Purification And Cloning Of An Extreme Thermophilic Polypeptide And Its Control Effect Against Rice Blast At Seedling Stage

Magnaporthe grisect is a worldwide plant disease, resulting in 10-20% yield loss in rice generally. Since the 1990s average yield loss in rice was climbed over 100 million kg, which was caused by M. grisea. Cultivating resistance varieties is an effective means to control M. grisea, but 3-5 successive plating will cause the loss of resistance. Chemical pesticide is another option to curb the disease, but the active agent in most of the current chemical pesticides is tricyclazole that leads to development of pesticide resistance, but also deals heavily a blow to the environment and threatens human being's health. However, all of these disadversities can be overcame by utilizing microbial pesticides, which provides a safer, effective, more endurable and environmentally friendly alternative for rice blast control. Extreme thermophilic proteins are commonly believed to be produced by thermophilic bacteria whose optimal growth temperature is 50-80℃. Studies on the mechanisms of thermostability will contribute to high-temperature-related industries, but there is no report arguing that extreme thermophilic proteins(or polypeptides) are produced by common bacteria. In the present study, we purified a polypeptide from ferments of strain LM-3, made an effort to clone the genes coding for this polypeptide, and evaluated its performance on seedling rice blast in field, aiming for set the foundation for breeding transgenic rice resistant to rice blast disease and for unveiling mechanisms of antagonism to M. grisea and extreme thermostability.â… . Purification of polypeptides from LM-3 ferments. Two extreme thermophilic polypeptides were isolated by heating from fermentation liquor of strain LM-3 which belongs to Paenibacillus polymyxa. The inhibition capacity of 5μl purified polypeptides against Magnaporthe grisea reached to 89.6% according to results on agar plates. These two polypeptides had a molecular weight between 6 000 and 7 000 Da by SDS-PAGE. They exhibited extreme thermophilic characteristic and were recovered from electro-lanes for detection of inhibition activity to M. grisea respectively. One polypeptide with strong inhibition activity to M. grisea, was named APPLM3(Antagonism Polypeptide from Paenibacillus polymyxa LM-3), and the other without such activity was named PPLM3(Polypeptide from Paenibacillus polymyxa LM-3). â…¡. N-terminal amino acid residues sequencing and their comparison. N-terminal amino acid residues of APPLM3 were then sequenced by Edman method and the sequence was H2N-ANDPR by which restriction similarity comparison of amino acid sequence was conducted on NCBI(National Center for Biotechnology Information) Website. Results showed that this sequence is may be related to these of 3 putative or hypothetical proteins from Burkholderia.â…¢. Detection of thermostability of APPLM3 polypeptide. The purified polypeptide was treated for 60 min under 60℃, 80℃and 100℃respectively, and 120℃for 20 min, 30 min and 60 min respectively. Purified polypeptide at 4℃was treated as control. The inhibition activity of APPLM3 was not reduced significantly at 60℃and 80℃for 60 min, and lost a little at 100℃for 20 min. When temperature increased to 120℃, APPLM3 maintained its 85.4% inhibition activity against M. grisea. This activity was dependent on heating time, and lost 60 min after heating at 120℃completely. These indicated that the thermostability of APPLM3 polypeptide was extremely high, but lost with the elongating of heating time and increasing of temperature acceleratively.â…£. Cloning of APPLM3 gene. The genomic DNA of LM-3 was extracted and subjected to partial digestion by Sau3AI. The fragments with a length of 500-4 000 bp were then recovered, inserted into the BamHI site of pUC19 phage, and transformed into Escheriachia coli. DH5a. a-complementation was used to select the positive clones which were screened again on PDA/LB agar plates for antagonistic clones. The cloning failed due to the un-uniformed temperature for growth and the short insert fragment of interest.â…¤. Field evaluation of 5 APPLM3-related microbial pesticides against rice blast in seedling stage. To evaluate its performance in field, APPLM3 was confected with methanol and compound emulsifier tween80/OP to confect 5 microbial pesticides. There were three controls, namely CK₁, CK₂ and CK₃. The weighted disease index of CK₁ and CK₂ was used as the disease index standard, increasing the experimental accuracy. As illustrated in table 5, control effects of all pesticides were superior to antagonistic agent without processing. With the increase of emulsifier OP control effects increased subsequently, and AB4 was the best whose control effect reached to 80.73%. However the excessive amount of emulsifier OP brought about impairing control effect. The treatment AB₄ was therefore considered as the best cross, and the weight ratio of its components was 50: 6.5: 1.75: 41.75(APPLM3: tween80: emulsifier OP: methanol).