CN110241121A - Application of soybean E3 ubiquitin ligase GmNLA1 coding gene - Google Patents

Abstract

The invention discloses the application of soybean E3 ubiquitin ligase GmNLA1. Soybean GmNLA1 protein coding gene GmNLA1, its nucleotide sequence is: SEQ ID NO.1. The constructed plant overexpression vectors pMDC83‑GmNLA1 and GmNLA1‑RNAi were transformed into soybean hairy roots, and after treatment with 1/2 Hoagland for 15 days, it was found that in GmNLA1‑OE transgenic hairy roots, GmNLA1‑OE transgenic hairy roots The P concentration was significantly reduced. P concentrations were significantly increased in GmNLA1‑RNAi transgenic hairy roots. Collectively, GmNLA1 may negatively regulate soybean P efficiency by negatively regulating P concentration in soybean transgenic hairy roots.

Description

Application of soybean E3 ubiquitin ligase GmNLA1 coding gene

technical field

The invention relates to the application of soybean E3 ubiquitin ligase GmNLA1 coding gene and belongs to the field of genetic engineering. Specifically, the soybean E3 ubiquitin ligase GmNLA1 gene affects the phosphorus concentration in soybean hairy roots, thereby changing the phosphorus utilization efficiency.

Background technique

Phosphorus (P) is one of the essential mineral elements in plant growth and reproduction (López-Arredondo et al. 2014), which is involved in many metabolic processes such as energy and cell membrane formation, nucleic acid synthesis, photosynthesis and respiration. Furthermore, the P content is 0.05%-0.5% of the dry weight of the plant (Vance et al. 2003). From 1961 to 2013, the total amount of agricultural fertilizer used in the world was 17.5 million tons per year. The use of phosphate fertilizer per unit area increased by about 3 times during the same period (Lu & Tian 2017). The application of phosphorus fertilizer can increase crop yield, but 80%-90% of the P fertilizer applied to the soil is absorbed by microorganisms or forms insoluble chelates with metal ions (Holford 1997). Only orthophosphate ions (H ₂ PO ₄ ^- and HPO ₄ ^2- ) can be directly taken up and utilized by plants (Hinsinger 2001), resulting in high P fertilizer in soil but low efficiency of P use by plants. The insoluble phosphorus fertilizer fixed in the soil is washed into the river by rainwater, resulting in eutrophication of the water body. Therefore, improving phosphorus efficiency is one of the effective and sustainable approaches to address food production and water eutrophication.

Soybean GmNLA1 is an E3 ubiquitin ligase. To date, only one E3 ubiquitin ligase gene belonging to the SPX-RING family, Nitrogen Limitation Adaptation (NLA), has been identified in Arabidopsis and rice mutants. Arabidopsis nla mutants were found to exhibit premature senescence under low nitrogen conditions due to P intoxication (Kant et al. 2011). Under normal P conditions, the P content in shoots and roots of nla mutants was twice that of WT (Lin et al. 2013; Park et al. 2014). And the homologous gene OsNLA1 was also identified in rice, and the leaf P content in the rice osnla1 mutant was significantly increased and was independent of nitrogen (Yue et al. 2017). However, there is no report on the function of GmNLA1 gene in soybean. Using genetic engineering technology, overexpression and RNA interference vectors were constructed respectively, and GmNLA1 was found to negatively regulate the phosphorus concentration in soybean hairy roots after transformation of soybean hairy roots. These results will help to understand the molecular mechanism of soybean phosphorus efficiency, and can accelerate the breeding process of soybean phosphorus efficient varieties.

Contents of the invention

The purpose of the present invention is to disclose the stress resistance genetic engineering application of soybean GmNLA1 as an E3 ubiquitin ligase. This gene can be introduced into soybean hairy roots as a target gene, and regulate the phosphorus utilization of soybean by affecting the phosphorus concentration in soybean hairy roots. efficiency.

The purpose of the present invention can be achieved through the following technical solutions:

Soybean E3 ubiquitin ligase GmNLA1, its nucleotide sequence is: SEQ ID NO.1.

Soybean E3 ubiquitin ligase GmNLA1, its amino acid sequence is: SEQ ID NO.2.

The application of the soybean E3 ubiquitin ligase gene GmNLA1 in regulating the phosphorus utilization efficiency of soybean, the nucleotide sequence of the soybean E3 ubiquitin ligase gene GmNLA1 is: SEQ ID NO.1.

The preferred application is to overexpress GmNLA1 in soybean hairy roots to reduce the phosphorus concentration in overexpressed hairy roots; or use RNA interference technology to silence GmNLA1 in soybean hairy roots to increase the phosphorus concentration in disturbed hairy roots.

When GmNLA1 is used to construct plant overexpression vectors or interference vectors, any enhanced promoter or inducible promoter can be added before the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a selectable marker gene (GUS gene, luciferase gene, etc.) in the plant. Considering the safety of the transgenic plants, the transformed plants can be screened through stress without adding any selectable marker gene.

After the soybean GmNLA1 protein coding gene GmNLA1 of the present invention is transformed into soybean hairy roots by genetic engineering, the gene is overexpressed to reduce the phosphorus concentration in the hairy roots; after RNA-mediated gene interference suppresses gene expression, the hairy roots are increased. Phosphorus concentration.

Plant overexpression vectors and interference vectors carrying the GmNLA1 of the present invention can transform plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, DNA direct transformation, microinjection, electrical conduction, and Agrobacterium-mediated , and growing transformed plant tissues into plants. The transformed plant host can be monocotyledonous plants such as sorghum, rice, wheat, and corn, and dicotyledonous plants such as peanut, soybean, rapeseed, tomato, poplar, lawn grass, and alfalfa.

Beneficial effect

Soybean GmNLA1 is a gene encoding E3 ubiquitin ligase, including SPX and RING domains, belonging to the SPX-RING family genes. Only NLA and PHO2 of the SPX-RING family have been reported to participate in plant phosphorus homeostasis in model crops such as Arabidopsis and rice. In soybean, we found that the expression level of GmNLA1 was negatively correlated with phosphorus concentration in soybean hairy roots. There was a significant difference in the expression level of GmNLA1 between phosphorus-efficient and phosphorus-sensitive materials. At the same time, through the overexpression of GmNLA1 gene and RNA interference, it was found that GmNLA1 affected the phosphorus use efficiency in soybean by affecting the phosphorus concentration in soybean hairy roots. Therefore, GmNLA1 can be used in the breeding of soybean phosphorus-efficient varieties.

Description of drawings

Figure 1G PCR amplification of the mNLA1 gene. Marker: DL5000

Fig. 2 Relative expression of GmNLA1 in Kefeng 1 and Nannong 1138-2.

Figure 3 Subcellular localization of GmNLA1.

(a): GFP; (d): GmNLA1-GFP; (b): GFP bright field image; (e): GmNLA1-GFP bright field image; (c): GFP fusion image; (f): GmNLA1-GFP fusion picture. Bars = 70 μm.

Figure 4. Hairy root phenotype and fresh weight of GmNLA1.

(a) Phenotype of GmNLA1-OE transgenic hairy root and its control 1 grown in 0.5mM KH ₂ PO ₄ nutrient solution for 15 days. (b) Phenotype of GmNLA1-RNAi transgenic hairy roots and its control 2 grown in 0.5mM KH ₂ PO ₄ nutrient solution for 15 days. (c) Fresh weight of GmNLA1-OE and GmNLA1-RNAi transgenic hairy roots and their control (Control 1/Control 2) grown in 0.5mM KH ₂ PO ₄ nutrient solution for 15 days.

Figure 5. Relative expression level of GmNLA1 and phosphorus concentration in soybean hairy roots.

(a) Relative expression of GmNLA1 in hairy roots of GmNLA1-OE and GmNLA1-RNAi transgenics. (b) P concentrations in GmNLA1-OE and GmNLA1-RNAi transgenic hairy roots and their non-transgenic shoots. GmNLA1-OE: soybean hairy root with pMDC83-GmNLA1, Control 1: soybean hairy root with pMDC83 empty load; GmNLA1-RNAi: soybean hairy root with pB7GWIWG2(II)-GmNLA1RNAi, Control 2: soybean hairy root with pB7GWIWG2( II) Unloaded soybean hairy roots. GmNLA1-OE and GmNLA1-RNAi hairy roots and their controls _were grown in 0.5 mM _KH2PO4 for 15 days. Three biological means ± standard error (SE). * and ** are significant at the 0.05 and 0.01 probability levels, respectively.

Figure 6. PCR detection of overexpressed hairy root (GmNLA1-OE). M: Marker DL2000, P: positive plasmid, H: ddH ₂ O, C: negative hairy root.

Figure 7. PCR detection of RNA interference hairy root (GmNLA1-RNAi). M: Marker DL1000, P: positive plasmid, H: ddH2O, C: negative hairy root.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

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

1) Cloning of soybean E3 ubiquitin ligase GmNLA1 gene

Take soybean cultivar Nannong 1138-2 as the sample object, take its root, grind it with a mortar, add it to a 1.5mL EP tube filled with lysate, shake it fully, transfer it to a 1.5mL EP tube, and extract total RNA (Total RNA Kit (Tiangen, Beijing, China). The total RNA quality was identified by formaldehyde denaturing gel electrophoresis, and the RNA content was measured by a spectrophotometer. The total RNA obtained was used as a template, and the reverse transcription kit ( TaKaRa Primer Script ^TM RT reagent kit, Japan) was used for reverse transcription to obtain the first strand of cDNA, followed by PCR amplification. The PCR program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, 72 Extended at ℃ for 1 min, a total of 35 cycles, and finally incubated at 72 °C for 5 min, followed by constant temperature at 12 °C. Then the PCR product was tapped and purified, ligated and transformed, and positive single clones were picked for sequencing. After sequencing, the length of the complete coding region was 948 bp The CDS sequence of the soybean GmNLA1 gene, wherein the sequence of the coding region is shown in SEQ ID NO.1, named GmNLA1, consisting of 948bp (Fig.

1).

2) Subcellular localization of GmNLA1

Design primers containing the complete ORF of the GmNLA1 gene (excluding stop codons). The primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10. The specific PCR process is the same as step 1). Then use XbaI and KpnI double enzyme digestion to homologously recombine the complete ORF of the GmNLA1 gene that does not contain the stop codon into the expression vector pSuper1300, so that the complete ORF of the GmNLA1 gene is combined with the 3' end of the reporter gene GFP on the expression vector pSuper1300 Fusion formed a chimeric gene of 35S-GmNLA1-GFP, and the subcellular localization vector pSuper1300-GmNLA1 was constructed. The target gene GmNLA1 was transformed into tobacco leaf cells using the Agrobacterium transformation method with the empty vector, and the results showed that the GmNLA1 protein was localized on the cell membrane (Fig. 3).

3) Relative expression of GmNLA1 in Kefeng 1 and Nannong 1138-2

Soybean phosphorus-sensitive variety (Nannong 1138-2) and low-phosphorus-tolerant variety (Kefeng 1) were treated with 1/2 Hoagland nutrient solution containing 0.5mM KH ₂ PO ₄ for 3d, 7d and 12d, and samples were taken. Stored at -80°C after quick freezing in liquid nitrogen. The extraction of total RNA is the same as step 1). Using soybean constitutively expressed Tubulin as an internal reference, the primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, and were derived from cultivated soybean phosphorus-sensitive varieties (Nannong 1138-2) and low-phosphorus-tolerant varieties (Kefeng 1 ) Under the different treatment conditions of the two materials, the total RNA in the subterranean part was used as a template, and after inversion into cDNA, a real-time fluorescent quantitative PCR reaction (Real-time RT-PCR) was carried out. See SEQ ID NO.5 and SEQ ID NO.6 for the primer sequences , to detect the expression level changes of GmNLA1 gene in different varieties.

When transferred to 1/2 Hoagland nutrient solution for 3d, 7d and 12d, the relative expression of GmNLA1 was significantly different in Nannong 1138-2 and Kefeng 1 with different P efficiency (Fig. 2). The relative expression level of GmNLA1 in Kefeng 1 was significantly lower than that in Nannong 1138-2. This indicated that the expression level of GmNLA1 was related to the phosphorus efficiency of different varieties.

Genetic engineering application of embodiment 2 gene GmNLA1

1) Cloning of soybean E3 ubiquitin ligase GmNLA1

The root total RNA of soybean (Glycine max) phosphorus-sensitive variety Nannong 1138-2 was used as a template, and the first strand of cDNA was synthesized by reverse transcription, followed by PCR amplification. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO. 4. The PCR program is as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 1 minute, a total of 35 cycles, and a final incubation at 72°C for 5 minutes, followed by constant temperature at 12°C. The product was cloned into PUC19-T Vector, and after sequencing, the CDS sequence of the 948bp soybean GmNLA1 gene with a complete coding region was obtained, wherein the sequence of the coding region is shown in SEQ ID NO.1.

2) Construction of plant expression vectors

The GmNLA1 gene sequence and Invitrogen's The pDONR221 vector in the Technology with Clonase ^TM II kit was subjected to BP reaction, and PCR sequencing of the bacterial liquid was performed for verification. The primer sequences are shown in SEQ ID NO.11 and SEQ ID NO.12. The specific PCR process was the same as step 1), and the entry clone was obtained ; The obtained entry clone was recombined and exchanged with the target expression vector pMDC83 developed by Invitrogen to obtain the pMDC83-GmNLA1 plant overexpression vector. The plant transformation vector pMDC83 contains a 2x 35S strong promoter, which can strongly induce the expression of the target gene GmNLA1 in the recipient . Then, the vector was transformed into Agrobacterium rhizogenes strain K599 by freeze-thaw method, and the empty pMDC83 was also transformed into K599 at the same time as the empty control.

When constructing an RNA interference vector, first use specific primers to amplify an interference fragment. The primer sequences are shown in SEQ ID NO.13 and SEQ ID NO.14, and the PCR product sequence is shown in SEQ ID NO.15. The next steps are the same as constructing an overexpression vector In the same way, the pMDC83 vector was replaced with the pB7GWIWG2(II) vector, and the constructed vector GmNLA1-RNAi was also transformed into K599. At the same time, the empty pB7GWIWG2(II) was transformed into K599 as an empty control.

3) Obtaining of transgenic root hairs

Using the soybean hairy root transformation method proposed by Kereszt et al. (2007), the Agrobacterium rhizogenes strain K599 bacteria solution containing pMDC83-GmNLA1 and GmNLA1-RNAi vectors and corresponding empty controls obtained in step 2) was injected for 7 days Below the cotyledon nodes of soybean seedlings, place them in a constant temperature light incubator, cultivate them in light for 12 hours and dark for 12 hours, and keep high humidity. After 2-3 weeks, hairy roots will grow from the injection site. The main roots of the seedlings were removed and cultured in 1/2 Hoagland nutrient solution for 15 days to obtain chimeras of soybean seedlings, including non-transgenic shoots and transgenic hairy roots. The chimera with hairy root overexpression is called GmNLA1-OE, and its empty control is Control 1; the chimera with hairy root interference is called GmNLA1-RNAi, and its empty control is Control 2. In order to detect whether it is a positive hairy root, specific primers are used to carry out PCR detection on the extracted DNA fragments. See SEQ ID NO.16 and SEQ ID NO.17 for the primer sequences for overexpression hairy root detection, and see Figure 6 for the gel map of PCR positive hairy root detection. RNA interference hairy root detection bar primers are SEQ ID NO.18 and SEQ ID NO.19, and PCR positive hairy root detection is shown in Figure 7. Real-time fluorescence quantitative qPCR found that the expression level of GmNLA1 gene in overexpressed hairy roots (GmNLA1-OE) was significantly higher than that of Control 1 (Fig. 5a). However, the expression level of GmNLA1 gene in RNA interference hairy roots (GmNLA1-RNAi) was significantly lower than that of Control 2 (Fig. 5a).

When the soybean hairy roots grew to a suitable size, the P concentration in the hairy roots of GmNLA1-OE and GmNLA1-RNAi transgenics was measured after being treated with 1/2 Hoagland for 15 days. The results showed that in GmNLA1-OE transgenic hairy roots, the P concentration in GmNLA1-OE was 0.77 times that of Control 1, indicating that the P concentration in GmNLA1-OE transgenic hairy roots was significantly reduced under the +P condition. But GmNLA1-OE non-transgenic shoots were not significantly different (Fig. 5b). In contrast, the P concentration in GmNLA1-RNAi was 1.66 times that of the transgenic root Control 2, indicating that the P concentration in the GmNLA1-RNAi transgenic hairy root was significantly increased under the +P condition. As with GmNLA1-OE transgenic hairs, GmNLA1-RNAi non-transgenic shoots were not significantly different (Fig. 5b). These results suggest that GmNLA1 negatively regulates P concentration in soybean transgenic hairy roots under +P conditions.

sequence listing

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Claims (3)

1. The application of the soybean E3 ubiquitin ligase gene GmNLA1 in regulating the phosphorus utilization efficiency of soybean, the nucleotide sequence of the soybean E3 ubiquitin ligase gene GmNLA1 is: SEQ ID NO.1. 2. The application according to claim 1, characterized in that the overexpression of GmNLA1 in the hairy root of soybean reduces the concentration of phosphorus in the hairy root of overexpression. 3. The application according to claim 1, characterized in that RNA interference technology is used to silence GmNLA1 in soybean hairy roots to increase the concentration of phosphorus in the disturbed hairy roots.