Function of purple acid phosphatase GmPAP33 gene for promoting reuse of phosphorus in soybean mycorrhiza

An acid phosphatase and gene technology, applied in the field of genetic engineering, can solve problems such as unclear specific functions, and achieve the effect of improving the ability of efficient phosphorus utilization and improving the phosphorus efficiency of plants

Inactive Publication Date: 2015-09-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently those skilled in the art only know that under the induction of AM fungi, the purple acid phosphatase gene GmPAP33 However, the specific function of this gene in phosphorus metabolism is not clear

Method used

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  • Function of purple acid phosphatase GmPAP33 gene for promoting reuse of phosphorus in soybean mycorrhiza
  • Function of purple acid phosphatase GmPAP33 gene for promoting reuse of phosphorus in soybean mycorrhiza
  • Function of purple acid phosphatase GmPAP33 gene for promoting reuse of phosphorus in soybean mycorrhiza

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Embodiment 1

[0032] GmPAP33 Gene expression pattern analysis: In the published soybean genome database, through homologous comparison, it was predicted that there are 35 members of soybean purple acid phosphatase, which were determined by quantitative PCR technology. GmPAP33 Genes are strongly expressed genes in mycorrhizae. GmPAP33 The length of the open reading frame of the gene is 1290 bp, encoding a protein of 429 amino acids. Studies have shown that this gene is closely related to soybean arbuscular mycorrhizal fungal infection, and its expression pattern was first analyzed.

[0033]Two soybean varieties were used, phosphorus low-efficiency local 2 (BD2) and phosphorus-high efficiency Yuechun 03-3 (YC03-3). Set 3 AMF inoculation treatments as non-inoculation (-AM) and inoculation with AM fungi Rhizophagus irregularis or AM fungi Glomus mosseae . Set up two phosphorus treatments, 5 μM KH 2 PO 4 As low phosphorus (-P) and 250 μM KH 2 PO 4 K as high phosphorus (+P), low phosph...

Embodiment 2

[0043] 1, GmPAP33 Gene cloning: using soybean flowering leaf cDNA as template, using upstream specific primer F1: 5'- GAGCTC ATGGGGATGCGAATG -3' (SEQ ID NO:5) and downstream specific primer R2: 5'- TCTAGA CTATTTACAGGAAGGAAT -3' (SEQ ID NO: 6) amplification GmPAP33 The ORF full-length sequence of the gene is 1290bp, and sequenced and compared, successfully obtained GmPAP33 A coding sequence that encodes a protein of 429 amino acids.

[0044] 2, GmPAP33 Gene promoter cloning, vector construction and tissue expression localization analysis: Promoter analysis Construction of expression vector: According to conventional methods, extract genome DNA of soybean YC03-3 genotype at the flowering stage, use soybean leaf genome DNA as template, and use upstream Specific primer F2: 5'-CCG GAATTC GAAAGCGTTGGGCTTAAAC-3' (SEQ ID NO: 7) and downstream specific primer R2: 5'-CTAG TCTAGA Amplification of TGTTTTCAAATTTCGGTGC-3' (SEQ ID NO:8) GmPAP33 The 2019 bp fragment of the pr...

Embodiment 3

[0048] 1. Overexpression GmPAP33 The construction of the vector: using the cDNA of the leaf part of the flowering stage of the soybean YC03-3 genotype as a template, using the upstream specific primer F1: 5'- GAGCTC ATGGGGATGCGAATG-3' (SEQ ID NO:5) and downstream specific primer R1: 5'- TCTAGA CTATTTACAGGAAGGAAT-3' (SEQ ID NO: 6) amplification GmPAP33 The full-length sequence of ORF is 1209 bp. After the PCR recovery fragment is sequenced correctly, pass Sac I and Xba After performing double enzyme digestion on the fragment and the target vector, the GmPAP33 The gene was connected to the destination vector pTF101.1. Transform Escherichia coli DH10B, transform Agrobacterium tumefaciens EHA101 after the sequence is correct, and use for Agrobacterium tumefaciens-mediated transformation of whole soybean cotyledon nodes.

[0049] 2. Interference expression GmPAP33 Construction of the vector: using the cDNA of the leaf at the flowering stage of the soybean YC03-3 genotyp...

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Abstract

The invention belongs to the field of gene engineering, particularly discloses a function of purple acid phosphatase GmPAP33 gene for promoting reuse of phosphorus in soybean mycorrhiza, and proves a function of purple acid phosphatase GmPAP33 gene in phosphorus metabolism for the first time. In a root system, GmPAP33 genes are mainly located in root cells containing mycorrhizal arbuscular structures. The GmPAP33 genes have the effect on the reuse of phosphorus in soybean mycorrhiza; through the over-expression of GmPAP33, the soybean biomass and the phosphorus absorption quantity can be increased, so that the plant phosphorus efficiency is improved accordingly. Through the transgenic technology of the GmPAP33 genes, the efficient phosphorus utilization capability of mycorrhizal crops can be improved, and phosphorus-efficient plant materials can be cultured, so that new purposes of fertilizer saving and high yield can be realized, and important theoretical and practical significances are provided for development of the environmental-friendly sustainable agriculture.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular, to the functional research and application of a gene, and more specifically, to the purple acid phosphatase gene GmPAP33 Function in promoting phosphorus reutilization in soybean mycorrhizae. Background technique [0002] Phosphorus is an essential nutrient element for plant growth and development, and it is also one of the important factors limiting crop production. Because phosphorus is easily fixed in the soil but difficult to absorb, soil phosphorus deficiency is very common. During the long-term evolutionary process, plants have developed many adaptive mechanisms to improve phosphorus utilization in the soil, including changes in root morphology and configuration, changes in the activity of acid phosphatase secreted in vivo and exocrine, and rhizosphere microbial symbiosis, etc. (Wang et al., 2010). Among them, acid phosphatase, as a multifunctional enzyme, plays ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/55A01H5/00
Inventor 王秀荣廖红周佳李成晨张海燕
Owner SOUTH CHINA AGRI UNIV
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