|
|
|
| Cloning and Prokaryotic Expression of Zaosu Pear NPR1 Gene |
| , , ,Yue-Jin Wang |
|
|
|
|
Abstract Abstract: The total RNA was extracted from leaves of Zaosu pear（Pyrus bretschneideri cv. Zaosu). Using RT-PCR method, the full-length cDNA of NPR1 gene was cloned from the Zaosu pear leaves by a pair of specific primers designed according to the Japanese pear （Pyrus pyrifolia ）NPR1 gene sequence released on the GenBank（EF079077）. Bioinformatics analysis showed that the obtained 1771bp fragment contained a 1761bp full open reading frame encoding 586 amino acid residues（GenBank accession No. FJ769372）. The homology rates of amino acid sequences of NPR1genes between Zaosu pear and Pyrus pyrifolia (ABA62792), Pyrus ussuriensis, Malus x domestica (ACC77697), Nicotiana tabacum (AAT57641) and Arabidopsis thaliana (AAM98084) were 99%、98%、98%、67% and 59% respectively. The gene was sub-cloned into the expression vector pGEX-4T-1 and expressed a fusion protein of approximately 91kD in Escherichia coli BL21 cells induced by IPTG. The result indicated that the fusion protein was expressed in inclusion body. In order to further analysis the expression profile of the NPR1 protein and study subcellular localization of the NPR1 protein in transformed pears, our future work will be the purification of the NPR1 protein and preparation of its polyclone antibodies.
|
|
Received: 05 March 2009
|
|
|
|
|
|
|
