Optimization of Protocol for Identifying Novel Genes for Structural Study

A. Samad^{1, 2, 3}, A. Bashir¹, S. A. Malik², M. Ahmad¹, M. Sajjaid¹, A. Mukbool¹, S. Moddassir¹, A. Aftab¹, E. Jindrova³, R. Ettrich³

¹National Institute for Biotechnology and Genetic Engineering NIBGE, P.O.Box No 577 Jhang Road Faisalabad, Pakistan.

²Quaid-I-Azam University Islamabad Pakistan.

³Laboratory of High Performance Computing, Institute of Systems Biology and Ecology ASCR

Institute of Physical Biology USB Zámek 136, CZ-373 33 Nové Hrady,

Czech Republic

Email: samad_aries@yahoo.com

A protocol for identifying novel genes of cotton (Gossypium hirsutum L.) fiber through differential display PCR [1,2], visualized by ethidium bromide staining, was optimized. The procedure was used to identify differentially expressed genes at 0 DAP (Day After Pollination) and 10 DAP of developing cotton fiber a single longest cell in plant kingdom [3]. Different populations of total RNA were reversely transcribed using single anchored oligo-dT primers. PCR amplification of relative cDNA was carried out using three single anchored oligo-dT primers (dd1: 5’ TTTTTTTTTTTG 3’, dd2: 5’ TTTTTTTTTTTC 3’,dd3: 5’ TTTTTTTTTTTA 3’) in combination with five arbitrary primers (random decamers). One percent agarose gel containing ethidium bromide for staining can resolve amplified transcripts over a large range of molecular weights. Several differentially expressed transcripts were successfully identified by this procedure. Isolated genes were used as a probe to screen cDNA library for their full-length genes and cloned into TA cloning vector for DNA sequencing. DNA sequence was blast searched for homology and translated into protein through translate tools (www.expasy.ch, www.justbio.com). Expanin, Tubulin, Lipid transfer proteins and several uncharacterized fiber proteins (e.g. DQ023530, DQ023531, DQ023532, DQ023533, DQ023534, DQ023535, DQ023536) were cloned in this study [4].

Structural information about these uncharacterized fiber proteins was not determined yet. These uncharacterized fiber proteins will be subcloned in pET 32a (+) for expression. Expressed protein will be purified by chromatography and measured through spectroscopy. Experimental results will be analyzed with modeling techniques to propose their three- dimensional structures. The future about these proteins appears bright and tremendously exciting.

[1] P. Liang, A.B. Pardee, Science, 257 (1992) 967-71.

[2] P. Liang. Genetic Engineering News, 20 {2000) 37.

[3] A.S. Basra, C.P. Malik, Int. Rev. Cytol, 89 (1984) 65–113.

[4] A. Samad. M. Phil Dissertation, Quaid-I-Azam University, Islamabad Pakistan (2005).