Studies On The CDNA Library Construction For Rice Blast, Activator Protein Gene Cloning And Its Protein Expression Study

Rice blast is a wide spread disease in production areas around the world. Enormous amounts of economic loss (up to 80% yield loss) are caused by rice blast when the weather is favor. At present, the main precaution method is the spraying of chemical fungicides. But it also causes a serious damage to the agricultural environment. Therefore, it is a long-term task for scientists to develop "green" biological fungicide.Activator protein is one promising new type "green" biological fungicide with obvious induced resistance to tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). To find out the molecular mechanism of induced resistance in plants and clone the activator protein gene, cDNA library consisting of 5.0 × 10~7 clones has been constructed by using solid phase method. According to the results of phage plaques bright selection of host bacterial strain XL1-Blue and PCR detection, the percentage of recombinant phages was 98% and the titer, 1.0× 10~7cfu/ml. With the partial sequence obtained, a pair of specific primers was designed. The cDNA of target gene has been obtained by RT-PCR with the phage DNA as template. And sequence analysis indicates that the coding region includes 951 base pairs, encoding 316 amino acids.Cloned activator protein gene was inserted into pGEX-5X-l to construct the recombinant prokaryotic expression vector, pGEX-XF-1. Then the vector was transformed into E. coli BL21 and ER2566. And high expression in vitro induced by IPTG was obtained and analyzed on SDS-PAGE gel. As a result, the molecular weight of the target protein is 36kD, which is consistent with the deduced one from nucleotide sequence, 33540.5 daltons.