Construction Of Selectable-Marker-Exercision Vectors For Genetic Engineering Of Saccharina Japonica

The kelp Saccharina japonica was an important cultivated seaweed with annual productivityranked frist in China and more than half of the total seaweed cultivation productivity around theworld. In addition to be industrial raw material and healthy food, progress has been made through,comprehensive processing and development of high value products with this kelp. Thus, it wassignificant that the establishment of genetic engineering techniques could develop or creat newtraits to this kelp, which would meet the needs of kelp, breeding ande utilization.Based on the life circle of S. japonica, genetic transformation in the gametophyte generationstage have made it possible for the stable expression of some introduced genes. However, theenvironmental release of transgenic organisms still needs to draw much attention. To eliminate thepossible environment and health hazard of the selectable marker gene which confers resistanceagainst antibiotics or herbicides, it has an important project to develop techniques about exercisingthe integrated selectable markers genes from the genome of transgenic kelp.Aiming at the safety issues above-mentioned, this research has constructed a series ofexpression vectors with an inducible self-deleting system by cloning the inducible promoter from S.japonica and combining with a self-deleting elements from higher plants. The work has made thefollowing progress:1. We cloned a1.6kb of fragment from the5’-flanking upstream sequence of hsp70gene(heat shock protein70) from S. japonica. We selected four different length promoter sequenceswith different elements after analysising the elements of the promoter sequence. The four promotersequences were cloned into phsp70-GUS vectors. To obtain the examining vectors of the differenthsp70promoters function, the phosphinothricin resistant gene BAR as a selective marker regulatedby CaMV35s promoter was also coned into the phsp70-GUS vectors. Those four new constructswill be used to functional verification of the hsp70promoter.2. Recombinase genes Cre driven by the four sequences from the5’ upstream of hsp70gene,with the fragment SV40-BAR castle, are respectively inserted into the fused site between the two homodromous loxP repeats, and the two homodromous loxp-FRT repeats, to obtain8expressedvectors used for deleting the selectively marked gene by the Cre/loxP system after induced. Thoseeight vectors will be used to test whether the japonica promoters can induce the deletion of BARafter heat shock, by which we will also compare the efficiency of deleting BAR gene in loxP vectorwith that in loxP-FRT vector.Construction of selectable marker-excision expression vectors of S. japonica is an importantbreakthrough in the security applications of transgenic S. japonica. The above work may offerbasic elements and various vectors, and laid a certain foundation to relatives studies.