CN110241121B - Application of soybean E3 ubiquitin ligase GmNLA1 coding gene - Google Patents
Application of soybean E3 ubiquitin ligase GmNLA1 coding gene Download PDFInfo
- Publication number
- CN110241121B CN110241121B CN201910424824.4A CN201910424824A CN110241121B CN 110241121 B CN110241121 B CN 110241121B CN 201910424824 A CN201910424824 A CN 201910424824A CN 110241121 B CN110241121 B CN 110241121B
- Authority
- CN
- China
- Prior art keywords
- gmnla1
- soybean
- hairy roots
- gene
- artificial sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses application of soybean E3 ubiquitin ligase GmNLA 1. The nucleotide sequence of the soybean GmNLA1 protein coding gene GmNLA1 is as follows: SEQ ID NO. 1. The constructed plant overexpression vectors pMDC83-GmNLA1 and GmNLA1-RNAi are transformed into soybean hairy roots, and after the soybean hairy roots are treated by 1/2Hoagland for 15d, the P concentration in the GmNLA1-OE transgenic hairy roots is obviously reduced in the GmNLA1-OE transgenic hairy roots. The P concentration in GmNLA1-RNAi transgenic hairy roots was significantly increased. In summary, GmNLA1 may negatively regulate soybean phosphorus efficiency by negatively regulating P concentration in transgenic hairy roots of soybeans.
Description
Technical Field
The invention relates to application of a soybean E3 ubiquitin ligase GmNLA1 coding gene, belongs to the field of genetic engineering, and particularly relates to application of a soybean E3 ubiquitin ligase GmNLA1 gene in influencing phosphorus concentration in soybean hairy roots so as to change phosphorus utilization efficiency.
Background
Phosphorus (P) is one of the essential mineral elements in plant growth and reproduction (L Lopez-Arredox et al 2014), and is involved in many metabolic processes, such as energy and cell membrane formation, nucleic acid synthesis, photosynthesis and respiration. Furthermore, the P content is between 0.05% and 0.5% of the dry weight of the plant (Vance et al 2003). From 1961 to 2013, the total amount of agricultural fertilizers used in the world is 1750 ten thousand tons per year. The usage amount of phosphate fertilizer per unit area is increased by about 3 times (Lu) in the same period&Tian 2017). The application of phosphate fertilizers can improve the crop yield, but 80% -90% of P fertilizers applied to soil are adsorbed by microorganisms or form insoluble chelates with metal ions (Holford 1997). Only orthophosphate ions (H)2PO4 -And HPO4 2-) Can be directly absorbed and utilized by plants (Hinsinger 2001), resulting in high phosphate fertilizer in soil, but the plant phosphorus utilization efficiency is low. The insoluble phosphate fertilizer fixed in the soil is flushed into the river by rainwater, so that the water body is rich in nutritionAnd (5) nourishing and transforming. Therefore, improving the phosphorus efficiency is one of the effective and sustainable development methods for solving the problems of grain 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. The arabidopsis nla mutant was found to exhibit premature senescence under low nitrogen conditions due to P poisoning (Kant et al.2011). Under normal P conditions, the P content in the aerial parts and roots of the nla mutant was twice that of WT (Lin et al.2013; Park et al.2014). And a homologous gene OsNLA1 is also identified in rice, and the content of leaf P in the rice OsNLA1 mutant is obviously increased and is independent of nitrogen (Yue et al 2017). However, there is no report on the function of the GmNLA1 gene in soybean. Overexpression and RNA interference vectors are respectively constructed by utilizing a genetic engineering technology, and after the soybean hairy root is transformed, the GmNLA1 can negatively regulate and control the phosphorus concentration in the soybean hairy root. These results will help to understand the molecular mechanism of phosphorus efficiency of soybean, and at the same time, can accelerate the breeding process of soybean phosphorus high-efficiency variety.
Disclosure of Invention
The invention aims to disclose stress resistance genetic engineering application of soybean GmNLA1 as E3 ubiquitin ligase, wherein the gene can be used as a target gene to be introduced into soybean hairy roots, and the phosphorus utilization efficiency of soybeans is regulated and controlled by influencing the phosphorus concentration in the soybean hairy roots.
The purpose of the invention can be realized by the following technical scheme:
soybean E3 ubiquitin ligase GmNLA1, the nucleotide sequence of which is: SEQ ID NO. 1.
Soybean E3 ubiquitin ligase GmNLA1, the amino acid sequence of which is: SEQ ID NO. 2.
The soybean E3 ubiquitin ligase gene GmNLA1 is applied to adjusting the phosphorus utilization efficiency of soybean, and the nucleotide sequence of the soybean E3 ubiquitin ligase gene GmNLA1 is as follows: SEQ ID NO. 1.
Preferably, the application of the method is that the GmNLA1 is over-expressed in the soybean hairy roots to reduce the phosphorus concentration in the over-expressed hairy roots; or silencing GmNLA1 in the soybean hairy roots by using an RNA interference technology to improve the phosphorus concentration in the interfering hairy roots.
When GmNLA1 is used to construct a plant overexpression vector or an interference vector, any of an enhanced promoter and an inducible promoter may be added in front of the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used may be processed, for example, by adding a selectable marker gene (GUS gene, luciferase gene, etc.) to the plant. From the viewpoint of safety of transgenic plants, transformed plants can be screened by stress without adding any selectable marker gene.
After the soybean GmNLA1 protein coding gene GmNLA1 is transformed into the soybean hairy root through genetic engineering, the gene is over-expressed, and the phosphorus concentration in the hairy root is reduced; following RNA-mediated gene interference to inhibit gene expression, the phosphorus concentration in hairy roots is increased.
Plant overexpression vectors and interference vectors carrying GmNLA1 of the present invention can be obtained by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and culturing the transformed plant tissues into plants. The transformed plant host can be monocotyledons such as sorghum, rice, wheat and corn, and can also be dicotyledons such as peanut, soybean, rape, tomato, poplar, lawn grass and alfalfa.
Advantageous effects
Soybean GmNLA1 is a gene encoding E3 ubiquitin ligase, contains SPX and RING domains, and belongs to the SPX-RING family of genes. The SPX-RING family only NLA and PHO2 have been reported to be involved in plant phosphorus homeostasis in model crops such as Arabidopsis and rice. In soybean, the expression level of GmNLA1 is found to be in negative correlation with the phosphorus concentration in soybean hairy roots. The expression amount of GmNLA1 in the phosphorus-efficient and phosphorus-sensitive materials is remarkably different. Meanwhile, GmNLA1 influences the phosphorus utilization efficiency in soybeans by influencing the phosphorus concentration in soybean hairy roots through overexpression and RNA interference of the GmNLA1 gene. Therefore, GmNLA1 can be used for breeding soybean phosphorus high-efficiency varieties.
Drawings
FIG. 1 PCR amplification of the GmNLA1 gene. Marker: DL5000
FIG. 2 is the relative expression level of GmNLA1 in Kefeng No.1 and Nannong 1138-2.
Fig. 3 subcellular localization of GmNLA 1.
(a) The method comprises the following steps GFP; (d) the method comprises the following steps GmNLA 1-GFP; (b) the method comprises the following steps GFP bright field map; (e) the method comprises the following steps GmNLA1-GFP brightfield map; (c) the method comprises the following steps GFP fusion map; (f) the method comprises the following steps GmNLA1-GFP fusion map. Bars 70 μm.
FIG. 4 hairy root phenotype and fresh weight of GmNLA 1.
(a) Phenotype of GmNLA1-OE transgenic hairy root and its empty Control 1 at 0.5mM KH2PO4Growing in nutrient solution for 15 days. (b) Phenotype of GmNLA1-RNAi transgenic hairy root and its empty Control 2 at 0.5mM KH2PO4Growing in nutrient solution for 15 days. (c) Fresh weights of GmNLA1-OE and GmNLA1-RNAi transgenic hairy roots and their controls (Control 1/Control 2) were at 0.5mM KH2PO4The growth in the nutrient solution is 15 days.
FIG. 5 relative expression levels of GmNLA1 and phosphorus concentration in soybean hairy roots.
(a) Relative expression levels of GmNLA1 in GmNLA1-OE and GmNLA1-RNAi transgenic hairy roots. (b) Concentration of P in GmNLA1-OE and GmNLA1-RNAi transgenic hairy roots and their non-transgenic aerial parts. GmNLA 1-OE: soybean hairy root with pMDC83-GmNLA1, Control 1: soybean hairy roots with pMDC83 empty; GmNLA 1-RNAi: soybean hairy root with pB7GWIWG2(II) -GmNLA1RNAi, Control 2: soybean hairy roots with pB7GWIWG2(II) empty load. GmNLA1-OE and GmNLA1-RNAi hairy roots and controls thereof at 0.5mM KH2PO4Medium growth for 15 days. Three biological means. + -. Standard Error (SE). And are significant at the 0.05 and 0.01 probability levels, respectively.
FIG. 6. PCR assay of overexpressed hairy roots (GmNLA 1-OE). M: marker DL2000, P: positive plasmid, H: ddH2O, C: negative hairy roots.
FIG. 7 RNA interference hairy root (GmNLA1-RNAi) PCR assay. M: marker DL1000, P: positive plasmid, H: ddH2O, C: negative hairy roots.
Detailed Description
The invention is further explained below with reference to the drawings and the embodiments.
The methods used in the following examples are conventional methods unless otherwise specified.
1) Cloning of soybean E3 ubiquitin ligase GmNLA1 gene
Taking soybean cultivar Nannong 1138-2 as a material-taking object, taking the root of the soybean cultivar, grinding the root by using a mortar, adding a 1.5mL EP tube containing a lysate, fully oscillating the tube, transferring the tube into the 1.5mL EP tube, extracting Total RNA (Total RNA Kit (Tiangen, Beijing, China), identifying the quality of the Total RNA by formaldehyde denaturing gel electrophoresis, measuring the RNA content by using a spectrophotometer, taking the obtained Total RNA as a template, and performing reverse transcription by using a reverse transcription Kit (TaKaRa Primer Script) provided by TaKaRa corporation of JapanTMRT reagent kit, japan) to obtain the first strand of cDNA, and PCR amplification was performed, with the following PCR procedure: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 15sec, extension at 72 ℃ for 1min for 35 cycles, final incubation at 72 ℃ for 5min, followed by constant temperature at 12 ℃. And then, performing tapping purification, connection and transformation on the PCR product, and selecting positive monoclonal sequencing. After sequencing, the CDS sequence of 948bp soybean GmNLA1 gene with complete coding region is obtained, wherein the coding region sequence is shown in SEQ ID NO.1, is named GmNLA1 and consists of 948bp (shown in the figure)
1)。
2) Subcellular localization study of GmNLA1
Primers (not containing stop codon) containing complete ORF of GmNLA1 gene were designed, the primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, and the specific PCR process is the same as in step 1). Then, the complete ORF of the GmNLA1 gene not containing a stop codon is homologously recombined into the expression vector pSuper1300 by utilizing double enzyme digestion of XbaI and KpnI, so that the complete ORF of the GmNLA1 gene is fused with the 3' end of the reporter gene GFP on the expression vector pSuper1300 to form a 35S-GmNLA1-GFP chimeric gene, and a subcellular localization vector pSuper1300-GmNLA1 is constructed. The target gene GmNLA1 is transferred into tobacco leaf cells by the agrobacterium transformation method and the empty vector respectively, and the result shows that the GmNLA1 protein is positioned on cell membranes (figure 3).
3) Relative expression quantity of GmNLA1 in Kefeng No.1 and Nannong 1138-2
Seedling of phosphorus-sensitive soybean variety (Nannong 1138-2) and low-phosphorus-resistant soybean variety (Kefeng No. 1) containing 0.5mM KH2PO4The 1/2Hoagland nutrient solution is treated for 3d, 7d and 12d, sampled, frozen by liquid nitrogen and stored at-80 ℃. The total RNA extraction was performed in the same manner as in step 1). The method is characterized in that a Tubulin constitutively expressed by soybean is used as an internal reference, primer sequences are shown in SEQ ID No.7 and SEQ ID No.8, underground total RNA from two materials of a phosphorus sensitive variety (Nannong 1138-2) and a low phosphorus resistant variety (Kefeng No. 1) for cultivating the soybean is used as a template under different treatment conditions, Real-time fluorescence quantitative PCR (Real-time RT-PCR) is carried out after the underground total RNA is inverted into cDNA, the primer sequences are shown in SEQ ID No.5 and SEQ ID No.6, and the expression quantity change of the GmNLA1 gene in different varieties is detected.
When transferred to 1/2Hoagland nutrient solutions at 3d, 7d, and 12d, the relative expression levels of GmNLA1 were significantly different in nannong 1138-2 and kefeng No.1 with different P efficiencies (fig. 2). The relative expression amount of GmNLA1 in the Kefeng No.1 is remarkably lower than that of Nannong 1138-2. This indicates that the expression level of GmNLA1 correlates with the phosphorus efficiency of different varieties.
Example 2 genetic engineering application of Gene GmNLA1
1) Cloning of Soybean E3 ubiquitin ligase GmNLA1
Taking root total RNA of a phosphorus-sensitive soybean (Glycine max) variety Nannong 1138-2 as a template, synthesizing a cDNA first chain through reverse transcription, and performing PCR amplification, wherein the primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, and the PCR program is as follows: pre-denaturation at 95 ℃ for 3 minutes, denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 15 seconds, extension at 72 ℃ for 1 minute, 35 cycles in total, finally heat preservation at 72 ℃ for 5 minutes, then constant temperature at 12 ℃, cloning the PCR product to PUC19-T Vector, and obtaining the CDS sequence of 948bp soybean GmNLA1 gene with a complete coding region after sequencing, wherein the coding region sequence is shown in SEQ ID NO. 1.
2) Construction of plant expression vectors
The GmNLA1 gene sequence was compared with that of Invitrogen corporation
Technology with ClonaseTMBP reaction is carried out on the pDONR221 vector in the II kit, and bacterial liquid is carried outPCR sequencing verification is carried out, primer sequences are shown in SEQ ID NO.11 and SEQ ID NO.12, the specific PCR process is the same as that in the step 1), and entry clone is obtained; the obtained entry clone is recombined and exchanged with a target expression vector pMDC83 developed by Invitrogen company to obtain a pMDC83-GmNLA1 plant over-expression vector, and the plant transformation vector pMDC83 contains a 2x 35S strong promoter and can strongly induce the expression of a target gene GmNLA1 in a receptor. The vector was then transferred to Agrobacterium rhizogenes strain K599 by freeze-thawing, while the empty pMDC83 was also transferred to K599 as an empty control.
When an RNA interference vector is constructed, firstly, a section of interference fragment is amplified by using a specific primer, the sequence of the primer is shown in SEQ ID NO.13 and SEQ ID NO.14, the sequence of a PCR product is shown in SEQ ID NO.15, the next steps are the same as the construction of an over-expression vector, only the pMDC83 vector is changed into a pB7GWIWG2(II) vector, and the constructed vector GmNLA1-RNAi is also transformed into K599. At the same time, unloaded pB7GWIWG2(II) was also transformed into K599 as an unloaded control.
3) Obtaining transgenic root hairs
Injecting the agrobacterium rhizogenes strain K599 bacterial fluid respectively containing pMDC83-GmNLA1 and GmNLA1-RNAi vectors and corresponding no-load control obtained in the step 2) into the lower part of cotyledonary nodes of 7-day soybean seedlings by using a soybean hairy root transformation method provided by Kereszt et al (2007), placing the bacterial fluid in a constant-temperature illumination incubator for 12h illumination and 12h dark culture, keeping high humidity, growing the hairy roots from the injection part after 2-3 weeks, reducing the main roots of the seedlings when 5-10cm is found, and placing the seedlings in 1/2Hoagland nutrient solution for 15 days to obtain soybean seedling chimeras comprising non-transgenic overground parts and transgenic hairy roots. The chimera with overexpressed hairy roots was called GmNLA1-OE, and its no-load Control was Control 1; the chimera with interfering hairy roots was called GmNLA1-RNAi, the empty Control was Control 2. In order to detect whether the hairy root is positive, PCR detection is carried out on the extracted DNA fragment by using a specific primer. The sequences of the detection primers for the over-expression hairy roots are shown in SEQ ID NO.16 and SEQ ID NO.17, and the detection gel picture of the PCR positive hairy roots is shown in FIG. 6. The bar primers for RNA interference hairy root detection are SEQ ID NO.18 and SEQ ID NO.19, and the PCR positive hairy root detection is shown in FIG. 7. Real-time fluorescent quantitative qPCR found that the expression level of GmNLA1 gene in over-expressed hairy roots (GmNLA1-OE) was significantly higher than that of Control 1 (FIG. 5 a). The expression level of GmNLA1 gene in RNA-interfered hairy roots (GmNLA1-RNAi) was significantly lower than that of Control 2 (FIG. 5 a).
P concentrations in GmNLA1-OE and GmNLA1-RNAi transgenic hairy roots were measured after treatment with 1/2Hoagland for 15d when the soybean hairy roots grew to the appropriate size. The results show that the P concentration in GmNLA1-OE transgenic hairy roots is 0.77 times that of Control 1 in GmNLA1-OE transgenic hairy roots, and that the P concentration in GmNLA1-OE transgenic hairy roots is remarkably reduced under the + P condition. However, there was no significant difference in the non-transgenic aerial parts of GmNLA1-OE (fig. 5 b). In contrast, the P concentration in GmNLA1-RNAi was 1.66 times that of transgenic root Control 2, indicating a significant increase in P concentration in GmNLA1-RNAi transgenic hairy roots under + P conditions. There was also no significant difference in the non-transgenic aerial parts of GmNLA1-RNAi as with the GmNLA1-OE transgenic hairs (FIG. 5 b). These results indicate that GmNLA1 negatively regulates P concentration in soybean transgenic hairy roots under + P conditions.
Sequence listing
<110> Nanjing university of agriculture
Application of <120> soybean E3 ubiquitin ligase GmNLA1 coding gene
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 948
<212> DNA
<213> Soybean (Glycine max)
<400> 1
atgaagttct gcaagaccta ccagcagtac atgcaaggac atggccacaa caagctccct 60
tctgttggct tcaagaacct aaaaaagatc attaaaagct gcaggagagc ctccactcaa 120
cctacctgcc ctgatcattg cccagtgtgc gatgggacct ttttcccttc ccttctcaat 180
gaaatgtcag atatagtagg gtgctttaat cagcgcgcgc agcaattgct ggaactacat 240
cttgcttctg gcttcagaaa gtactttctc atgttgaaag gaaaattaca caagaatcat 300
actgctctaa tcgaagaagg aaaagatcta gtcatatatg cactcataaa ttccatcgca 360
attcgaaaaa tcttgaagaa atatgataag attcattatt ccaagcaagg ccaattattc 420
aagtcgaaag tccagaccat gcacaaggaa attcttcaaa gtccctggct ttgtgagctt 480
attgccttac acattaactt aagggaaaca aaatccaagc caagggaggc atccgcactg 540
tttgatggat gttatctcac attcacggat ggaaaaccat cacttacttg tgagctcttt 600
gattccgtca aaattgatat tgacttgacc tgctctatat gcttggatac agtgtttgat 660
tcagtttctc tgacatgcgg ccacatattc tgctacacct gtgcttgctc aactgcatca 720
gttaccattg tcgatggact taaggcagca aatcctaaag aaaaatgtcc tctatgccga 780
gagggaagag tttacgaaga tgctgtgcat ttggaagaat taaatattct gctaggccga 840
agctgcaggg agtactggga gcaaaggctt cagatggaga gggtagagag ggttaagcaa 900
gttaaggagc actgggaaac gcagtgtagg gcgttcatgg gcatctaa 948
<210> 2
<211> 315
<212> PRT
<213> Soybean (Glycine max)
<400> 2
Met Lys Phe Cys Lys Thr Tyr Gln Gln Tyr Met Gln Gly His Gly His
1 5 10 15
Asn Lys Leu Pro Ser Val Gly Phe Lys Asn Leu Lys Lys Ile Ile Lys
20 25 30
Ser Cys Arg Arg Ala Ser Thr Gln Pro Thr Cys Pro Asp His Cys Pro
35 40 45
Val Cys Asp Gly Thr Phe Phe Pro Ser Leu Leu Asn Glu Met Ser Asp
50 55 60
Ile Val Gly Cys Phe Asn Gln Arg Ala Gln Gln Leu Leu Glu Leu His
65 70 75 80
Leu Ala Ser Gly Phe Arg Lys Tyr Phe Leu Met Leu Lys Gly Lys Leu
85 90 95
His Lys Asn His Thr Ala Leu Ile Glu Glu Gly Lys Asp Leu Val Ile
100 105 110
Tyr Ala Leu Ile Asn Ser Ile Ala Ile Arg Lys Ile Leu Lys Lys Tyr
115 120 125
Asp Lys Ile His Tyr Ser Lys Gln Gly Gln Leu Phe Lys Ser Lys Val
130 135 140
Gln Thr Met His Lys Glu Ile Leu Gln Ser Pro Trp Leu Cys Glu Leu
145 150 155 160
Ile Ala Leu His Ile Asn Leu Arg Glu Thr Lys Ser Lys Pro Arg Glu
165 170 175
Ala Ser Ala Leu Phe Asp Gly Cys Tyr Leu Thr Phe Thr Asp Gly Lys
180 185 190
Pro Ser Leu Thr Cys Glu Leu Phe Asp Ser Val Lys Ile Asp Ile Asp
195 200 205
Leu Thr Cys Ser Ile Cys Leu Asp Thr Val Phe Asp Ser Val Ser Leu
210 215 220
Thr Cys Gly His Ile Phe Cys Tyr Thr Cys Ala Cys Ser Thr Ala Ser
225 230 235 240
Val Thr Ile Val Asp Gly Leu Lys Ala Ala Asn Pro Lys Glu Lys Cys
245 250 255
Pro Leu Cys Arg Glu Gly Arg Val Tyr Glu Asp Ala Val His Leu Glu
260 265 270
Glu Leu Asn Ile Leu Leu Gly Arg Ser Cys Arg Glu Tyr Trp Glu Gln
275 280 285
Arg Leu Gln Met Glu Arg Val Glu Arg Val Lys Gln Val Lys Glu His
290 295 300
Trp Glu Thr Gln Cys Arg Ala Phe Met Gly Ile
305 310 315
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggatcttcca gagatatgaa gttctgcaag acctaccagc 40
<210> 4
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctgccgttcg acgatttaga tgcccatgaa cgccctacac 40
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggagttcaca gaggcagag 19
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ctagtctaga atgaagttct gcaagaccta ccagc 35
<210> 10
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cggggtaccg atgcccatga acgccctaca c 31
<210> 11
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggggacaagt ttgtacaaaa aagcaggctc catgaagttc tgcaagacct accagc 56
<210> 12
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggggaccact ttgtacaaga aagctgggtg ttagatgccc atgaacgccc tacac 55
<210> 13
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ggggacaagt ttgtacaaaa aagcaggctc cagtgtgcga tgggaccttt t 51
<210> 14
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggggaccact ttgtacaaga aagctgggtg ccatcaaaca gtgcggatgc 50
<210> 15
<211> 405
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agtgtgcgat gggacctttt tcccttccct tctcaatgaa atgtcagata tagtagggtg 60
ctttaatcag cgcgcgcagc aattgctgga actacatctt gcttctggct tcagaaagta 120
ctttctcatg ttgaaaggaa aattacacaa gaatcatact gctctaatcg aagaaggaaa 180
agatctagtc atatatgcac tcataaattc catcgcaatt cgaaaaatct tgaagaaata 240
tgataagatt cattattcca agcaaggcca attattcaag tcgaaagtcc agaccatgca 300
caaggaaatt cttcaaagtc cctggctttg tgagcttatt gccttacaca ttaacttaag 360
ggaaacaaaa tccaagccaa gggaggcatc cgcactgttt gatgg 405
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
tcagtaacat agatgacacc gc 22
<210> 17
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ggggaccact ttgtacaaga aagctgggtg ttagatgccc atgaacgccc tacac 55
<210> 18
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
acgtcatgcc agttcccgt 19
Claims (3)
1. The soybean E3 ubiquitin ligase gene GmNLA1 is applied to adjusting the phosphorus utilization efficiency of soybean, and the nucleotide sequence of the soybean E3 ubiquitin ligase gene GmNLA1 is as follows: SEQ ID NO. 1.
2. The use according to claim 1, wherein GmNLA1 is overexpressed in soybean hairy roots, reducing the phosphorus concentration in the overexpressed hairy roots.
3. The use according to claim 1, wherein GmNLA1 is silenced in soybean hairy roots by RNA interference technology to increase phosphorus concentration in the interfering hairy roots.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910424824.4A CN110241121B (en) | 2019-05-21 | 2019-05-21 | Application of soybean E3 ubiquitin ligase GmNLA1 coding gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910424824.4A CN110241121B (en) | 2019-05-21 | 2019-05-21 | Application of soybean E3 ubiquitin ligase GmNLA1 coding gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110241121A CN110241121A (en) | 2019-09-17 |
| CN110241121B true CN110241121B (en) | 2022-03-29 |
Family
ID=67884687
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910424824.4A Active CN110241121B (en) | 2019-05-21 | 2019-05-21 | Application of soybean E3 ubiquitin ligase GmNLA1 coding gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110241121B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249395B (en) * | 2021-05-14 | 2022-04-29 | 南京农业大学 | Application of soybean agglutinin receptor kinase Rsc7-1 coding gene |
| CN116355948B (en) * | 2023-03-27 | 2024-03-22 | 南京农业大学 | Application of soybean E2 ubiquitin conjugated enzyme GmUBC2 coding gene |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007314469A (en) * | 2006-05-25 | 2007-12-06 | Univ Of Tokushima | Ubiquitin ligase inhibitor |
| CN101501195A (en) * | 2006-06-13 | 2009-08-05 | 圭尔夫大学 | Nitrogen limitation adaptibility gene and protein and modulation thereof |
| CN101602800A (en) * | 2009-07-16 | 2009-12-16 | 合肥工业大学 | A kind of albumen and encoding gene and application of regulating plant tolerant to low-phosphorus stress |
| CN102234328A (en) * | 2010-04-29 | 2011-11-09 | 中国农业大学 | Plant low phosphorus stress tolerance correlated protein AtLPT2, its coding gene and application |
| CN102317312A (en) * | 2008-12-17 | 2012-01-11 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-related traits and/or abiotic stress tolerance and a method for making the same |
| CN104178496A (en) * | 2013-05-27 | 2014-12-03 | 南开大学 | Plant low-phosphorus response regulation and control unit and expression vector construction technology |
| CN105829536A (en) * | 2013-08-22 | 2016-08-03 | 纳幕尔杜邦公司 | Methods for producing genetic modifications in a plant genome without incorporating a selectable transgene marker, and compositions thereof |
| WO2016181013A1 (en) * | 2015-05-14 | 2016-11-17 | Universidad Politécnica de Madrid | Use of oligosaccharides as stimulators of plant growth in already germinated plants and method for obtaining said oligosaccharides |
| CN107058339A (en) * | 2013-10-12 | 2017-08-18 | 中国农业科学院作物科学研究所 | Soybean GmCIB1 genes and GmCRY2 genes and its regulation and control bloom and aging effect |
| JP2017216882A (en) * | 2016-06-02 | 2017-12-14 | 国立大学法人金沢大学 | Method of detecting the existence of osteopathy, osteopathy therapeutic agent, and method for screening osteopathy therapeutic agent |
| CN107988248A (en) * | 2017-11-14 | 2018-05-04 | 浙江大学 | A kind of method of easy, efficient fusion His label SPX albumen pronucleus expressions, purifying and renaturation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7943825B2 (en) * | 2007-04-12 | 2011-05-17 | Iowa State University Research Foundation, Inc. | Metacaspase II in engineering soybean for disease resistance |
-
2019
- 2019-05-21 CN CN201910424824.4A patent/CN110241121B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007314469A (en) * | 2006-05-25 | 2007-12-06 | Univ Of Tokushima | Ubiquitin ligase inhibitor |
| CN101501195A (en) * | 2006-06-13 | 2009-08-05 | 圭尔夫大学 | Nitrogen limitation adaptibility gene and protein and modulation thereof |
| CN102317312A (en) * | 2008-12-17 | 2012-01-11 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-related traits and/or abiotic stress tolerance and a method for making the same |
| CN101602800A (en) * | 2009-07-16 | 2009-12-16 | 合肥工业大学 | A kind of albumen and encoding gene and application of regulating plant tolerant to low-phosphorus stress |
| CN102234328A (en) * | 2010-04-29 | 2011-11-09 | 中国农业大学 | Plant low phosphorus stress tolerance correlated protein AtLPT2, its coding gene and application |
| CN104178496A (en) * | 2013-05-27 | 2014-12-03 | 南开大学 | Plant low-phosphorus response regulation and control unit and expression vector construction technology |
| CN105829536A (en) * | 2013-08-22 | 2016-08-03 | 纳幕尔杜邦公司 | Methods for producing genetic modifications in a plant genome without incorporating a selectable transgene marker, and compositions thereof |
| CN107058339A (en) * | 2013-10-12 | 2017-08-18 | 中国农业科学院作物科学研究所 | Soybean GmCIB1 genes and GmCRY2 genes and its regulation and control bloom and aging effect |
| WO2016181013A1 (en) * | 2015-05-14 | 2016-11-17 | Universidad Politécnica de Madrid | Use of oligosaccharides as stimulators of plant growth in already germinated plants and method for obtaining said oligosaccharides |
| JP2017216882A (en) * | 2016-06-02 | 2017-12-14 | 国立大学法人金沢大学 | Method of detecting the existence of osteopathy, osteopathy therapeutic agent, and method for screening osteopathy therapeutic agent |
| CN107988248A (en) * | 2017-11-14 | 2018-05-04 | 浙江大学 | A kind of method of easy, efficient fusion His label SPX albumen pronucleus expressions, purifying and renaturation |
Non-Patent Citations (8)
| Title |
|---|
| Arabidopsis RING E3ubiquitin ligase AtATL80 is negatively involved in phosphate mobilization and cold stress response in sufficient phosphate growth conditions;Ji Yeon Suh等;《Biochem biophys Res Commun》;20150615;第463卷(第4期);第793-799页 * |
| E3 ubiquitin-protein ligase BAH1 isoform X1[Glycine max];NCBI;《Genbank Database》;20180831;Accession No.XP_003536160.1 * |
| Identification of loci and candidate gene GmSPX-RING1 responsible for phosphorus efficiency in soybean via genome-wide association analysis;Wenkai Du等;《BMC Genomics》;20201019;第21卷(第1期);725 * |
| The emerging importanca of the SPX domain-containing proteins in phosphate homeostasis;David Secco等;《New Phytol》;20120331;第193卷(第4期);第842-851页 * |
| Two RING –finger ubiquitin E3 ligase regulate the degradation of SPX4,an internal pjosphate sensor,for phosphate homeostasis and signaling in rice;Wenyuan Ruan等;《Mol Plant》;20190417;第12卷(第8期);第1060-1074页 * |
| 大豆与疫霉菌非亲和互作早期差异显示基因的表达分析;李永刚等;《植物保护学报》;20111015(第05期);第31-36页 * |
| 水稻SUMO化E3连接酶SIZ1调控缺磷条件下根的发育和根构型形成;周红敏等;《中国水稻科学》;20150110(第01期);第38-47页 * |
| 玉米泛素结合酶基因家族的生物信息学及表达分析;陈曙等;《南方农业学报》;20180927(第08期);第16-23页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110241121A (en) | 2019-09-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110714013B (en) | Application of soybean E2 ubiquitin-conjugating enzyme gene GmUBC1 | |
| CN110872598B (en) | Cotton drought-resistant related gene GhDT1 and application thereof | |
| CN108998470B (en) | Application of soybean MYB32 transcription factor coding gene GmMYB32 | |
| CN109576283B (en) | Application of soybean GER protein coding gene GmGER12 | |
| CN110241121B (en) | Application of soybean E3 ubiquitin ligase GmNLA1 coding gene | |
| CN114752579A (en) | Application of ZmMAPK protein and coding gene thereof in regulation and control of low-temperature stress tolerance of plants | |
| CN109666677A (en) | The application of soybean PHR transcription factor encoding gene GmPHRa | |
| CN111979253B (en) | TrFQR1 gene, cloning thereof, expression vector construction method and application | |
| CN106867979B (en) | Application of NtRLK2 gene in bacterial wilt resistance of tobacco | |
| CN115073573B (en) | Sweet potato stress resistance related protein IbNAC087, and coding gene and application thereof | |
| CN114774437B (en) | Genetic engineering application of wild soybean NADPH oxidase gene GsRbohA1 | |
| CN114014922B (en) | Protein for regulating and controlling plant salt tolerance, coding gene and application thereof | |
| CN109609510A (en) | The application of soybean PHR transcription factor encoding gene GmPHRb | |
| CN114525298B (en) | Application of soybean protein GmFVE in regulation and control of salt tolerance of plants | |
| CN113136398B (en) | GsHA24 protein and application of related biological material thereof in regulation and control of stress tolerance of plants | |
| CN111676227B (en) | Genetic engineering application of soybean ribosomal protein coding gene GmRPL12 | |
| CN114773444A (en) | Application of thionin protein gene OsThi9 in plant breeding regulation | |
| CN116640193A (en) | Soybean stress resistance related protein GmSQLE1 and application of encoding gene thereof in regulation and control of plant stress resistance | |
| CN111073905B (en) | Application of soybean mitogen-activated protein kinase GmMMK1 coding gene | |
| CN108504664B (en) | Application of soybean cation-excreting protein GmCDF1 coding gene | |
| CN110205325B (en) | Application of soybean VQ motif coding gene GmVQ58 | |
| CN110184253B (en) | Application of CiCPK32 gene of caragana intermedia in regulation and control of plant stress resistance | |
| CN109790547B (en) | Constructs and methods for controlling stomatal closure in plants | |
| CN106349353B (en) | Plant starch synthesis related protein OsFSE (OsFSE) regulation and control, and coding gene and application thereof | |
| CN116355948B (en) | Application of soybean E2 ubiquitin conjugated enzyme GmUBC2 coding gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |