Specific promoter of soybean seeds and use thereof

A soybean seed-specific technology, applied in the field of plant genetic engineering, can solve problems such as the lack of soybean seed-specific promoters

Inactive Publication Date: 2010-09-01
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention provides a soybean seed-specific promote...

Method used

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  • Specific promoter of soybean seeds and use thereof
  • Specific promoter of soybean seeds and use thereof
  • Specific promoter of soybean seeds and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Cloning of the upstream distal fragment SAP1-b of the soyAP1 gene and sequence analysis of SAP1

[0036] 1. Design and synthesis of primers

[0037]According to a 785bp known sequence upstream of the ATG of the soyAP1 gene in the soybean genome, its nucleotide sequence is as described in Sequence NO.2, referred to as SAP1-a, three nested primers SP1-SP3 were designed and synthesized; refer to the paper "Soybean Seed Specific Cloning and sequence analysis of promoters" (Caiyin Qinggele, Li Mingchun, Cai Yi. Acta Crops, 2005, 31(1): 11-17) and "Rapid isolation and functional analysis of promoter sequences of the nitrateresuctase gene from Chlorella ellipsoidea" (Peng Wang, Yongru Sun, Xia Li. Journal of Applied Phycology, 2004, 16: 11-16.) described in the TAIL-PCR method, synthetic random degenerate primers AD1 ~ AD4:

[0038] SP1: 5'-GAAACGCGGTTAGGAAAACAGATGAG-3'

[0039] SP2: 5'-GAGGAATAGGTTTACCGATACGTGGAGA-3'

[0040] SP3: 5'-CAGTAGTGTCACACCTTCCTTCCTCTTT...

Embodiment 2

[0093] Example 2: Cloning of SAP1 fragment and its 5' end deletion fragment

[0094] Sequence analysis of SAP1:

[0095] The known sequence SAP1-a at the proximal 785bp upstream of the soyAP1 gene ATG, and the amplified 865bp upstream distal sequence SAP1-b, totaling 1650bp, were named SAP1. The online software Neural Network Promote Prediction performs basic promoter prediction on soybean SAP1, whose nucleotide sequence is as described in Sequence NO.4. There is a basic promoter sequence at the position of 1144bp-1194bp, and the probabilities are 0.82, 0.78, 0.52, 0.62, 0.77 and 0.75, respectively, and the prediction value of the first two basic promoter regions is higher. According to the basic characteristics of eukaryotic gene promoters, it is speculated that the possible transcription start site is A at 553bp.

[0096] After analyzing the SAP1 sequence, design the PCR primers for this fragment and its 5' end deletion fragment sequence: upstream primer Y1, Y2, Y3, Y4, do...

Embodiment 3

[0115] Example 3: Construction of Transient Expression Vector and Agrobacterium-mediated Infection of Soybean Roots, Stems, Leaves, and Seeds

[0116] The pMD18-T-SAP1, pMD18-T-SAP2, pMD18-T-soyAP1-p, pMD18-T-SAP4 plasmid vectors and pCAMBIA1301 vectors were double-digested with PstI and NcoI respectively, and the first 4 plasmid vectors were digested to obtain The small fragments were ligated with the large fragments obtained by digesting the pCAMBIA1301 vector, respectively, and the expression vectors pCAM-SAP1, pCAM-SAP2, pCAM-soyAP1-p and pCAM-SAP4 which were fused with SAP1, SAP2, soyAP1-p, SAP4 and GUS genes were respectively obtained . The four vector plasmids were also digested and identified with two enzymes, PstI and NcoI, and the results Figure 5 As shown, M: 2000bp DNAMarker; 1, 2, 3, 4: PstI and NcoI double-enzyme-digested expression vectors pCAM-SAP1, pCAM-SAP2, pCAM-soyAP1-p and pCAM-SAP4 respectively, proving that pCAM-SAP1, pCAM -SAP2, pCAM-soyAP1-p and pCA...

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Abstract

The invention relates to a specific promoter of soybean seeds, which belongs to the sequence and use of a soybean aspartic protease gene promoter. The nucleotide sequence of the promoter is represented by the sequence No.1. The preparation method of the promoter comprises the following steps of: cloning the upstream distal sequence of gene soyAP1, analyzing the upstream sequence of the ATG of the gene soyAP1 and determining the deleted fragments SAP1, SAP2, soyAP1-p and SAP4; and amplifying the soyAP1-p fragment, linking the amplified fragment of the soyAP1-p with a cloning vector pMD 18-T, performing identification, measuring the sequence, and verifying the sequence. In the invention, the soyAP1-p promoter of soybean is cloned and expressed in the soybean seeds specifically, and the promoter obtained can be used as a favorable tool in researches on soybean transgenosis and particularly creates conditions for researching and developing soybean seed bioreactors.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to the sequence of soybean aspartic acid protease gene promoter and its application. Background technique [0002] Soybean is an important economic crop in my country. The grain is rich in protein, lipid and various nutrients. The phosphorus, iron and calcium minerals are dozens of times higher than other crops, and contain many vitamins, especially soybean contains The 8 essential amino acids synthesized are unmatched by other cereal crops. Soybean is not only the main source of protein and lipids for human beings, but also has a very obvious role in health care. Therefore, using soybean seeds as bioreactors and using plant genetic engineering methods to produce new soybean varieties with industrial uses and medicinal values ​​from transgenic soybean seeds has become a research hotspot. [0003] To increase or decrease the content of certain substances in transgenic soybea...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10
Inventor 王庆钰赵艳李景文王英钱丹丹程浩张庆林王洪预潘肃
Owner JILIN UNIV
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