Highly toxic and broad-spectrum insecticidal localBacillusstrains engineered using protoplast fusion

Abstract

Protoplast fusion was performed between a local Bacillus thuringiensis UV-resistant mutant 66/1a (Bt) and Bacillus sphaericus GHAI (Bs) to produce new Bacillus strains with a wider spectrum of action against different insects. Bt is characterized as sensitive to polymyxin and streptomycin and resistant to rifampicin and has shown 87% mortality against Spodoptera littoralis larvae at concentration of 1.5 × 10(7) cells/mL after 7 days of feeding; Bs is characterized as resistant to polymyxin and streptomycin and sensitive to rifampicin and has been shown to have 100% mortality against Culex pipiens after 1 day of feeding at the same concentration as that of Bt. Among a total of 64 Bt::Bs fusants produced on the selective medium containing polymyxin, streptomycin, and rifampicin, 17 fusants were selected because of their high mortality percentages against S. littoralis (Lepidoptera) and C. pipiens (Diptera). While Bt harboured 3 plasmids (600, 350, and 173 bp) and Bs had 2 plasmids (544 and 291 bp), all the selected fusants acquired plasmids from both parental strains. SDS-PAGE protein analysis of the 17 selected fusants and their parental strains confirmed that all fusant strains acquired and expressed many specific protein bands from the 2 parental strains, especially the larvicidal proteins to both lepidopteran and dipteran species with molecular masses of 65, 70, 80, 88, 100, and 135 kDa. Four protein bands with high molecular masses of 281, 263, 220, and 190 kDa, which existed in the Bt parental strain and did not exist in the Bs parental strain, and 2 other protein bands with high molecular masses of 185 and 180 kDa, which existed in the Bs parental strain and did not exist in the Bt parental strain, were expressed in most fusants. The results indicated the expression of some cry genes encoded for insecticidal crystal proteins from Bt and the binary toxin genes from Bs in all fusant strains. The recombinant fusants have more efficient and potential values for agricultural application compared with both the insecticidal Bt and the mosquitocidal Bs strains alone against S. littoralis and C. pipiens larvae, respectively.

Molecular Genetic Characterization of Fusants from Protoplast Fusion of S. Cerevisiae and P. Stipitis ATCC 58785

“…The similar bands from parents that were expressed in the fusants are indications of protoplast fusion between two yeasts S. Cerevisiae and P. Stipitis ATCC 58785 (Figure 5). These findings of acquisition of different proteins by the fusant from the parental strains as observed in this study agree with those reported by [26], who found that 17 selected fusants obtained in their study and their parental strains confirmed that all fusant strains acquired and expressed many specific protein bands from the parental strains [26]. [30] used SDS-PAGE analysis for screening of the fused cultures from protoplast fusion of the polypeptide profile yeast cultures S. Cerevisiae and K. Marxianus for enhancing bioethanol production from cheese industry waste [31][32][33][34].…”

“…The polypeptide profile was seen with reference to a protein ladder (GangNam-STAIN™ Prestained), with 12 bands between 10 to 245 kDa. SDS-AGE protein analysis of the five selected fusants and their parental strains confirmed that all fusant strains acquired and expressed many specific protein bands from the parental strains as similarly reported in an earlier study [26]. Protein bands with high molecular masses of 100 kDa, which existed in S. Cerevisiae parental strain and not in P. Stipitis ATCC 58785 parental strain was expressed in the fusants F22 and F24.…”

“…The sequencing of a region of the rDNA gene unit, which includes two non-coding regions designated as the internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of fusants F12, F18, F22 and F24 as shown in (Appendix 1-4) receptively. Molecular study using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to identify the genetic inter-relationship between fusants and their parents [26][27][28].…”

Comparative Analysis of Genetic Diversity Among Bacillus Thuringiensis and Bacillus Sphaericus and Their Fusants Using Molecular Markers

“…Ten fusants stains; six of them produced from Bs GHAI: Bt 66 Ia experiment (fusants no. 1, 2, 16, 26, 31 and 34) [13] and four fusants produced from Bs GHAI Bt I 977experiment (fusants no. 1, 2, 17 and 25) [14].…”

“…In this study, two marker systems; ISSR and SCoT, were successfully used to differentiate between Bt and Bs parents and their ten fusants stains; six of them produced from Bs GHAI:: Bt 66 1a experiment (fusants no. 1, 2, 16, 26, 31 and 34) [13] and four fusants produced from Bs GHAI:: Bt I 977 experiment (fusants no.1, 2, 17 and 25) [14]. A total of 11 primers were used and 55 bands for ISSR and 97 bands for SCoT were produced, with a mean of 13.750 and 13.857 bands per primer, respectively, among which 92.696 % and 81.948% were polymorphic for ISSR and SCoT, respectively.…”