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Soybean purple acid phosphatase GmPAP36 and encoding gene and application thereof

A technology of acid phosphatase and soybean, which is applied in the field of genetic engineering and can solve the problems that functional research has not been carried out in depth

Active Publication Date: 2016-06-08
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, GmPAP1 and GmPAP2 are induced and expressed under low phosphorus conditions, but their functional studies have not been carried out in depth (LeBansky et al., 1992; Schenketal., 1999; Liaoetal., 2003)

Method used

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  • Soybean purple acid phosphatase GmPAP36 and encoding gene and application thereof
  • Soybean purple acid phosphatase GmPAP36 and encoding gene and application thereof
  • Soybean purple acid phosphatase GmPAP36 and encoding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Molecular cloning of embodiment 1GmPAP36 gene

[0041] 1. Soybean seedling cultivation and phosphorus treatment: select plump and uniform soybean variety 'Zhonghuang 15' seeds for germination, after germination, select materials with consistent germination and sow them on quartz sand, and transplant them to vermiculite after 3 days. After the opposite true leaves were unfolded, phytate phosphorus (1.0mmol / L) was treated, and KH was used as the control 2 PO 4 Treatment, the concentration is 1.0mmol / L. Samples were taken after 21 days of phosphorus treatment, and the samples were quickly frozen in liquid nitrogen and stored in a -80°C low-temperature refrigerator until use.

[0042] 2. Material processing for real-time quantitative PCR analysis: select plump, uniform seeds of high-efficiency phosphorus soybean 'Zhonghuang 15' and low-efficiency phosphorus soybean 'Niufahuang' to sow in vermiculite, select plant root samples for analysis after 7 days, and record 0d; fol...

Embodiment 2

[0057] Expression analysis of embodiment 2GmPAP36 gene in different soybean varieties

[0058] With GmPAP36 as the target gene and soybean constitutively expressed Actin11 gene as the internal control, real-time quantitative PCR primers GmPAP36-F2 and GmPAP36-R2 were designed, using Ⅰ Fluorescent dye method for real-time quantitative PCR, by comparing C t GmPAP36 gene expression analysis was performed.

[0059] GmPAP36-F2: 5'-ATGACAGAACCACAGCCAAAGTAT-3'

[0060] GmPAP36-R2: 5'-CAGCAACTCCATCTTGATTTCG-3'

[0061] Relative expression of GmPAP36 gene in roots of different soybean varieties=2 –△△Ct ,

[0062] where ΔΔC t =(C tGmPAP36 –C tActin11 ) 植酸磷处理 –(C tGmPAP36 –C tActin11 ) 对照处理

[0063] The results of differential expression of GmPAP36 gene in the roots of 'Zhonghuang 15' (a soybean variety with high phosphorus efficiency) and 'Niu Maohuang' (a soybean variety with low phosphorus efficiency) under phytate phosphorus treatment are shown in figure 2 .

[0064] ...

Embodiment 3

[0065] The prokaryotic expression of embodiment 3GmPAP36 gene

[0066] 1. Analyze the sequence of GmPAP36 and prokaryotic expression vector pET-32a(+), design primers GmPAP36-F3 and GmPAP36-R3 (GmPAP36-F3: 5'-GGATCCGACAGCGATGTCTTTGC-3', GmPAP36-R3: 5'-GAGCTCTGAAACATGAGCAGTGGAA-3' ), to amplify the GmPAP36 sequence containing a suitable restriction site, connect the sequence to the pMD-T vector to form the intermediate recombinant pMD-GmPAP36, and sequence it to determine its sequence correctness. Use BamHI and SacI to digest pMD-GmPAP36 and prokaryotic expression vector pET-32a(+), respectively, and recover the target fragment. The enzyme digestion reaction system is as follows:

[0067]

[0068]

[0069] 2. Ligate the recovered pET-32a(+) expression vector with the GmPAP36 restriction enzyme fragment, and the reaction system is as follows:

[0070]

[0071] After mixing, connect at 16°C for 12-16 hours.

[0072] 3. Induced expression of fusion recombinant protein: ...

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Abstract

The invention provides soybean purple acid phosphatase GmPAP36 and an encoding gene and application thereof. A low-phosphorous induction soybean root system cDNA subtractive library is established based on the suppression subtractive hybridization technique, low-phosphorous stress inducible expression acid phosphatase candidate EST is selected from the subtractive library, and on this basis, an acid phosphatase candidate gene sequence is cloned and the expression difference of a candidate gene under the phytate phosphorus processing condition is analyzed with the real-time quantitative PCR technique; a gene prokaryotic expression vector is established to achieve gene prokaryotic expression; meanwhile, a gene eukaryotic expression vector is established, arabidopsis and soybean conversion is conducted to obtain transgenosis positive plants, the biological function of the gene GmPAP36 is analyzed, and functional genes are provided for efficient transgenosis breeding of plant phosphorus.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to soybean purple acid phosphatase GmPAP36, its coding gene and application. Background technique [0002] 50%-80% of soil phosphorus exists in organic form, and phytate phosphorus accounts for more than half; and most of phytate phosphorus is insoluble in water and cannot be directly absorbed and utilized by plants (Lungetal., 2006; Bilyeutal., 2008; Georgeetal., 2008). At the same time, most of the phosphorus fertilizer applied to the soil every year will be fixed in the soil due to adsorption, sedimentation and transformation into organic form, which cannot be absorbed by the plants of the season (Baek et al., 2013; Wang et al., 2013). According to statistics, my country uses about 11 million tons of pure phosphorus every year, accounting for about 30% of the world's total consumption, but the seasonal utilization rate of phosphorus fertilizers is only 10% to 15%, wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/82A01H5/00
CPCC12N9/16C12N15/8242C12Y301/03002
Inventor 张彩英李喜焕孙星段鹏博孔佑宾杜汇李文龙
Owner HEBEI AGRICULTURAL UNIV.
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