Down-Regulation Of Myrosinase Gene Expression In Brassica Parachinensis Bailey By RNA Interference

In plants of the cruciferous family,which includes the model plant Arabidopsis thaliana and brassica crops,the myrosinase-glucosinolate system serves as a major chemical defense mechanism against phytophagous insects and pathogens,and is also called "mustard oil bomb".The potential resources of insect-resistant germplasm are relatively limited in Brassica parachinensis Bailey,it is therefore very important to sensibly use the myrosinase-glucosinolate system for breeding of insect-resistant varieties.The defense mechanism should be sufficiently understood before the system can be well applied in practice.One fragment of myrosinase gene of 890 bp was cloned from the total RNA of Brassica parachinensis Bailey using RT-PCR method,and inserted into the two sides of the intron in pJM007 vector in a reverse way to produce the MY(RNAi)cassette. This cassette was excised from pJM007 by PstI endonuclease and inserted into the binary vector pCAMBIA1301 to produce MY(RNAi)vector,which was transferred into Agrobacterium tumefaciens GV3101 and then transformation was conducted. Under the selection of hygromycin,transgenic plantlets were obtained.Four transgenic lines were identified by PCR and Southern blot,and myrosinase genes were effectively suppressed in the transgenic plants tested by Northern blot.In the meantime,we studied the effect of 12 different combinations of 6-BA and NAA on plant regeneration of Brassica parachinensis Bailey in inducing media.And determined the suitable hygromycin selection concentration for Brassica parachinensis Bailey transformation.Finally,a suitable genetic transformation system of Brassica parachinensis Bailey was developed.