| Bacillus thuringiensis is widely used in agriculture,horticulture,forestry,storage pest control.More and more Bt biological insecticide and crops of transgenic Bt have been widely developed and applied,and achieved good effect that in the control of harmful insects.At present,many countries of the world are an effective way to delay insect resistance generated in the research,separation and tap new Bt insecticidal crystal proteins with broad-spectrum insecticide resistance is the primary task.But long-term use of a single insecticidal toxins such harmful insect resistance gradually produce,and secondary pests becoming major pests that prevention of harmful insects cause great distress.In this study,the ecological environment in Yunnan region of separation Bt resources and to identify the genotype and analysis of biological activity.This paper also studied on Growth and Development of corn borer,beet armyworm,bollworm which fed on artificial diet containing different Bt strains.The conclusions are as follows:1.Isolation and identification of strains,genotyping and biometrics:from this study were collected 1487 soil samples from different ecological regions in Yunnan,and got 1741 strains,using an optical microscope,which identified 406 strains of Bt,the average separation rate of more than 20%.Using quick and easy method to identify the isolated Bt strains by PCR-RFLP and found 18 genotypes:cry1,cry2,cryll cry4,cry6,cry7,cry9,cry15,cry20,cry26,cry28,cry30,cry40,cry54,cry56,cry73,cyt2 and vip3.Where the cry 1 genotype and cry2 most,achieve the more than 65%;second is cry4,cry28,cry30 genotype,while cry9 type genotype relatively less than normal.These genotypes identified,we also found that kill lepidopteran pests genotype(crylA,cry2A)ones,kill Diptera pests genotype(cry4B,cry20A)less than normal,there are some nematicides(cry6A)and Coleoptera(cry7A)genotype.Select a representative sample of the strain by SDS-PAGE protein electrophoresis analysis and found that these strains mainly expressed stripe size of 130 kDa,90 kDa,80 kDa,75 kDa,66 kDa,55 kDa,35kDa,30kDa and 15kDa.This also shows that Bt strain isolated gene type is very rich.In addition,we also carry out these strains bioassay found that these strains have broad insecticidal activity against lepidopteran pests have a good effect.2,The affect on growth and development of corn borer by feeding artificial diet that containing the Bt bacterial protein:This study selected from strains(LJ76-2,LJ86-1,DL80-1,KM54-1 and DQ62-1)to has a further study.The five strains producing spherical crystal.that can inhibit the growth of corn borer.Using PCR-RFLP and protein gel electrophoresis analysis and identification and found that these strains have multiple genotypes and expression of various protein bands,indicating a possible new genotypes containing these strains.We using artificial diet while containing different strains of Bt proteins to fed corn borer,and found:the five strains can hindered the growth and development,of corn protein.Such as:the rlarvae average body length becomes shorter,the average survival rate is low,less the number of pupation,State insect duration increase,etc.But most can be successfully pupate corn borer can be successful emergence.Therefore,this study feeding semi-artificial diet while containing different protein Bt bacteria to corn borer larvae,and confirmed that the several strains of Bt have some growth inhibitory effect to corn borer in varying degrees.3.The affect on growth and development of beet armyworm by feeding artificial diet that containing the Bt bacterial protein:This research,semi-prepared artificial diet used to feed beet armyworm and observe their growth and development.Analysis of the data shows that these types of bacterial protein on the beet armyworm effects as compared with the control group,there are different differences.The DQ62-1 effects beet armyworm with the control group inoriginal is insignificant,the gradual emergence of differences in the late,but also the integrity of the development later than other groups;LJ76-2 and DL80-1 has a best role to Beet armyworm,both the average length of the worm,and the average survival rate have significant differencesto the control group in the seventh day,in addition,these two sets of pupation rate is the lowest.4.The affect on growth and development of cotton bollworm by feeding artificial diet that containing the Bt bacterial protein:This experiment also studied with different Bt proteins in semi-artificial diet on the growth and development of cotton bollworm,Found the average survival rate in different growth stages is gradually reduced,the growth and development of cotton bollworm affected.By significant analysis showed that in addition to the various experimental groups LJ86-1 differences with other groups,the difference between the rest of the experimental group is not obvious When we observe the growth of the larvae found Earworm initial period of poor feeding phenomenon occurs,and the larvae body length change is not obvious.After some larvae adaptation Bt toxin effects by adjusting its physiological metabolism,to continue the growth and development,and some larvae failed to adjust gradually died.In summary,the in Yunnan Area has a wealth of Bt resources,and there is a wide variety of types.Therefore,in-depth excavation work Bt strains has important significance.Study on growth and development of lepidopteran pests,the results can be used to refer to the Bt protein harmful insects effects,biological effects of harmful insects prevention research and transgenic crops,but also for the provision of new data stand by. |
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