Soybean gene gmacp2, encoded protein and its application for tolerance to low phosphorus

A low-phosphorus, soybean-resistant technology, applied in the field of genetic engineering

Inactive Publication Date: 2020-01-31
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, in soybean, copies of other members of the ACP gene family have not been reported

Method used

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  • Soybean gene gmacp2, encoded protein and its application for tolerance to low phosphorus
  • Soybean gene gmacp2, encoded protein and its application for tolerance to low phosphorus
  • Soybean gene gmacp2, encoded protein and its application for tolerance to low phosphorus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Cloning of soybean low phosphorus tolerance related gene GmACP2

[0038] (1) Design primers, extract RNA, reverse cDNA:

[0039] Plant total RNA extraction kit (DP432, Tiangen) was used to extract total RNA from leaves of soybean material Nannong 94-156 (low phosphorus tolerant soybean germplasm), and the integrity of RNA was detected by 1% agarose gel electrophoresis. Synthesis of cDNA refers to TaKaRa PrimerScript TM The RT reagent kit with gDNA Eraser kit explains the operation. Design primers as follows:

[0040] Seq ID NO.3: GmACP2-F 5'-ATGTCTGGAATTGTGGTT-3';

[0041] Seq ID NO.4: GmACP2-R 5'-TTAAGCAGAGACAGACAG-3'.

[0042] (2) PCR amplification, the specific steps are as follows:

[0043] Step 1: Prepare PCR reaction solution (50μl system) according to the following components: 10×PCR Buffer (25μl), ddH 2 O (9 μl), dNTP (10 μl), GmACP2-F (1.5 μl), GmACP2-R (1.5 μl), cDNA (2 μl), KOD FX enzyme (1 μl);

[0044] Step 2: The reaction was carried out ...

Embodiment 2

[0046] Example 2: Fluorescent quantitative analysis of soybean low phosphorus tolerance related gene GmACP2

[0047] (1) Soybean low-phosphorus-tolerant varieties Nannong 94-156 and B20 and soybean low-phosphorus stress-sensitive materials Bogao and B18 were subjected to two treatments of normal growth and low-phosphorus stress under hydroponic growth conditions, and the normal condition was 1 / 2 Hogland nutrient solution, stress treatment is 1 / 2 Hogland nutrient solution (wherein replace KH with KCl 2 PO 4 ), 7 days later, the root and leaf samples were collected for quick freezing in liquid nitrogen and stored at -80°C; the extraction of total RNA and the inversion of cDNA were the same as in Example 1;

[0048] (2) Design primers

[0049] The fluorescent quantitative specific primers designed for the GmACP2 gene sequence are:

[0050] Seq ID NO.5: Upstream primer 5'-GGTGGACATCTATCAAAAACAAATACAT-3';

[0051] Seq ID NO.6: downstream primer 5'-TTTATCCTTTTTGTGGCAATTCCTTAT-3'. ...

Embodiment 3

[0059] Embodiment 3: the acquisition of the soybean hairy root of transgenic GmACP2

[0060] (1) Select the XhoI and XbaI restriction sites, and insert the full-length sequence of GmACP2 cloned in Example 1 forward into pFGC5941 (such as figure 2 Shown) behind the CaMV35S promoter, construct the recombinant plant expression vector pFGC5941-GmACP2 (such as image 3 shown).

[0061] (2) Transform pFGC5941-GmACP2 and empty plasmid pFGC5941 into Agrobacterium rhizogenes strain K599 (BioVector NTCC Inc.) by freeze-thaw method, and pFGC5941-GmACP2 and pFGC5941 were transformed by Agrobacterium rhizogenes K599-mediated genetic transformation Systematic transformation of soybeans, the specific method is as follows:

[0062] Step 1: Seedling cultivation: select uniform soybean seeds, sterilize them with chlorine gas for 15 hours, and place them in a light incubator at 25°C for 12 hours / d to raise seedlings with vermiculite;

[0063] Step 2: Induction of hairy roots: Streak the stra...

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Abstract

The invention belongs to the technical field of genetic engineering and particularly discloses a soybean low-phosphorus-resistance related gene GmACP2, encoded protein and application of the GmACP2. The nucleotide sequence of the gene is shown by the Seq ID NO. 1. The expression quantity of the gene GmACP2 in the low-phosphorus-resistance material is remarkably higher than that of the low phosphorus sensitive material; the carrier which can guide expression of an exogenous gene in the plant is used, the gene GmACP2 is guided into the soybean hairy root, and the soybean phosphorus content, the dry matter accumulation and the phosphorus absorption and utilization ratio can be remarkably improved. The disclosed gene can be guided into the plant as a target gene, the phosphorus in-vitro metabolism balance capability of a transgenic plant is improved, and great significance is provided for cultivating soybean varieties with efficient phosphate use.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a gene GmACP2 related to resistance to low phosphorus, encoded protein and application thereof. Background technique [0002] Soybean is the main source of human edible oil and vegetable protein. However, soybean is also a crop that requires a large amount of phosphorus, and the phosphorus content of its grain is much higher than that of rice, wheat and corn. Phosphorus deficiency in soybeans will not only affect the growth and development of plants, increase the shedding of flower pods, but also affect the formation of root nodules, thereby reducing the efficiency of nitrogen fixation, and ultimately affecting its yield and quality. Therefore, the lack of soil phosphorus has become an important factor limiting the development of soybean production. In order to adapt to the low-phosphorus environment, plants have formed a series of adaptive mechanisms to c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/16C12N15/82C12N15/11A01H5/00A01H6/54
CPCC12N9/16C12N15/8201C12N15/8271C12Y301/03002
Inventor 张丹褚姗姗马兴立
Owner HENAN AGRICULTURAL UNIVERSITY
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