Identification Of Transcription Factors Invovled In The Crystal Cell Differentiation In Bacillus Thuringiensis Strain LM1212

Bacillus thuringiensis?Bt?LM1212,whose spores and crystal are forming in different cells,has the phenomenon of cell differentiation.In this study,the whole genome sequence of LM1212 with 6320549bp were determined,using Pacbio RSII sequencing platforms.Sequence analysis showed that LM1212 had a circle genome and 8 plasmids;According to the new genome sequence of LM1212,twelve cry/cyt genes were located in pLM248,pLM158 and pLM113.The gene of endogenous large plasmids in LM?p35'Z?which carrying cry35-like gene promoter and lacZ gene fusion plasmid p35'Z were cured by high temperature treatment.Two plasmid gene deletion mutants LM?p35'Z?-W and LM?p35'Z?-DB strains were obtained.The activity of cry35-like gene promoter p_cry35-like was affected in these two strains.Observation by laser confocal scanning microscope and optical microscope,calculation of spore formation rate,SDS-PAGE and LC-MS/MS?Q-TOF?mass spectrometry were utilized to determine the effect of plasmid gene deletion on the cell differentiation and expression of cry genes in LM1212 strain.Cell morphology observation showed that more crystal-producers were found in LM?p35'Z?-DB than in both LM?p35'Z?-W and LM?p35'Z?.Crystal protein yield in LM?p35'Z?-DB increased,not in LM?p35'Z?-W.In order to verify the functionality of the orf28-32 fragment of pLM248 in LM1212,we selected the LM?p35'Z?-W strain in which the orf28-32 fragment was deleted as experimental strain.And plasmid p35'Z was cured by high temperature and without antibiotics treatment in LM?p35'Z?-W,we transferd pHT304-orf28-29 and pHT304-orf28-32 into LM?p35'Z?-W?p35'Z,the results showed that the orf28-29 and orf28-32 could increase the proportion of crystal cells;SDS-PAGE analysis showed that the orf28-29 and orf28-32 could increase the crystal protein yield.The transcription factor encoded by orf28 is named as CpcR?Crystal producer cell regulator?.GFP protein of T0 to T4 in LM?p35'gfp?which carrying cry35-like gene promoter and gfp gene fusion plasmid p35'gfp was observed by laser confocal scanning microscope.The results showed that cell differentiation of LM1212 has already begun at T2.We extracted the total RNA from T0 to T4 of LM?p35'gfp?,LM?p35'Z?-W and LM?p35'Z?-DB strains,then sequenced them and all the transcriptome dataset was obtained.It will contributed to analysising genes regulated by transcription factor CpcR and screening the binding proteins of differentially expressed genes and then we can identified other transcription factors invovled in cell differentiation of LM1212.