Application of soybean GmDCR2L-3 coding gene in regulating soybean tolerance to low phosphorus

Abstract

The application discloses application of a soybean GmDCR2L-3 coding gene in regulation of low-phosphorus tolerance of soybean. The nucleotide sequence of the gene is as shown in SEQ ID NO. 1. A constructed plant overexpression vector pBA002-GmDCR2L-3 and a knockout vector pSC-M-GmDCR2L-3 are transformed into soybean. After 14 days of low-phosphorus nutrient solution water culture treatment, seedling stage aerial parts and underground parts of wild type WT, overexpression line and knockout line are taken, total root length, root volume and root tip number of the roots are scanned by a root scanning instrument, then the roots are killed green in an oven at 105 DEG C for 30 min, and then the aerial parts and the roots are dried at 55 DEG C until constant weight, and then aerial part and root dry weights are measured. The results show that compared with the control, the dry weight of the plant, the root system characteristics and the phosphorus content of the knockout line are significantly improved; compared with the control, the overexpression transgenic line is slightly reduced. In general, GmDCR2L-3 negatively regulates low-phosphorus tolerance of soybean.

Description

Technical Field

[0001] This invention relates to the application of the soybean GmDCR2L-3 encoding gene, belonging to the field of genetic engineering. Specifically, the soybean GmDCR2L-3 gene negatively regulates the soybean's tolerance to low phosphorus. Background Technology

[0002] Soybean [Glycine max (L.) Merr.] is an important dual-purpose crop for grain and oil in my country. Soil phosphorus deficiency is a significant abiotic stress factor limiting soybean yield. Phosphorus is an essential mineral element for plant nucleic acid synthesis, energy metabolism, and signal transduction. While most arable land in my country has high total phosphorus content, the proportion of available phosphorus that can be directly absorbed and utilized by plants is very low. Soybeans in low-phosphorus soils exhibit a series of low-phosphorus stress symptoms, including inhibited root development, stunted growth, reduced biomass, and decreased grain yield. Identifying key genes for low-phosphorus tolerance and improving soybean phosphorus stress adaptability through gene editing is of significant practical importance for breeding new low-phosphorus-tolerant soybean varieties, reducing phosphate fertilizer application, and achieving green and high-yield soybean production.

[0003] Currently, studies have identified a number of genes, including phosphorus transporters and transcription factors, involved in the soybean low-phosphorus response. For example, patent CN107435047A discloses the key regulatory gene GmPHR25 in the plant phosphorus signaling network and its applications; overexpression of GmPHR25 can increase the concentration of soluble and total phosphorus in soybeans and improve biomass under low-phosphorus conditions. Furthermore, the GmPHR1-GmRALF22 module promotes soybean phosphorus absorption by upregulating the expression of high-affinity phosphorus transporter genes, and the GmMyb73-GmGDPD2-GmGA2ox1 transcriptional regulatory module participates in the regulation of soybean low-phosphorus tolerance. However, the current understanding of the molecular regulatory network of the soybean low-phosphorus response is still incomplete, and many genes regulating low-phosphorus tolerance have yet to be discovered.

[0004] Purple acid phosphatases (PAPs), a superfamily of metallophosphatases, have been reported to play important roles in soybean's response to low phosphorus stress, primarily by secreting organic phosphorus into the extracellular environment to release inorganic phosphorus. However, the function of DCR2-type phosphatases within this superfamily in regulating soybean's low phosphorus tolerance has not been reported. The specific molecular mechanisms and regulatory networks by which these genes participate in soybean's response to low phosphorus stress remain to be investigated. Therefore, clarifying the molecular functions of DCR2-type phosphatases in soybean's response to low phosphorus stress is of great value for improving the molecular regulatory network of soybean's response to low phosphorus stress and for the molecular breeding of low-phosphorus-tolerant soybean varieties.

[0005] GmDCR2L-3 is a soybean DCR2 phosphatase-related gene, but its involvement in soybean's response to low phosphorus stress has not been reported. Therefore, identifying the biological function of GmDCR2L-3 in soybean's tolerance to low phosphorus stress can improve the genetic regulatory network of soybean's low phosphorus response and provide new gene resources and theoretical basis for molecular breeding of low phosphorus-tolerant soybean varieties. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an application of the soybean GmDCR2L-3 encoding gene in regulating soybean tolerance to low phosphorus.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] The application of knocking out or inhibiting the soybean phosphatase gene GmDCR2L-3 in improving the tolerance of leguminous plants to low phosphorus stress, wherein the nucleotide sequence of the soybean phosphatase gene GmDCR2L-3 is shown in SEQ ID NO.1.

[0009] Preferably, the legume is soybean.

[0010] A sgRNA targeting the soybean phosphatase gene GmDCR2L-3, wherein the target sequence of the sgRNA is selected from SEQ ID NO.19 or SEQ ID NO.20.

[0011] A CRISPR / Cas9 gene editing vector containing a nucleotide sequence encoding the sgRNA.

[0012] Preferably, the vector is obtained by annealing the primers shown in SEQ ID NO.11 and SEQ ID NO.12 or the primers shown in SEQ ID NO.13 and SEQ ID NO.14 and then ligating them to the pSC-M vector.

[0013] The application of the sgRNA or the CRISPR / Cas9 gene editing vector in improving the tolerance of leguminous plants to low phosphorus stress.

[0014] Preferably, the legume is soybean.

[0015] A method for improving the tolerance of legumes to low phosphorus stress, wherein the method involves introducing a knockout vector targeting the soybean GmDCR2L-3 gene into a host legume through plant genetic transformation, thereby knocking out the expression of the GmDCR2L-3 gene in soybean to obtain transgenic soybeans with improved tolerance to low phosphorus; the nucleotide sequence of the soybean phosphatase gene GmDCR2L-3 is shown in SEQ ID NO.1.

[0016] Preferably, the improved tolerance of the legume to low phosphorus stress is manifested in any one or more of the following:

[0017] (1) Increase in stem dry weight or total root length.

[0018] (2) Increase in root dry weight or root volume.

[0019] (3) Increase in root-to-crown ratio or number of root tips.

[0020] Preferably, the knockout vector targeting the soybean GmDCR2L-3 gene is the CRISPR / Cas9 gene editing vector described in this invention.

[0021] Beneficial effects:

[0022] GmDCR2L-3 is a soybean DCR2 phosphatase-related gene. In soybean, we found that the expression level of GmDCR2L-3 was significantly induced by low phosphorus stress. Hydroponic experiments on stable transgenic soybean seedlings showed that, compared to wild-type controls, GmDCR2L-3 overexpressing transgenic soybean plants had significantly lower biomass and root structure. However, CRISPR / Cas9-mediated GmDCR2L-3 knockout mutants exhibited significantly better biomass and root structure than wild-type plants. These results demonstrate that GmDCR2L-3 negatively regulates soybean's tolerance to low phosphorus. The GmDCR2L-3 gene identified in this study can serve as an excellent target and has significant application value for creating new low-phosphorus-tolerant soybean varieties. Attached Figure Description

[0023] Figure 1 PCR and bar strip identification of soybean plants overexpressing the GmDCR2L-3 gene.

[0024] Figure 2 DNA sequencing results of CRISPR-mediated GmDCR2L-3 knockout materials. KO-3 showed a 4-base deletion mutation at sgRNA3; the KO-4 line showed a 1-base deletion mutation at sgRNA4, and the sequencing peaks were single peaks.

[0025] Figure 3 Relative expression levels of the GmDCR2L-3 gene in overexpression and knockout materials. OE-4 and OE-9 represent two GmDCR2L-3 overexpression lines, and KO-3 and KO-4 represent GmDCR2L-3 knockout mutant lines. Three biological means ± standard error (SE), *** indicates significance at the 0.001 probability level.

[0026] Figure 4The aboveground and root dry weight, root-shoot ratio, total root length, root volume and root tip number of GmDCR2L-3 overexpression and knockout materials. Based on Duncan's multiple range test, if the letters are different between groups, it indicates that the difference is statistically significant (P<0.05). DETAILED DESCRIPTION

[0027] The terms used in the present application have the meanings generally understood by those of ordinary skill in the art, unless otherwise specified.

[0028] The present application is described in further detail below in connection with specific preparation examples and application examples, and with reference to the data. It should be understood that these examples are merely intended to illustrate the present application, and not to limit the scope of the present application in any way.

[0029] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The primers used are all indicated at the first occurrence, and the same primers used thereafter are all the same as the first indicated.

[0030] The present application is described in further detail below in connection with specific preparation examples and application examples, and with reference to the data. It should be understood that these examples are merely intended to illustrate the present application, and not to limit the scope of the present application in any way.

[0031] Example 1, cloning of soybean GmDCR2L-3 gene

[0032] The soybean variety JACK was used as the material, and its leaves were taken and crushed with a mortar. The crushed leaves were added to a 1.5 mL EP tube containing lysis solution, shaken thoroughly, and then transferred to a 1.5 mL EP tube. Total RNA was extracted using a TianGen RNA extraction kit (Total RNA Kit, Beijing, China). The quality of the total RNA was identified by formaldehyde denaturing gel electrophoresis, and the RNA content was determined by spectrophotometer. The obtained total RNA was used as a template, and reverse transcription was performed according to the instructions of the Evo M-MLV RT Kit with gDNA Cleanfor qPCR II (Changsha, China) provided by Aikeli Company. After obtaining the first strand of cDNA, PCR amplification was performed, and the primers were SEQ ID NO. 3 and SEQ ID NO. 4. The PCR program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 sec, 58°C annealing for 15 sec, 72°C extension for 1 min, a total of 35 cycles, and finally 72°C for 5 min, followed by 12°C constant temperature. Subsequently, the PCR product was gel purified, ligated with PUC19-T Vector, and transformed. Positive single clones were selected for sequencing. After sequencing, a CDS sequence of 363 bp of soybean GmDCR2L-3 gene with complete coding region was obtained, wherein the coding region sequence is SEQ ID NO. 1, and the name is GmDCR2L-3 ( Figure 1 ).

[0033] Example 2, Construction of GmDCR2L-3 gene plant expression vector

[0034] The GmDCR2L-3 gene sequence was amplified from PUC19-T Vector by PCR using primers shown in SEQ ID NO. 5 and SEQ ID NO. 6, and GmDCR2L-3 was ligated into pBA002 vector by recombination reaction to obtain pBA002-GmDCR2L-3 plant overexpression vector. The plant transformation vector pBA002 contains a 2x35S strong promoter that can strongly induce the expression of the target gene GmCPN60 in the recipient. Then the vector was transformed into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method, and soybean was transformed by soybean cotyledon node transformation method.

[0035] According to the sgRNA designed by the website CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / ), specific target sites were designed in the exon region of GmDCR2L-3 gene, and 4 sgRNAs were selected for GmDCR2L-3, thereby designing 4 pairs of targeting sequences, and the primer sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, NO. 11 and SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14. The primers of the four sgRNAs were annealed and ligated into pSC-M vector, and the ligation product was transformed into E. coli, and single colonies were selected by plating and bacterial detection. The band was detected by bacterial detection, and sequencing showed that the four sgRNA sequences were correct, indicating that the vector pSC-M-GmDCR2L-3 was successfully constructed. Then the vector was transformed into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method, and soybean was transformed by soybean cotyledon node transformation method.

[0036] Example 3, Relative expression amount of GmDCR2L-3 gene in overexpression and knockout materials

[0037] Stable transformed positive plants were obtained by tissue culture method. Two overexpression lines were obtained, named OE-4 and OE-9, and PCR and bar test strip were used to detect overexpression positive seedlings (Fig. 2). Figure 1 Two knockout lines KO-3 and KO-4 were obtained by tissue culture, and the sgRNA fragment of the target gene of the sample was amplified and sequenced, and the sequencing results were analyzed combined with the peak graph. KO-3 had 4 base deletion mutations at sgRNA3 (Fig. 3). Figure 2 KO-4 strain had 1 base deletion mutation at sgRNA4 (Fig. 4). Figure 2The sequencing results above all showed single peaks, indicating homozygous mutations. GmDCR2L-3 overexpression and knockout soybean materials were grown in a constant temperature and light incubator. After 20 days, leaves were harvested, flash-frozen in liquid nitrogen, and stored at -80℃. Total RNA extraction was performed as described in step 1. Using constitutively expressed Tubulin in soybean as an internal reference (primer sequences are shown in SEQ ID NO.15 and SEQ ID NO.16), total RNA from GmDCR2L-3 overexpression and knockout soybean materials was used as a template. After being reverse-engineered into cDNA, real-time quantitative PCR (RT-PCR) was performed (primer sequences are shown in SEQ ID NO.17 and SEQ ID NO.18). The expression level of the GmDCR2L-3 gene in different varieties was detected. The figure shows that the expression level of GmDCR2L-3 in overexpression lines was significantly higher than that in wild-type, while the expression level in knockout lines was significantly lower. Figure 3 ).

[0038] Example 4: Phenotypic Identification of GmDCR2L-3 Transgenic Material

[0039] Soybean seeds were germinated using vermiculite. After the cotyledons were fully expanded, soybean seedlings were transferred to a hydroponic tank with a low-phosphorus nutrient solution for hydroponic treatment. After 14 days of treatment, the aboveground and root parts of wild-type WT, overexpression lines, and knockout lines were collected. The total root length, root volume, and number of root tips were scanned using a root-scanning instrument. The roots were then placed in an oven at 105℃ for 30 min to kill the enzymes, followed by drying at 55℃ to constant weight, and the dry weight of the aboveground and root parts was measured. The results showed that, compared with the control, the knockout lines had significantly improved plant dry weight, root-to-shoot ratio, and root traits. Figure 4 The overexpression transgenic lines showed varying degrees of decline compared to the control. Figure 4 Therefore, we believe that GmDCR2L-3 plays an important role in regulating the low phosphorus tolerance of soybeans. New low-phosphorus tolerant soybean materials obtained using the GmDCR2L-3 gene can be used for breeding applications in leguminous plants.

Claims

1. The application of knocking out or inhibiting the soybean phosphatase gene GmDCR2L-3 in improving the tolerance of leguminous plants to low phosphorus stress, characterized in that, The nucleotide sequence of the soybean phosphatase gene GmDCR2L-3 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The legume mentioned is soybean.

3. An sgRNA targeting the soybean phosphatase gene GmDCR2L-3 as described in claim 1, characterized in that, The target sequence of the sgRNA is selected from either SEQ ID NO.19 or SEQ ID NO.

20.

4. A CRISPR / Cas9 gene editing vector, characterized in that, The vector contains a nucleotide sequence encoding the sgRNA of claim 3.

5. The CRISPR / Cas9 gene editing vector according to claim 4, characterized in that, The vector is obtained by annealing the primers shown in SEQ ID NO.11 and SEQ ID NO.12 or the primers shown in SEQ ID NO.13 and SEQ ID NO.14 and then ligating them to the pSC-M vector.

6. The application of the sgRNA of claim 3 or the CRISPR / Cas9 gene editing vector of claim 4 or 5 in improving the tolerance of leguminous plants to low phosphorus stress.

7. The application according to claim 6, characterized in that, The legume in question is soybean.

8. A method for improving the tolerance of leguminous plants to low phosphorus stress, characterized in that, The method involves introducing a knockout vector targeting the soybean GmDCR2L-3 gene into a host leguminous plant via plant genetic transformation, thereby knocking out the expression of the GmDCR2L-3 gene in soybean to obtain transgenic soybeans with improved tolerance to low phosphorus levels; the nucleotide sequence of the soybean phosphatase gene GmDCR2L-3 is shown in SEQ ID NO.

1.

9. The method according to claim 8, characterized in that, The improved tolerance of leguminous plants to low phosphorus stress is manifested in any one or more of the following: (1) Increase in stem dry weight or total root length; (2) Increase in root dry weight or root volume; (3) Increase in root-to-crown ratio or number of root tips.

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