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Seed-specific promoter separated from soybean and applications thereof

A soybean seed, specific technology, applied in the biological field, can solve the problem of lack of soybean seed specific promoter and the like

Inactive Publication Date: 2011-08-03
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention provides a seed-specific promoter sequence isolated from soybean and its application to solve the problem of lack of soybean seed-specific promoter at present

Method used

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  • Seed-specific promoter separated from soybean and applications thereof
  • Seed-specific promoter separated from soybean and applications thereof
  • Seed-specific promoter separated from soybean and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Cloning of the upstream distal fragment of the stearic acid-ACP desaturase gene and sequence analysis of SACPD-Cp

[0045] 1. Design and synthesis of primers

[0046] According to a known sequence upstream of the stearic acid-ACP desaturase gene ATG in the soybean genome (GeneBank assessment AK245255), three nested primers B1, B2, and B3 were designed and synthesized; refer to the paper "Clone of Soybean Seed-Specific Promoter and sequence analysis" (Caiyin Qinggele, Li Mingchun, Cai Yi. Journal of Crops, 2005, 31 (1): 11-17) and "Rapid isolation and functional analysis of promoter sequences of the nitrate resuctase gene from Chlorella ellipsoidea "(Peng Wang, Yongru Sun, Xia Li. Journal of Applied Phycology , 2004,16:11-16.) in the TAIL-PCR method, synthesize random degenerate primers AD1~AD6:

[0047] B1: 5'-TGGAACTGTGGAGAGGTTGGAG-3'

[0048] B2: 5'-GGTGTGACTAGTGATTAGCCCTTG-3'

[0049] B3: 5'-GTGGAAATTTGGCCCGATACCT-3'

[0050] AD1: 5'- NTCGA(G / C) T...

Embodiment 2

[0099] Example 2: SACPD-Cp Fragment analysis and cloning of 5'-deleted fragments

[0100] Sequence analysis of SACPD-Cp:

[0101] SACPD-C gene ATG upstream sequence 2081bp, named SACPD-Cp. The online software Neural Network Promote Prediction performs bioinformatics analysis on soybean SACPD-Cp, its nucleotide sequence analysis is as shown in Figure 4, and the basic promoter prediction is carried out, at 411bp-461bp, 1146bp-1196bp, 1530bp-1580bp, 1988bp- There are basic promoter sequences at 2038bp and 2021bp-2071bp, the probabilities are 0.95, 0.83, 0.90, 0.97 and 0.94, respectively, and the latter two basic promoter regions have higher predicted values. According to the basic characteristics of eukaryotic gene promoters, it is speculated that the possible transcription start site is A at 2062bp.

[0102] After analyzing the promoter element of the SACPD-Cp sequence, five deletion vectors were designed, such as Figure 5 Shown: respectively, design five PCR primers wit...

Embodiment 3

[0126] Embodiment 3: The construction of the expression vector of each deletion fragment of SACPD-Cp fragment

[0127] The pMD18-T-SACPD-Cp, pMD18-T-AC1, pMD18-T-AC2, pMD18-T-AC3, pMD18-T-AC4, pMD18-T-AC5 plasmid vectors and pCAMBIA1301 vectors were carried out with EcoRI and NocI respectively enzyme digestion, the small fragments obtained by digestion of the first 6 plasmid vectors were respectively connected with the large fragments obtained by digestion of pCAMBIA1301 vector, and SACPD-Cp, AC1, AC2, AC3, AC4, AC5 and GUS Gene fusion expression vectors pCAM-SACPD-Cp, pCAM-AC1, pCAM-AC2, pCAM-AC3, pCAM-AC4 and pCAM-AC5. These 6 vector plasmids were digested and identified by bacterial liquid PCR and double enzyme digestion respectively, and the results of bacterial liquid PCR Figure 7 As shown, M: 2000bp DNA Marker; 1, 2, 3, 4, 5, 6 are expression vectors pCAM-SACPD-Cp, pCAM-AC1, pCAM-AC2, pCAM-AC3, pCAM-AC4 and pCAM-AC5 respectively; Digestion results Figure 8 Shown:...

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Abstract

The invention relates to the technical field of biology, in particular to a seed-specific promoter sequence separated from soybean and applications thereof. The nucleotide sequence is shown in SEQ No.1. The seed-specific promoter sequence is used as the seed-specific promoter of soybean. In the invention, the stearic acid-acyl carrier protein (ACP) desaturase promoter of soybean is cloned and the tissue expression characteristics and expression efficiency of the promoter in soybean are researched, thus an evidence for the effective use of the promoter is provided; and the promoter provides a good tool for the transgenic research of soybean and creates conditions for the researches and developments by utilizing the soybean seed as bioreactor.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the sequence cloning and application of soybean stearic acid-ACP desaturase gene promoter. Background technique [0002] Soybean is an important food crop and oil crop, and there are more than 70 million hectares of planting area in the world. Soybean grains are rich in protein, lipids and a variety of nutrients. The phosphorus, iron, and calcium minerals are dozens of times higher than other crops, and contain a variety of vitamins. In particular, soybeans contain 8 essential amino acids that cannot be synthesized by the human body. It is unmatched by other cereal crops. Soybean is not only the main source of human protein and lipid, but also plays an important role in health care. [0003] Although there are many controversies about genetically modified soybeans, they have many advantages in application compared with traditional soybeans. Increase or reduce the content of certai...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10A01H5/00
Inventor 王庆钰张庆林赵艳王英李景文李晓薇翟莹张艳程浩
Owner JILIN UNIV
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