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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2009, Vol. 3 Issue (1) : 55-59     DOI: 10.1007/s11703-009-0017-y
Cloning of strawberry FaEtr2 gene and its plant expression vector construction for antisense RNA
Chunli SONG1, Pingping ZHOU1, Junlian MA2, Xia TANG2, Zide ZHANG2(), Zhixia HOU3
1. College of Chinese Veterinary,Agricultural University of Hebei,Dingzhou 073000,China; 2. College of Food Science and Technology,Agricultural University of Hebei,Baoding 071001,China; 3. The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China
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An ethylene receptor FaEtr2 gene was amplified by Polymerase Chain Reaction (PCR) from ripening strawberry fruit. A 1049-bp PCR product (All Star-Etr2) was cloned. Sequence analysis showed that the All Star-Etr2 nucleotide sequence had 100% identity with Chandler-Etr2 from the GenBank. A pair of primers containing restriction enzyme sites were designed and used to amplify the sequenced plasmid. The PCR product was digested by the corresponding restricted enzymes and inserted between the CaMV 35S promoter and NOS terminator of expression vector pBI121 directionally. The constructed expression vector was transformed into Agrobacterium fumefeciens LBA4404 in the follow-up research to silence a ripening-related ethylene receptor FaEtr2 gene in strawberry fruits.

Keywords strawberry      ethylene receptor      FaEtr2 gene      plant expression vector     
Corresponding Authors: ZHANG Zide,   
Issue Date: 05 March 2009
URL:     OR
Fig.1  Agarose gel electrophoretogram of PCR products
Note: M: 100 bp DNA Ladder-3K marker; 1—5: PCR products; CK: negative comparison.
Fig.2  Electrophoresis pattern of recombinant plasmid digested by R I
Note: M: 1 kb ladder marker; 1, 2: recombinant plasmid digested by R I.
Fig.3  The nucleotide sequence of all star strawberry
Note: Underlined sequences were the primer sequences.
Fig.4  Agrose gel electrophoretogram of PCR products
Note: M: 100 bp ladder marker; 1, 2: PCR products of antisense; CK: negative comparison of PCR products.
Fig.5  Agrose gel electrophoretogram of antisense PCR digested by enzymatic
Note: M: 100 bp ladder marker; 1: antisense PCR digested by I + H I; 2: PCR products of antisense.
Fig.6  Agrose gel electrophoretogram of pBI121 plasmid digested by I and H I
Note: M: 1 kb ladder marker; 1: pBI121 plasmid digested by I and H I; 2: pBI121 plasmid.
Fig.7  Detection of the antisense expression vector by enzymatic digestion
Note: 1: I/R I; 2: I/H I; 3: d III/ I; 4: d III/ H I; 5: pBI-Anti-Etr2; M: 1 kb ladder marker.
Fig.8  Agrose gel electrophoretogram of antisense expression vector transformed LBA4404
Note: M: 100 bp ladder marker; CK: comparison of negative contral; 1—3: PCR products of bacterium liquid; CK: PCR product of the plasmid.
1 Cancel J D Larsen P B (2002). Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis. Plant Physiology , 129: 1557-1567
doi: 10.1104/pp.003780
2 El-Kereamy A., Chervin C, Roustan J P, Cheynier V, Souquet J M, Moutounet M, Raynal J Ford C, Latche A, Pech J C, Bouzayen M (2003). Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries . Physiologia Plantarum, 119: 175-182
doi: 10.1034/j.1399-3054.2003.00165.x
3 Ferrarese L, Trainotti L, Moretto P, Polverinode L P, Rascio N, Casadoro G (1995). Differential ethylene-inducible expression of cellulase in pepper plants . Plant Molecular Biology,29:735-747
doi: 10.1007/BF00041164
4 Giovannoni J (2001). Molecular biology of fruit maturation and ripening. Annual Review of Plant Molecular Biology, 52: 725-749
doi: 10.1146/annurev.arplant.52.1.725
5 Giovannoni J J (2004). Genetic regulation of fruit development and ripening . The Plant Cell, 16: S170-S180
doi: 10.1105/tpc.019158
6 Harpster M H, Brummell D A, Dunsmuir P (1998). Expression analysis of a ripening-specific, auxin-repressed endo-1,4-beta-glucanase gene in strawberry . Plant Physiol, 118: 1307-1316
doi: 10.1104/pp.118.4.1307
7 Harpster M H, Lee K Y, Dunsmuir P (1997). Isolation and characterization of a gene encoding endo- β -1,4 -glucanase from pepper ( Capsicum annuum L.) . Plant Molecular Biology, 33: 47-59
doi: 10.1023/A:1005795028489
8 Jimenez-Bermudez S, Redondo-Nevado J, Munoz-Blanco J, Caballero J L, Lopez-Aranda J M, Valpuesta v, Pliego-Alfaro F, Quesada M A, Mercado J A (2002). Manipulation of strawberry fruit softening by antisense expression of pectate lyase gene . Plant Physiol, 128: 751-759
doi: 10.1104/pp.010671
9 Katz E, Lagunes P M, Riov J, Weiss D, Goldschmidt E E (2004). Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in non-climacteric Citrus fruit . Planta, 219: 243-252
doi: 10.1007/s00425-004-1228-3
10 Klee H J (2002). Control of ethylene-mediated processes in tomato at level of receptors . J Exp Bot, 53: 2057-2063
doi: 10.1093/jxb/erf062
11 Knee M, Sargent J A, Osborne D J (1977). Cell wall metabolism in developing strawberry fruits . J Exp Bot, 28: 377-396
doi: 10.1093/jxb/28.2.377
12 Luo Y B, Sheng J P, Shen L (1995). Control of tomato ethylene biosynthesis by the use of antisense gene . Journal of Agricultural Biotechnology, 3(2): 37-44 (in Chinese)
13 Marty I, Douat C, Tichit L, Jungsup K, Leustek T, Albagnac G (2000). The cystathionine-gamma-synthase involved in methionine biosynthesis is highly expressed and auxin-repressed during wild strawberry ( Fragaria vesca L . ) fruit ripening . Theor Appl Genet, 100: 1129-1136
doi: 10.1007/s001220051396
14 Medina-Escobar N, Cardenas J, Munoz-Blanco J, Caballero J L (1998). Cloning and molecular characterization of a strawberry fruit ripening-related cDNA corresponding a mRNA for low-molecular-weight heat-shock protein . Plant Mol Biol, 36: 33-42
doi: 10.1023/A:1005994800671
15 Sambrook J, Russsell D W, Huang P T (2001). Molecular Cloning: A Laboratory Manual. 3rd ed (in Chinese, trans. Peitang HUNG) . Beijing: Academic Press, 2002, 68-70
16 Tesniere C, Pradal M, El-Kereamy A, Torregrosa L, Chatelet P, Roustan J P, Chervin C (2004). Involvement of ethylene signaling in a non-climacteric fruit: new elements regarding the regulation of ADH expression in grapevine . Journal of Experimental Botany, 55: 2235-2240
doi: 10.1093/jxb/erh244
17 Trainotti L, Pavanello A, Casadoro G (2005). Different ethylene receptors show an increased expression during the ripening of strawberries: Does such an increment imply a role for ethylene in the ripening of these non-climacteric fruits . Journal of Experimental Botany, 56(418): 2037-2046
doi: 10.1093/jxb/eri202
18 Trainotti L, Spolaore S, Pavanello A, Baldan B, Casadoro G (1999). A novel E-type endo-beta-1,4-glucanase with a putative cellulose-binding domain is highly expressed in ripening strawberry fruits . Plant Mol Biol, 40: 323-332
doi: 10.1023/A:1006299821980
19 Waterhouse P M., Graham M W, Wang M B (1998). Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA . Proc Natl Acad Sci, 95: 13959-13964
doi: 10.1073/pnas.95.23.13959
20 Wooly L C, James D J, Manning K (2001). Purification and properties of an endo-beta-1,4-glucanase from strawberry and down-regulation of the corresponding gene . Cell Planta, 214: 11-21
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