| The serious loss of crops was caused by agricultural harmful living things every year. The insects of the orders of Lepidoptera, Coleoptera, Diptera and plant pathogens of fungi and nematode etc. are severe ones. The microbial pesticide of Bacillus thuringiensis (Bt) was successfully used for pests control, as well as have made some progress in plant disease control. It is very significant and necessary to screen new Bt strains with higher toxicity, and clone it's novel action genes for constructing of both genetically engineered bacteria and transgenic plants. In this study, 51 strains of Bacillus thuringiensis from 178 soil samples coming from 20 provinces, autonomous regions and municipalities all over China were isolated with nutrition agar media and identification with parasporal crystal dyeing methods. All of the 51 Bt were analyzed by the PCR method with general primers of cry1, cry2, cry3, cry4, cry-n, cry7, cry8, cry11, cry13 and cyt genes. The results showed that 4 strains of them contain cry4 type gene,12 contain cry7 ,4 contain cry8, 4 have cry1 respectively; 2 of them contain both cry1 and cry2. 27 strains did not give any PCR products. None of them contain cry3, cry-n, cry11, cry13 and cyt type gene. Four isolates with cry4 gene were studied systematically. It was showed that BD-2-1,BD-2-2,HC-7-1 and STG-21-7 contained mainly spherical and some irregular shapes parasporal crystal observed with scanning electron microscope. Eleven physiological and biochemical characteristic of the former 3 strains were unanimous, but different from STG-21-27. ICPs (Insecticidal Crystal Proteins) were encoded in the 4 isolates of Bacillus thuringiensis with SDS-PAGE analysis. Their crystal proteins composed with 66~72kDa, 27~31kDa and 20kDa. Partial gene sequence of cry4 revealed that BD-2-1 and BD-2-2 was the same isolates, they are different from the other 2 strains. The 4 isolates of Bacillus thuringiensis had high degree of homology (90%) each other, and they are all homologous with cry40-like (88%). Four isolates, YN-1-1,SH-1-2,HZJ-35-17 and HZJ-3-3, contained cry8 gene. Their parasporal crystal shapes were mainly rhomboid and some irregular observed with scanning electron microscope. Biochemical reaction indicated that YN-1-1, SH-1-2, HZJ-35-17 and HZJ-3-3 was not identical with standard strain of Bt. 70~75kDa ICPs was encoded in YN-1-1, SH-1-2 and HZJ-3-3, But 130kDa and 70~75kDa ICPs were in HZJ-35-17 with SDS-PAGE method. Partial sequence of cry8 analysis showed that YN-1-1, SH-1-2, HZJ-35-17 and HZJ-3-3 with higher degree of homology (90%) to each others, belonging to cry8B gene group.YN-1-1 and SH-1-2 with higher degree of homology(99.67%). 97% homology of HZJ-3-3 cry8 gene to cry8Bb1. 95% sequence similarities of HZJ-35-17, YN-1-1 and SH-1-2. The four strains'cry8 have 89% homology to cry8B, and 83% to cry8A. Bioassay of 51 strains　of Bacillus thuringiensis to Bursaphelenchus xylophilus have been studied in laboratory. Two of them had higher toxicity to B. xylophilus. Bioassay of extraction of endotoxin and exotoxin of the two isolates to B. xylophilus showed the action of exotoxin higher than that of endotoxin. Control effect of pot test in greenhouse to tomato root-knot nematode (Meloidogyne spp) revealed that the 2 Bt strains could reduced certain galls of tomato root. The strain of the XZ-4-5 was studied on biological and molecular characteristics systematically, because it contained cry7 gene which are toxic to Coleopteran insect larva and plant parasitic nematodes. Crystal shape of the isolate was bipyramidal observed with scanning electron microscope. Biochemical reaction indicated that XZ-4-5 was identical with standard strain of B. t. var. aizawai. 130~145kDa ICP was encoded in XZ-4-5 by SDS-PAGE analysis. Gene cry7 of XZ-4-5 was located on plasmids. 1466 base pair nucleotide of cry7 gene was cloned with PCR amplification, and the sequence had been registered in GenBank (the registered No. is AY952319). |
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