Studies On Defence Gene-Myrosinase Gene From Cruciferae Vegetable

Glucosinolates-myrosinase is an important defence system in brassica of cruciferous vegetables, also referred to as "the mustard oil bomb" The myrosinase genes in 49 Brassica parachinensis Bailey, Brassica oleracea L. Var. capitata (L.) and Brassica aboglabra Baily were cloned and analyzed in this paper.Three methods of RNA extraction were compared and the results showed that the methods of Phenol-Chloroform and Trizol were better than the method of UNIQ-10. The abundance of RNA in cruciferous vegetable was also related to the attacks from different insects.Specific primers based on the the conservative domain of myrosinasewere designed to clone myrosinase genes in 49 Brassica parachinensis Bailey, Brassica oleracea L. Var. capitata (L.) and Brassica aboglabra Baily by RT-PCR. One nucleotide acid fragment (350bp) was obtained in Brassica parachinensis Bailey and Brassica oleracea L. Var. capitata (L.) respectively, but two fragments (350bp and 466bp) in Brassica aboglabra Baily. It was found that that the fragments of 350bp were highly homologous with the cloned myrosinase genes in Genbank. Therefore, these fragments of 350bp were part of myrosinase genes in these plants. The fragment of 466bp in Brassica aboglabra Baily had an additional nucleotide acid between 310 and 426 compared with myrosinase genes in Genbank. Amino acid sequences were deduced from these fragments and analyzed.Primers for RACE were designed based on the nucleotide acid sequences of the fragment that has been cloned in Brassica parachinensis Bailey to gain the full gene of myrosinase (full cDNA). The full myrosinase gene is of 1841 bp with an open reading frame of 1647bp. The secondary structure of myrosinase mRNA was predicted with 83 loops that affect the stability of mRNA and the least structure energyof -33.61kcal.The protein sequence of 548 amino acids was deduced from this gene. This protein was predicted to be a hydrophilic and dissoluble protein according to the hydrophobicity analyse.