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Soybean phosphorus starvation transcription factor GmWRKY75, encoded protein and application of phosphorus starvation transcription factor GmWRKY75

A technology of transcription factors and phosphorus starvation, applied in the field of genetic engineering, can solve problems affecting Pi absorption and other issues

Inactive Publication Date: 2016-12-21
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is further speculated that WRKY75 acts as a constitutive negative regulator of root development to affect Pi uptake

Method used

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  • Soybean phosphorus starvation transcription factor GmWRKY75, encoded protein and application of phosphorus starvation transcription factor GmWRKY75
  • Soybean phosphorus starvation transcription factor GmWRKY75, encoded protein and application of phosphorus starvation transcription factor GmWRKY75
  • Soybean phosphorus starvation transcription factor GmWRKY75, encoded protein and application of phosphorus starvation transcription factor GmWRKY75

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Structural analysis of GmWRKY75

[0044] GmWRKY75 is located on chromosome 16 of soybean at Gm16 (5 308 516-5 311 399), with 2 exons and 1 intron. Protein structure analysis (SMART, http: / / smart.embl-heidelberg.de / ) found that, such as figure 1 As shown in A, the N-terminus of GmWRKY75 has a WRKYGQK domain and a C 2 h 2 -type zinc finger structure.

[0045] Using the amino acid sequence of GmWRKY75 as a probe, using the BLASTp tool of NCBI, the WRKY75 proteins in Arabidopsis, Gossypium raimondii, Medicago truncatula, Oryza sativa and Phaseolus vulgaris were obtained: AT5G13080, Gorai013G179400, Medtr7g028415, Os0280Phulv0680. Such as figure 1 As shown in B, multiple alignments found that these WRKY75 proteins all contain a WRKYGQK domain and a C-X 4 -C-X 23 -H-X 1 -H zinc finger structure. After searching the literature, all WRKY proteins that have been reported to be involved in the regulation of plant phosphate starvation were retrieved: GmWRKY75, A...

Embodiment 2

[0046] Example 2: Cloning of Soybean Phosphorus Starvation Transcription Factor GmWRKY75

[0047] (1) Design primers, extract RNA, reverse cDNA:

[0048] RNAiso Reagent (purchased from TaKaRa) was used to extract total RNA from the root of soybean variety Kefeng No. 1 treated with low phosphorus for 5 days, and the integrity of RNA was detected by 1% agarose electrophoresis; cDNA was synthesized according to the SYBR PrimeScript RT-PCR Kit II reagent of TaKaRa Company box instructions. The primer sequence of the amplified gene GmWRKY75 is:

[0049] Seq ID NO.3: GmWRKY75-F 5'-ATGGAGAATTATTCTATGTTGTTCC-3';

[0050] Seq ID NO.4: GmWRKY75-R 5'-AAAGGGAGTGTATATTTTCATC-3'.

[0051] (2) PCR amplification, the specific steps are as follows:

[0052] Step 1: Prepare PCR reaction solution (50 μL system) according to the following components: ExTaq enzyme mix (25 μL), GmWRKY75-F (1 μL), GmWRKY75-R (1 μL), cDNA (1 μL), ddH 2 O (22 μL);

[0053] Step 2: Set the reaction program as fol...

Embodiment 3

[0055] Example 3: Fluorescence quantitative analysis of soybean phosphorus starvation transcription factor GmWRKY75

[0056] (1) Select the cDNA samples of soybean roots and leaves with processing time of 0h, 12h, 1d, 2d and 3d as materials, and the fluorescence quantitative PCR (qRT-PCR) kit selected is IQ SYBR Green (Bio-Rad, Hercules ,CA,USA);

[0057] (2) Design primers

[0058] The fluorescent quantitative specific primers designed for the GmWRKY75 gene sequence are:

[0059] Seq ID NO.5: Upstream primer 5'-TGTGAGTGATGAGTTAGGTGGTTCC-3';

[0060] Seq ID NO.6: Downstream primer 5'-AACAGCTTTTTGGCCATACTTCCTC-3'.

[0061] The internal reference gene was Tubulin, and the primer sequences were as follows:

[0062] Seq ID NO.7: Upstream primer 5'-GGAGTTCACAGAGGCAGAG-3';

[0063] Seq ID NO.8: Downstream primer 5'-CACTTACGCATCACATAGCA-3'.

[0064] (3) Fluorescent quantitative PCR amplification, the specific steps are as follows:

[0065] Step 1: Prepare a PCR reaction system...

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular discloses a phosphorus starvation transcription factor GmWRKY75, an encoded protein and an application of the phosphorus starvation transcription factor GmWRKY75. The nucleotide sequence of the gene is as shown in Seq ID NO.1. According to the invention, it discovers that by transforming plant cells by virtue of any one plant expression vector in which the exogenous gene GmWRKY75 can be imported, a transgenic plant for over-expression of the GmWRKY75 can be obtained, and in comparison with a non-transgenic plant, the total phosphorus content of the transgenic plant is significantly increased and the development of lateral roots and root hair is obviously inhibited. The gene disclosed by the invention, as a target gene, can be imported into plants, so that the absorption of phosphorus element to transgenic plants is enhanced; therefore, the gene has an important significance for breeding a soybean variety which achieves the efficient utilization of phosphorus.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a soybean phosphorus starvation transcription factor GmWRKY75, encoded protein and application thereof. Background technique [0002] Phosphorus is a very important element among the nutrients necessary for plants, and it participates in important physiological and biochemical processes such as plant biosynthesis, signal transduction, and energy transfer. Soybean roots can only absorb phosphorus that exists in the form of inorganic phosphorus ions in the soil. However, due to weathering, erosion, and oxidation and fixation of metal ions, the available phosphorus content in soil that can be used by soybeans is relatively low. The growth and development of plants is actually a dynamic equilibrium process in which endogenous signals and environmental factors are constantly interacting and utilizing each other; this depends on the plant's perception of the ext...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00C12N15/11
CPCC07K14/415C12N15/11C12N15/8227C12N15/8271C12N2310/10C12N2800/60
Inventor 杨守萍张璟曜徐影周汐
Owner NANJING AGRICULTURAL UNIVERSITY
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