Soybean phosphate-starvation negative regulatory gene GmSPX1, and coding protein and application thereof

A technology of phosphorus starvation and negative regulation, applied in the field of genetic engineering, can solve the problems of increased plant total phosphorus content, increased PSI gene expression, and low affinity

Inactive Publication Date: 2016-04-20
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] Similar results were obtained in the study of rice OsSPX; although the expression level of OsSPX4 did not change significantly under the condition of phosphorus starvation stress, it was found that the expression level of the PSI gene in the OsSPX4 mutant was significantly increased, and the plant total phosphorus The content will also increase significantly; experiments such as yeast two-hybrid experiment and BiFC found that OsSPX4 can interact with OsPHR2, and under normal phosphorus environment, OsSPX4 can inhibit the activity of OsPHR2, but under low phosphorus enviro

Method used

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  • Soybean phosphate-starvation negative regulatory gene GmSPX1, and coding protein and application thereof
  • Soybean phosphate-starvation negative regulatory gene GmSPX1, and coding protein and application thereof
  • Soybean phosphate-starvation negative regulatory gene GmSPX1, and coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Cloning of Soybean Phosphorus Starvation Negative Regulatory Gene GmSPX1

[0042] (1) Design primers, extract RNA, reverse cDNA:

[0043] RNAisoReagent (purchased from TaKaRa) was used to extract total RNA from the roots of soybean variety Williams82 treated with low phosphorus for 5 days, and the integrity of the RNA was detected by 1% agarose electrophoresis; The primer sequence of the amplified gene GmSPX1 is:

[0044] Seq ID NO.3: GmSPX1-F5'-ATGAAGTTTGGGAAGAGACT-3';

[0045] Seq ID NO.4: GmSPX1-R5'-ACAATAGGAACTGCAGCATTG-3'.

[0046] (2) PCR amplification, the specific steps are as follows:

[0047] Step 1: Prepare PCR reaction solution (50 μL system) according to the following components: ExTaq enzyme mix (25 μL), GmSPX1-F (1 μL), GmSPX1-R (1 μL), cDNA (1 μL), ddH 2 O (22 μL);

[0048] Step 2: Set the reaction program as follows: 95°C, 5min; (94°C, 30sec; 58°C, 30sec; 72°C, 60sec; 30 cycles); 72°C, 10min; store at 4°C;

[0049] Step 3: PCR products ...

Embodiment 2

[0050] Example 2: Fluorescent quantitative analysis of soybean phosphorus starvation negative regulator gene GmSPX1

[0051] (1) The cDNA samples of soybean roots and leaves with treatment times of 0d, 1d, 5d, 10d and R-11d (recovery treatment 1 day) were selected as materials, and the fluorescent quantitative PCR (qRT-PCR) kit selected was IQSYBRGreen (Bio-Rad, Hercules, CA, USA);

[0052] (2) Design primers

[0053] The fluorescent quantitative specific primers designed for the GmSPX1 gene sequence are:

[0054] SeqID NO.5: Upstream primer 5'-TGAATGATCCTGCTCCAGTTT-3';

[0055] Seq ID NO.6: Downstream primer 5'-TATCTTCAACGGTGGCAATG-3'.

[0056] The internal reference gene was Tubulin, and the primer sequences were as follows:

[0057] SeqID NO.7: Upstream primer 5'-GGAGTTCACAGAGGCAGAG-3';

[0058] Seq ID NO.8: Downstream primer 5'-CACTTACGCATCACATAGCA-3'.

[0059] (3) Fluorescent quantitative PCR amplification, the specific steps are as follows:

[0060] Step 1: Prepar...

Embodiment 3

[0064] Example 3: Expression analysis of soybean GmSPX1 gene in Arabidopsis

[0065] (1) Utilize Gateway technology to construct plant expression vector pEarleygate103-GmSPX1 (such as image 3 A, B shown). Design specific primers according to the instructions of Gateway;

[0066] SeqIDNO.9: The upstream primer is as follows

[0067] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAAGTTTGGGAAGAGACT-3'

[0068] SeqIDNO.10: Downstream primers are as follows

[0069] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCACAATAGGAACTGCAGCATTG-3';

[0070] (2) Using the PCR amplification product obtained in Example 1 as a template, carry out PCR amplification, and the specific operation is the same as in Example 1, and then carry out BP reaction and LR reaction successively;

[0071] BP reaction: Prepare the reaction solution according to the following components and add it to a 200 μL EP tube: GmSPX1 (3 μL), pDONR221 (1 μL), ddH 2 O (4 μL); quickly vortex BPClonase enzyme once, add 2 μL to the above reactio...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses a phosphate-starvation negative regulatory gene GmSPX1, and a coding protein and application thereof. The nucleotide sequence of the gene is disclosed as Seq ID NO.1. The invention detects that any plant expression vector capable of inserting the exogenous gene GmSPX1 can be used for transforming plant cells to obtain the GmSPX1-overexpressed transgenic plant. Compared with the non-transgenic plant, the complete phosphorus content of the transgenic plant is obviously reduced, the phosphate-starvation related gene is obviously lowered, the root system morphology is obviously changed, and the phosphorus poisoning phenomenon of the mutant is relieved. The gene disclosed by the invention can be inserted into the plant as a target gene, enhances the phosphorus in-vivo metabolic balance capacity of the transgenic plant, and thus, has important meanings for culturing phosphorus-efficient-utilization soybean varieties.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a soybean phosphorus starvation negative regulation gene GmSPX1, encoded protein and application thereof. Background technique [0002] Among the 17 elements necessary for plant growth and development, phosphorus (P) is a very important one. P element is inseparable from plant cell activities including ribonucleic acid synthesis, cell membrane construction and ATP synthesis. Even many complexes involved in enzymatic reactions and signal transduction processes must contain P elements. Plant roots can only obtain the necessary P element by absorbing inorganic phosphate (Pi) in the soil. However, due to the negative charge of the soil, Pi is easily leached, and most of the Pi in the soil is either converted into organic compounds by microorganisms, or becomes insoluble in water by interacting with cations, resulting in a large amount of P being fixed. Cann...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/11A01H5/00
CPCC07K14/415C12N15/8243
Inventor 杨守萍张璟曜周汐韩少怀姚敏磊
Owner NANJING AGRICULTURAL UNIVERSITY
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