An efficient method for generating cDNAs in Brassica carinata A. Br.

摘要

Recombinant DNA technology makes it possible to identify and isolate genes from any biological system. Previously genes were isolated using genomic/cDNA libraries. Dealing with low abundance transcripts can be frustrating as conventional cDNA library screening is labour intensive and there are chances of loosing low abundance transcript during sample handling, poor efficiency of first and second strand cDNA synthesis and the proportion of recombinant clones to be screened. The development of PCR to detect rare transcripts has revolutionized the sensitivity of gene expression analysis as well as identification of these transcripts/genes. Therefore, RT-PCR approach is relatively simple and efficient in identification and isolation of cDNA of a particular gene, large scale functional genomics and sequencing projects. An efficient method was developed for large scale RNA isolation and RT-PCR to generate cDNAs for isolation of Myb transcription factor genes related to drought in Brassica species. The total RNA was isolated from drought exposed two weeks old seedlings grown in vitro on MS medium. Lyophilized and liquid nitrogen frozen seedlings were used in RNA isolation. Trizol method, traditional Guanidine isothiocyanate and commercial kits were used for RNA isolation. It was found that Trizol method was quite efficient and yielded 2.25 mg per g fresh weight of the plant tissue. Electrophoretic analysis on formaldehyde gel revealed two distinct ribosomal bands. The mRNA was isolated from total RNA using Genelute mRNA miniprep kit and yielded 150-200 μg per g fresh weight ofthe plant tissue. Two primer sets were used to generate cDNA from isolated mRNA/total RNA. Total RNA did not yield amplified product, however, mRNA showed amplification in all the three sets of primers which were designed on the basis of Atmyb2 conserved domain sequence. The optimized procedure gives good quality and quantity of RNA for generating cDNAs in Brassica carinata which is crucial in gene isolation and transcript level studies.