cDNA Clone, fusion expression and purification of the novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E. coli |
| |
Authors: | Ling Lin Xiping Wang Yuejin Wang |
| |
Institution: | (1) Key Laboratory of Northwest Horticulture Plant Germplasm and Genetic Improvement of Ministry of Agriculture of People’s Republic of China, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, 712100, The People’s Republic of China |
| |
Abstract: | Powdery mildew, caused by Uncinula necator Burr, is one of the most seriously damaging diseases of grapevine all over the world. To gain the novel gene and investigate
the resistance mechanism in Chinese Wild Vitis pseudoreticulata clone Baihe-35-1, mRNA differential display was employed to study the differential expression of the resistant gene to the
disease of it when inoculated by Uncinula necator under natural field conditions, 5′ RACE and 3′ RACE have been used to clone the whole cDNA sequences of VpAPX, the novel
gene related to Ascorbate Peroxidase which involved in resistant to the disease, is composed of specific sequence 1077 bp and has an open reading frame of 750 bp
coding for 250 amino acid residues with a molecular weight of 27.566 kDa. The VpAPX gene was obtained by polymerase chain
reaction (PCR) with the special primers synthesized according to the sequences of cDNA, and further cloned it into the pGEM-T
easy vector. The cloned VpAPX gene was cut out again with two restriction enzymes and was inserted into the prokaryotic expression
vector pGEX-4T-1, then transferred into E. coli BL21. As result, GST-VpAPX fusion protein was successfully expressed by induction of IPTG and purified by GST affinity resin.
After injecting rabbit, the polyclonal antibodies were produced. Western blot analyses showed that the antibody reacted specifically
to GST-VpAPX fusion protein and the titer for this antibody is 105. This research made the foundation to transform the VpAPX gene into grape plants for follow research in processing.
Ling Lin, Xiping Wang: These two authors contributed equally to this work. |
| |
Keywords: | cDNA clone Chinese wild Vitis species fusion protein expression mRNA differential display polyclonal antibodies |
本文献已被 PubMed SpringerLink 等数据库收录! |
|