaagcatac tcaatcagga aaatacaatg acattctca gaggcact 1920 gatttaatgg aacttgtagg tgcacaga gaagctttgt cttcaattaa gtccttagag 1980 agaaagccaa catttaaaat atcaagtaca tccgaggagg acccaaatttc attaagtgat 2040 tttgaacttg agaaaaatgt ggaaaacaca atgatcaa ctgataagtc aaatgacaca 2100 gttgagcccc aaggacagct tgttcagaa gagaacgag aaagggtag agttggtttt 2160 aaagtatact ggaaatacat cacacagct tatggaggta ctcttgtacc cttcatatta 2220 ctttcacaga cactcaccat cagtttcag attgcagca attattggat gactgtggca 2280 acacctgttt cagcaactgc agaacctgat attagaagct attacactt ggttgtctat 2340 gttgctttgg cagttggaag ttccatttc accttgcca gagcattctt tgctgcgata 2400 gctggataca aacagccac tgtgctctc aaaaatgc atttgagtgt tttcgagca 2460 ccaatatcat ttttgatgc caccccagt ggtcgaatcc ttaatagagc ttcacagac 2520 caagcacac tggatatgta cattgcagat atattatggg cagttaccct caatctggtt 2580 actctcttcg gaaatatttt tgtgatgtct caagctgcat ggcaggtgtt tatagtattg 2640 attccagtca tggcagcatg catatggtac cagcgatact attctgcatc agcaagagaa 2700 ttggcacgat tagttggtac atgccaagct ccagttatac aacatttttc ggaaactatc 2760 tctggatcaa caaccataag aagttttgag caagaatcaa gatttattga cataaatatg 2820 aaaatgatag acagatattc ccaacccaaa ttatacagtg ctactgcaat cgaatggctg 2880 aatttcagat tggatatttt atctactctc acatttgcct gctgtttggt tttcttgata 2940 tcttttccaa gttcaatgac tgctcctgga attgcgggat tggctgtgac atatggactt 3000 aatctaaatg ctgtacaaac taaagtaatc tggttttctt gcaatttgga gaacaaaatt 3060 atatctgttg aaagaatgct ccaatacaca tccctcccaa gtgaagctcc tcttgtaata 3120 aaagataacc aaccagatta ttcttggcca tcatttggag aagttcatat ccgggattta 3180 caggttcaat atgctcctca cttgcctatt gttttacgag gtcttacttg cacttttact 3240 gctggagcaa aaactggtat tgtgggaaga acaggaagtg gaaaatcaac tcttgtgcaa 3300 acacttttcc gacttattga acctgttgct ggacaaatat tgatagatag cattaacatc 3360 tctttcattg gaattcatga tttgcggtcc cgactgagca ttattcctca agatccaaca 3420 atgtttgaag ggacaataag aaccaacctg gatccactgg aagagtacac agatgaacag 3480 atttgggagg ctctatatat gtgccaactt ggagatgaag taaggaaaaa agaagggaag 3540 cttgactccg tagttacaga gaatggagaa aattggagta tgggccaaag gcaattggtc 3600 tgcctcggcc gtgttctact taagaaaagc aagatcttgg tgcttgatga agctactgca 3660 tcagttgata cagccacaga taatattatt cagcaaacag ttaagcaaca tttttccgaa 3720 tgcacagtca ttaccattgc tcataggata acttcaatcc ttgatagtga catggttttg 3780 tttttaaatc aagggttgat tgaggaatat gattcaccaa agaaactgct taagaacaag 3840 tcttcatctc tagctcaact agttgaagaa tacacaagga ggtcaaattc tggttttgga 3900 aattaa 3906 <210> 2 <211> 1301 <212> PRT <213> Glycine max <400> 2 Met Ala Lys Leu Ala Ser Leu Glu Glu Pro Leu Leu Asn Gly Asp Ser 1 5 10 15 Lys Val Gln Asn Asn Ser Asp Pro Ser Lys Thr Lys Gly Asn Asn Tyr 20 25 30 Ser Ile Ala Gly Val Phe Ser Ile Leu Thr Phe Ser Trp Ile Ser Pro 35 40 45 Ile Ile Thr Leu Gly Asn Glu Lys Thr Leu Glu His Glu Asp Leu Pro 50 55 60 Leu Leu Ala Thr Asp Asp Ser Ala Tyr Gly Val Phe Pro Thr Phe Arg 65 70 75 80 Asn Lys Leu Glu Ser Glu Cys Gly Ser Val Arg Asn Val Thr Thr Leu 85 90 95 Lys Leu Val Lys Val Leu Phe Leu Ser Thr Trp Gln Gly Ile Leu Leu 100 105 110 Ser Gly Leu Phe Ala Leu Leu Tyr Thr Cys Ala Ser Tyr Val Gly Pro 115 120 125 Phe Leu Ile Glu Ile Phe Val Gln Tyr Leu Asn Gly Glu Gln Lys Phe 130 135 140 Lys Asn Glu Gly Tyr Val Leu Ala Met Ala Phe Val Ala Ala Lys Leu 145 150 155 160 Val Glu Cys Leu Ser Gln Arg His Trp Met Phe Arg Phe Gln Gln Val 165 170 175 Gly Val Arg Met Gln Ser Lys Leu Val Ala Met Ile Tyr Ala Lys Gly 180 185 190 Leu Thr Leu Ser Cys Gln Ser Lys Glu Val Arg Ser Thr Gly Glu Ile 195 200 205 Ile Asn Leu Met Thr Val Asp Ala Glu Arg Ile Gly Glu Phe Cys Trp 210 215 220 Tyr Met His Asp Pro Trp Met Cys Val Leu Gln Val Ala Leu Ala Leu 225 230 235 240 Leu Ile Leu Tyr Arg Ser Val Gly Val Ala Ser Ile Ala Ala Leu Ala 245 250 255 Ala Thr Val Ile Val Met Leu Leu Asn Phe Pro Val Ser Ser Leu Gln 260 265 270 Glu Lys Phe Gln Gly Lys Val Met Glu Phe Lys Asp Lys Arg Met Lys 275 280 285 Ala Thr Ser Glu Ile Leu Lys Asn Ile Arg Ile Leu Lys Leu Gln Ala 290 295 300 Trp Glu Met Lys Phe Leu Ser Lys Ile Ile Gln Leu Arg Lys Thr Glu 305 310 315 320 Glu Ile Trp Leu Lys Lys Phe Leu Ala Ser Thr Ala Ile Ile Lys Phe 325 330 335 Leu Phe His Asn Ala Pro Thr Phe Ile Ala Val Val Thr Phe Gly Ala 340 345 350 Cys Ala Leu Ile Gly Ile Pro Leu Glu Ser Gly Lys Val Leu Ser Ala 355 360 365 Leu Ala Thr Phe Arg Ile Leu Gln Met Pro Ile Tyr Gly Leu Pro Asp 370 375 380 Thr Ile Ser Met Ile Ala Gln Thr Lys Val Ser Leu Glu Arg Ile Ala 385 390 395 400 Ser Phe Leu Arg Leu Glu Glu Leu Gln Thr Asp Val Val Glu Lys Leu 405 410 415 Pro Trp Gly Ser Ser Asp Lys Ala Ile Glu Leu Val Asp Gly Tyr Phe 420 425 430 Ser Trp Asp Leu Ser Ser Pro Asn Thr Thr Leu Lys Asn Ile Asn Leu 435 440 445 Thr Ile Phe His Gly Met Arg Val Ala Val Cys Gly Thr Val Gly Ser 450 455 460 Gly Lys Ser Ser Leu Leu Ser Cys Ile Ile Gly Glu Val Pro Lys Ile 465 470 475 480 Ser Gly Thr Leu Lys Ile Cys Gly Thr Lys Ala Tyr Val Ser Gln Ser 485 490 495 Pro Trp Ile Gln Gly Gly Lys Ile Glu Asp Asn Ile Leu Phe Gly Lys 500 505 510 Glu Met Asp Arg Gly Lys Tyr Lys Lys Val Leu Glu Ala Cys Ser Leu 515 520 525 Thr Lys Asp Leu Glu Ile Leu Pro Phe Gly Asp Gln Thr Ile Ile Gly 530 535 540 Glu Lys Gly Ile Asn Leu Ser Gly Gly Gln Lys Gln Arg Val Gln Ile 545 550 555 560 Ala Arg Ala Leu Tyr Gln Asp Ala Asp Val Tyr Leu Phe Asp Asp Pro 565 570 575 Phe Ser Ala Val Asp Ala His Thr Gly Ser His Leu Phe Lys Glu Cys 580 585 590 Leu Leu Gly Leu Leu Lys Ser Lys Thr Val Ile Tyr Ile Thr His Gln 595 600 605 Val Glu Phe Leu Pro Asp Ala Asp Leu Ile Leu Val Met Arg Glu Gly 610 615 620 Ser Ile Thr Gln Ser Gly Lys Tyr Asn Asp Ile Leu Lys Thr Gly Thr 625 630 635 640 Asp Leu Met Glu Leu Val Gly Ala His Arg Glu Ala Leu Ser Ser Ile 645 650 655 Lys Ser Leu Glu Arg Lys Pro Thr Phe Lys Ile Ser Ser Thr Ser Glu 660 665 670 Glu Asp Pro Asn Ser Leu Ser Asp Phe Glu Leu Glu Lys Asn Val Glu 675 680 685 Asn Thr Asn Asp Gln Thr Asp Lys Ser Asn Asp Thr Val Glu Pro Gln 690 695 700 Gly Gln Leu Val Gln Glu Glu Glu Arg Glu Lys Gly Arg Val Gly Phe 705 710 715 720 Lys Val Tyr Trp Lys Tyr Ile Thr Thr Ala Tyr Gly Gly Thr Leu Val 725 730 735 Pro Phe Ile Leu Leu Ser Gln Thr Leu Thr Ile Ser Phe Gln Ile Ala 740 745 750 Ser Asn Tyr Trp Met Thr Val Ala Thr Pro Val Ser Ala Thr Ala Glu 755 760 765 Pro Asp Ile Arg Ser Phe Thr Leu Met Val Val Tyr Val Ala Leu Ala 770 775 780 Val Gly Ser Ser Ile Phe Thr Phe Ala Arg Ala Phe Leu Ala Ala Ile 785 790 795 800 Ala Gly Tyr Lys Thr Ala Thr Val Leu Phe Asn Lys Met His Leu Ser 805 810 815 Val Phe Arg Ala Pro Ile Ser Phe Phe Asp Ala Thr Pro Ser Gly Arg 820 825 830 Ile Leu Asn Arg Ala Ser Thr Asp Gln Ser Thr Leu Asp Met Tyr Ile 835 840 845 Ala Asp Ile Leu Trp Ala Val Thr Leu Asn Leu Val Thr Leu Phe Gly 850 855 860 Asn Ile Phe Val Met Ser Gln Ala Ala Trp Gln Val Phe Ile Val Leu 865 870 875 880 Ile Pro Val Met Ala Ala Cys Ile Trp Tyr Gln Arg Tyr Tyr Ser Ala 885 890 895 Ser Ala Arg Glu Leu Ala Arg Leu Val Gly Thr Cys Gln Ala Pro Val 900 905 910 Ile Gln His Phe Ser Glu Thr Ile Ser Gly Ser Thr Thr Ile Arg Ser 915 920 925 Phe Glu Gln Glu Ser Arg Phe Ile Asp Ile Asn Met Lys Met Ile Asp 930 935 940 Arg Tyr Ser Gln Pro Lys Leu Tyr Ser Ala Thr Ala Ile Glu Trp Leu 945 950 955 960 Asn Phe Arg Leu Asp Ile Leu Ser Thr Leu Thr Phe Ala Cys Cys Leu 965 970 975 Val Phe Leu Ile Ser Phe Pro Ser Ser Met Thr Ala Pro Gly Ile Ala 980 985 990 Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn Ala Val Gln Thr Lys 995 1000 1005 Val Ile Trp Phe Ser Cys Asn Leu Glu Asn Lys Ile Ile Ser Val Glu 1010 1015 1020 Arg Met Leu Gln Tyr Thr Ser Leu Pro Ser Glu Ala Pro Leu Val Ile 1025 1030 1035 1040 Lys Asp Asn Gln Pro Asp Tyr Ser Trp Pro Ser Phe Gly Glu Val His 1045 1050 1055 Ile Arg Asp Leu Gln Val Gln Tyr Ala Pro His Leu Pro Ile Val Leu 1060 1065 1070 Arg Gly Leu Thr Cys Thr Phe Thr Ala Gly Ala Lys Thr Gly Ile Val 1075 1080 1085 Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Val Gln Thr Leu Phe Arg 1090 1095 1100 Leu Ile Glu Pro Val Ala Gly Gln Ile Leu Ile Asp Ser Ile Asn Ile 1105 1110 1115 1120 Ser Phe Ile Gly Ile His Asp Leu Arg Ser Arg Leu Ser Ile Ile Pro 1125 1130 1135 Gln Asp Pro Thr Met Phe Glu Gly Thr Ile Arg Thr Asn Leu Asp Pro 1140 1145 1150 Leu Glu Glu Tyr Thr Asp Glu Gln Ile Trp Glu Ala Leu Tyr Met Cys 1155 1160 1165 Gln Leu Gly Asp Glu Val Arg Lys Lys Glu Gly Lys Leu Asp Ser Val 1170 1175 1180 Val Thr Glu Asn Gly Glu Asn Trp Ser Met Gly Gln Arg Gln Leu Val 1185 1190 1195 1200 Cys Leu Gly Arg Val Leu Leu Lys Lys Ser Lys Ile Leu Val Leu Asp 1205 1210 1215 Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn Ile Ile Gln Gln 1220 1225 1230 Thr Val Lys Gln His Phe Ser Glu Cys Thr Val Ile Thr Ile Ala His 1235 1240 1245 Arg Ile Thr Ser Ile Leu Asp Ser Asp Met Val Leu Phe Leu Asn Gln 1250 1255 1260 Gly Leu Ile Glu Glu Tyr Asp Ser Pro Lys Lys Leu Leu Lys Asn Lys 1265 1270 1275 1280 Ser Ser Ser Leu Ala Gln Leu Val Glu Glu Tyr Thr Arg Arg Ser Asn 1285 1290 1295 Ser Gly Phe Gly Asn 1300 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <400> 3 cttgttcttg ccttgttatt gactt 25 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <400> 4 atgtgaagca gagtgagtaa aatctc 26 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 aatccaagga ggttcgca 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 gcaatgatga cacgggaa 18 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 tgcaaaggag gctgctaact 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 cagcatcacc gttcttcaaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 tgcaaaggag gctgctaact 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 cagcatcacc gttcttcaaa 20 <210> 11 <211> 2077 <212> DNA <213> Artificial Sequence <400> 11 cctctcctct ccatcctcac tccagctgtc cagcactcat gtagaccttg gatcaatttt 60 gttttagttt tttttttttt acataatcga aaatgtaatg ttaaaaatta aattaatcca 120 gttgaaaaaaaaaaaaaaaaaaaaaaaaaaaaatcaaa 180 ttatgtttta attttatat acattctaaa aacaatcaa tttaatatta attatgagt 240 tcatttaaga ggttgatctc tcatgataa gtttaggatt gatttaata ctttatatt 300 aagtttagga ttgaatttat acctttaata ttagagaca aaatttatta tcaatgtaat 360 ctcattttat tattatatac catttgcttt tgggatgagc ataatagaca agttttttctt 420 aatcgcatga ttgatcgaag gatctacaac aatgctggca taattcagga gaggatccag 480 ataggtgat taaacgcaac atatgaataa cctttaagta tttttttt attaaaatca 540 aaatttat tatatatac aattttttat tagataatg tataaaaaaa atctttacat 600 cggttcataa aacattctc tcaacgataa aaatttatag attcaaaac atctttaatc 660 ttttataaaa aaaaatttctt gtgatatatt attatttatt gtatcgatat tattttcttc 720 tcgggtgtat attgggacag agtctcatt aattttga aaaataataa gttttgcaca 780 ctcatacgtt aaacatac cgttaaaact tatatattta ataattttat atttttttct 840 tatcacgtca tatcatatat tatatatcat acatggatta catttttctt ttttatttgt 900 atttctttta acattttttt tgctatgata atttttaatg ggattctttc attccaatca 960 tattttttat tcataagatt tgaacgtgac atcttactta aaaaaatcga atttaatatc 1020 gcttaaataa taacttgtta attttaatcg tttgaacgag aaaaaaaata aaaagaaaat 1080 aaattagata gaaacaaaaa atgtgagttg atttgaaaga ggaaaaagaa aagatgaaga 1140 aaaaaaattc acacttaaat ttccattcta tttctcactt tttgttatta ttatttttat 1200 tcctttcttt ttttctctta atccatacac ctttgttttc atcatatttt tcatatattt 1260 aatctatttg attgaaagtg gaattcacca tgattgcatg tctacgttga acacgctatc 1320 tcctacttat ctatcactct ctcacaccgg ctcactcgct agccacttct tgttaaaaga 1380 actctcactt tgcagcaaag tcaacaagtc aaccattgct catcatcgag aaaaatggac 1440 cccaagtgaa gcttcttgct tggttttgtt tcgtttaaag taaggtatat gataccatgt 1500 tcttactctt cttccacgat gcactatgga actgattttc tcctccaatc cattttctca 1560 cgaggggtat ctgcttcgtt ccacctagtc ttgttaattg tgcttgcttg tgtaatcgct 1620 attgaagaaa ttaaagtgca agtgaaccat agataaaaga gtgaaaaaat tagattccat 1680 aattctgctt tcttgtacta aaaacatagc ctggtttgta gcttagttat ttctgtattc 1740 aaccttgtgt tgggcttact aagttacttt tacttgtcta aacatgatgg cttacaggag 1800 catatcacag tttctgattt ggctctaaaa acagttgttt gggggtgcta tttgtgctta 1860 tttacattct aggaactctg aggcacagga cccgagtttc ccatgtttgt tgagaatttg 1920 gtgctggccc taagcatttg tttcttgttc ttgccttgtt attgactttg tagtctatgg 1980 aaagaatgtt tttttgccaa ttacaaacct tgtttctgac ataggttcct ctatttcggg 2040 tttaattctg tgtaatgttg gatgttctcc taaaaat 2077 <210> 12 <211> 44 <212> DNA <213> Artificial Sequence <400> 12 acgggggact cttgaccatg gctaagcttg cttctcttga agag 44 <210> 13 <211> 45 <212> DNA <213> Artificial Sequence <400> 13 ggggaaattc gagctggtca ccttaatttc caaaaccaga atttg 45 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <400> 14 caccatggct aagcttgcttctcttga 27 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <400> 15 ttaatttcca aaaccagaat ttgacctc 28 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <400> 16 tatgaccatg attacgaatt ccctccccca tcctcactcc 40 <210> 17 <211> 48 <212> DNA <213> Artificial Sequence <400> 17 ttaccctcag atctaccatg gatttttagg agaacatcca acataaca 48 <210> 18 <211> 44 <212> DNA <213> Artificial Sequence <400> 18 tatgaccatg attacgaatt ccctctcctc tccatcctca ctcc 44 <210> 19 <211> 48 <212> DNA <213> Artificial Sequence <400> 19 ttaccctcag atctaccatg gatttttagg agaacatcca acattaca 48 <210> 20 <211> 44 <212> DNA <213> Artificial Sequence <400> 20 atttacgaac gatagggtac catggctaag cttgcttctc ttga 44 <210> twenty one <211> 45 <212> DNA <213> Artificial Sequence <400> twenty one gcccttgctc accatggatc catttccaaa accagaattt gacct 45 <210> twenty two <211> 40 <212> DNA <213> Artificial Sequence <400> twenty two gttaattaag aattcgagct ccctccccca tcctcactcc 40 <210> twenty three <211> 48 <212> DNA <213> Artificial Sequence <400> twenty three gttgtgttga gaattctcga gatttttagg agaacatcca acataaca 48 <210> twenty four <211> 44 <212> DNA <213> Artificial Sequence <400> twenty four gttaattaag aattcgagct ccctctcctc tccatcctca ctcc 44 <210> 25 <211> 48 <212> DNA <213> Artificial Sequence <400> 25 gttgtgttga gaattctcga gatttttagg agaacatcca acattaca 48 <210> 26 <211> 44 <212> DNA <213> Artificial Sequence <400> 26 gggaatatta agcttggtac catggctaag cttgcttctc ttga 44 <210> 27 <211> 47 <212> DNA <213> Artificial Sequence <400> 27 gcggccgtta ctagtggatc cttaatttcc aaaaccagaa tttgacc 47

Claims

1. Co-promoter of the gene encoding soybean ABC total transporter protein GmABCC3 GmABCC Pro2 In its application to improving the aluminum toxicity tolerance of soybeans, the gene encoding the soybean ABC total transporter protein GmABCC3 is shown in SEQ ID NO.1, with the promoter... GmABCC Pro2 As shown in SEQ ID NO.

11.

2. The application according to claim 1, characterized in that, Overexpression of the gene encoding GmABCC3 can significantly improve the aluminum toxicity tolerance of soybeans and reduce the aluminum ion concentration in soybean roots.

3. Application of expression vectors overexpressing the gene encoding soybean ABC total transporter GmABCC3 in improving soybean aluminum toxicity resistance, wherein the superior haplotype of the soybean ABC total transporter GmABCC3 gene is described. GmABCC3 CDS2 As shown in SEQ ID NO. 1; the expression vector uses the sequence shown in SEQ ID NO. 11 as an excellent aluminum-inducible promoter. GmABCC Pro2 .

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