Functional And Characteristic Analysis Of Constitutive Promoter Pmagpd And Specific Promoter Pmagas1in Metarhizium Acridum

Metarhizium have been used as important biocontrol agents against various insects.There are around more than two hundred insect species can be infected by Metarhiziumspecies according to incomplete statistics. Like other mycoinsectcides, Metarhiziumhave the adventages of contact poisoning activity, non-polluting and no-resurgence ofinsect. However, the common weakness of bioinsectcide, such as slow acting, subject tothe environmental and unstable, limited the application of Metarhizium. Therefore, tomodifiy the wild type strains with genetic engineering techniques would bring greatchalleages and oppotunities to improve the efficiency and resistance to unfavorableconditions of the fungal insecticide.To date, a number of virulence, stress resistance-related genes have been cloned,including genes involved in surface attachment, hydrolysising or penetrating the insect’scuticle and colonization in the host. These findings lay a good foundation for theimprovement of entomopathogenic fungal strains. Transformation techniques plays animportant roles in genetically modifying bio-engineering fungal. Constitve expressingtarget genes is necessary for molecular genetic alteration of the microorganism. Thus, itwas meaningful to employing a strong promoter to express endogenous or exogenousgenes in scientific research and application aspect. So, it is an effective method thatusing a strong promoter to express the above genes to build high toxin strains.Metarhizium was used as experimental metarials, and different5’ deficiencyfragment of the promoter has been cloned on the basis of the known sequence, thencombined with reporter genes GUS and EGFP. The structure and function of theconstitutive promoter PMagpd and PMagas1were detected by the reporter gene of thetransforment strains.The main results were followed:1. Functional and characteristic analysis of PMagpd1.1Sequence analysis of the PMagpdA1.7-kb upstream sequence from the start codon (ATG) of the Magpd gene wasretrieved from the genomic DNA sequence of M. Acridum be denominated PMagpd. Ithas been found that no consensus-regulatory boxes TATA and CAAT were found in thePMagpd promoter, compared with other gpd promoters. Alighment with the PgpdAsequence showed that some typical boxes of gpd promoters were found in the5’ flanking sequence of Magpd, such as gpd-box, pgk-box, qa-box, qut-box and ct-richregion. Also, similar to other gpd promoters, a considerable number of direct-orinverted-repeat sequences, possibly binding sites of some regulatory factors, were alsofound in these boxes and other regions of PMagpd.1.2Analysis of transformantsVarious lengths (1691bp,1463bp,946bp,684bp,405bp) of DNA fragments ofthe Magpd promoter region were amplified, then, combined with GUS gene and clonedinto pK2vector. The transformants were analyzed by PCR, and then further confirmedby Southern blot. Results showed that the copy number of PMagpd-GUS-bar cassettesvaried from1to3.1.3Deletion analysis of the PMagpdThe enzyme activity of GUS were determined, the results showed that deletions ofthe regions upstream of-946bp and upstream of-684bp caused34%and39%decreases of GUS activity, respectively, suggesting that strong positive cis-actingelements were located in this region. Compared with the PgpdA, PMagpd(Del2)exhibited1.3times of PgpdA for GUS activity. To confirm the activity of eachshortened PMagpd, transcription level of the GUS gene was also analyzed by RT-qPCR.Results showed a similar trend with the GUS activity in all these deletion groups ofPMagpd.2. Functional and characteristic analysis of PMagas12.1Sequence analysis of the PMagas1A1.2-kb upstream sequence from the start codon (ATG) of the Magas1gene wasretrieved from the genomic DNA sequence of M. acridum and be denominatedPMagas1. The sequence analysis results showed that some general elements such asCAAT-box,GC-box and TATA-box, fungal promoter characteristic element ct-box andsome dirct-or re-dirct-repeat sequences were found in PMagas1.2.2Analysis of transformantsVarious lengths (1228bp,897bp,611bp,377bp) of DNA fragments of the Magas1promoter region were amplified, then, combined with EGFP gene and cloned into pK2vector. The transformants were analyzed by PCR, and then further confirmed bySouthern blot. Results showed that the copy number of PMagpd-GUS-bar cassettesvaried from1to3.2.3Phase localization analysis of PMagas1The EGFP fluorescence detection of meterhizium transforments contained the1228 bp integral promoter in various phases were made individually, the results showed thatthe signal can only be detected in the appresorium phase; therefore, no fluorescencesignal can be found in other phase such as conidium, germination, hyhae and polypidein the hemolymph of locust.2.4Deletion analysis of the PMagas1We analyzed the relative transcription level of egfp gene of the transformants with aseries truncated PMagas1. The results showed that deletion of the region form1228bpto-897bp cannot affect the activity of PMagas1. However, the activity decreasedalmost70％when further to truncate the region from-897bp to611bp, and the localdeletion of region from-392bp to-328bp caused a decrease of82％.We concluded thatthe present of this two regions is important for the activity of Magas1promoter.