Improvement And Application Of The TILLING Technology In Screening The Mutants Of Silkworm Fibroin Genes

With the development of genomes sequencing,the focus is shiftting to discover and explore gene function in the post-genomic era.Construction of large-scale mutation library is an important part of functional genomics.Mutant populations are indispensable genetic resources for functional genomics in all organisms.There are many ways to obtain mutants,such as physical and chemical mutagenesis,gene knock-out,gene silence,insertional mutagenesis,etc,each of which has its special features.TILLING(Targeting Induced Local Lesions In Genomes)is a newly developed reverse genetic technology.Its principle is that:the experimental materials were induced by chemical mutagen and produced mutation population,then the mutation signals may be zoomed by PCR with gene special primers,after heating and annealing,heteroduplexes were recognized and cut at mismatched sites by the endonuclease(eg.CEL I),cut strands were visualized using denaturing gel electrophoresis,lastly target gene mutants can be screened.Compared with other methods this technique has many advantages such as high-throughput,easy operation,economic cost,which overcome the shortage of other mutation methods.TILLING is widely used in functional genomic researches of multiple organisms.So it is believed to have potential application in functional analysis of silkworm.Based on the former researches,we mutagenized the silkworm pupa by chemical mutagens N-methyl-N-nitrosourea(MNU)to construct a mutation library,then we improved the detection conditions and chose fibroin genes as target for the primary application of the TILLING in silkworm.The results are as follows:1.We investigated the lethal dosage 50%(LD₅₀)of the 5th day pupa treated with MNU,the result showed that when the injection volume was 50μL,the median lethal concentration respectively was 1730μg/mL in female pupa,1416.7μg/mL in male pupa;that is LD₅₀(female)=86.5μg/pupa,LD₅₀ (male)=70.8μg/pupa.We also examined the eclosion rate of M₀ generation and hatch rate of M₁ egg.When the MNU concentration was 1000μg/mL the eclosion rate was low and M₁ egg mostly dead in embryo.More live mutagenized individuals could be obtained when the concentration of MNU was 100μg/mL.The silkworm larva of M₂ generation in M18-21 exhibited a quarter-death rate in the 3rd molt,and it still remained in the M₃ generation,which suggested that a recessive lethal mutation caused by MNU might exist.2.A high-throughput method for genomic DNA extraction was constructed.815 genomic DNA samples were obtained and arrayed in 96-well pates.Two 4×DNA pools were constructed for TILLING by one dimensional pooling method,which supplied the basis for applying TILLING in silkworm Bombyx mori.3.Templete quantities in PCR reaction and enzyme reaction system in TILLING were optimized in this research.The current optimal reaction conditions were as follows:the optimal amount of genomic DNA for PCR reaction was～20ng,the best incubation time of heteroduplex digestion by CELI was 15min,the optimum amount of CELI was 0.51μL of diluted in ten-fold solution for every 20μL digestion reaction system.The TILLING detection system established in this study should be helpful to improve the efficiency of screening mutation of silkworm.4.To screen the mutation of Fib-1 and P25 gene,815 MNU-mutagenized M₁ individuals were investigated by TILLING.For Fib-1,a total of 810kb were screened,and two mutations were found and named C3-2,M18-21.For P25,no mutant was found presently after screening of a total of 1500kb.The sequencing results showed that in M18-21,there are two signals in the 107th bp of amplicon Fib-1-C:T and C.The site was located in intron which could not bring change to protein coding.And also,the gel image of TILLING provided support to the mutation of this site.The mutation sample of C3-2 shoud be detected and validated farther.These had suggested that TILLING could be used in silkworm.In conclusion,to apply TILLING to silkworm,we developed a mutagenized silkworm population by treatment with the chemical mutagen MNU.We also optimized the amount of genomic DNA template in PCR reaction and enzyme reaction system.In addition,we detected and identified Fib-1 and P25 genes mutation from mutation library by TILLING At pesent,one mutant induced by MNU was obtained.It is believed that TILLING would become a popular method applied in the functional genomic research of silkworm.