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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front. Agric. China    2007, Vol. 1 Issue (1) : 17-23     DOI: 10.1007/s11703-007-0003-1
Research article |
Isolation and sequencing analysis on the seed-specific promoter from soybean
Qinggele CAIYIN,Mingchun LI,Dongsheng WEI,Yi CAI,Laijun XING()
Tianjin Key Laboratory of Microbial Functional Genomics, Department of Microbiology, Nankai University, Tianjin300071, China
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Keywords soybean      seed-specific promoter      motif      TAIL PCR     
Issue Date: 22 February 2016
 Cite this article:   
Qinggele CAIYIN,Mingchun LI,Dongsheng WEI, et al. Isolation and sequencing analysis on the seed-specific promoter from soybean[J]. Front. Agric. China, 2007, 1(1): 17-23.
Reagent Amount in primary reaction mixture/µL Amount in secondary reaction mixture/µL Amount in tertiary reaction mixture/µL
10xPCR buffer 2 2 5
2 mmol/L dNTPs 2 2 5
10 mmol/L AD 4 4 10
1 mmol/L GSP 4 4 10
ddH2O 6.5 6.5 17
Taq Pol (2 U/µL) 0.5 0.5 2
Template 1(100 nmol) 1(1/50 primary PCR product) 1(1/50 secondary PCR product)
Table 1  The compose of TAIL PCR reaction mixture
Reaction File No. Thermal cycling condition Cycle No.
Primary 1 95°C 2 min, 7°C 1 min 1
2 95°C 15s, 65°C 15s, 72°C 30s 5
3 95°C 15s, 5°C 3 min, ramping to 72°C over 3 min, 72°C 2 min 1
4 95°C 15s, 44°C 15s, 72°C 30s 3
95°C 10s, 65°C 15s, 72°C 30s
5 95°C 10s, 65°C 15s, 72°C 30s 12
95°C 10s, 44°C 15s, 72°C 30s
6 72°C 7 min 1
95°C 10s, 61°C 15s, 72°C 30s
7 95°C 10s, 61°C 15s, 72°C 30s 15
Secondary 95°C 10s, 48°C 15s, 72°C 30s
8 72°C 7 min 1
95°C 10s, 65°C 15s, 72°C 30s
Tertiary 9 95°C 10s, 65°C 15s, 72°C 30s 15
95°C 10s, 48°C 15s, 72°C 30s
10 72°C 7 min 1
Table 2  TAIL PCR procedure
Fig. 1  Cloning of BCSP489

1: PCR product BCSP4892: DNA Marker

Fig. 2  TAIL PCR product

1: DNA Marker 2: Primary TAIL PCR product 3: Secondary TAIL PCR product 4:Tertiary TAIL PCR product

Fig. 3  Cloning of BCSP666

1: PCR product BCSP6662: DNA Marker

Fig. 4  Sequence analysis of soybean seed-specific promoter BCSP666

A: RY repeat motif; B: ACGT motif; C: AGCCCA motif; D: TACACAT motif; E: E-box; F: CAAT box; G: TATA box

Fig. 5  Schematic diagram summarizing distribution of seed-specific promoter related motifs on nucleotide sequences of the a’-subunit gene promoter and BCSP666

□: TATA box; ■: CAAT box; ○: RY repeat elements; △: AG/CCCCA motifs; ●: TACACAT motifs;▲: ACGT motifs; ◇: E-box

Fig. 6  Construction of seed-specific expression vector pBI121-666
Fig. 7  Construction and identification of the seed-specific vector pBI121-666

1:The DNA Marker; 2:The PCR product BCSP489; 3:Double digestion product pT-BCSP666/HindIII/XbaI; 4:Double digestion product pGEM-T/HindIII/XbaI; 5:Double digestion product pBI121-666/HindIII/XbaI; 6:Double digestion product pBI121/ HindIII/XbaI

Fig. 8  Southern Blot Analysis of the transgenic plants

1: Wild-type plants as a negative control; 2-5: Transgenic plant strains; 6: PCR products of GUS gene as a positive control 3.4 Seed-specific expression of GUS gene in the transgenic Arabidopsis thaliana plants

Fig. 9  Fluorescence analysis of transgenic plants and wild-type plants

1: Leaves of wild type plants; 2: Seeds of wild type plants; 3: Leaves of transgenic plants; 4: Seeds of transgenic plants

Fig. 10  Histochemical analysis of the transgenic plants

A and B: Transgenic plant strains; C and D: Wild-type plants as negative control

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