The Establishment Of The W-linked Transgenic Line And The A Tentative Exploration On Gene Targeting By ZFN In Silkworm

The sterile insect technique (SIT), Which play an important part in Area Wide integrated pest control(AM-IPC), is species-specific and eco-friendly. We employed too much organic pesticides and contributed a bad environment in the past. The wide use of the SIT can reduce the dependence of organic pesticides and creating a friendly environment. In most cases, release males only is desirable, mixed-sex releases can cause the assortative mating, thus reduce the efficiency of the programs. Furthermore, many sterile females may cause damage themselves. Separate the male and female pest or to eliminate the females in an efficient way via Genetic Sexing Strains are important in a successful SIT program.Bombyx mori, economically important and model organism of Lepidopteran. Besides SIT program in Lepidoptera, developing genetic sexing strains in silkworm can brings significant economical benefit. In sericulture facilities, males are easy to raise, take less mulberry leaves and not vulnerable to the disease. Males also produce 20% more silk and better quality in silk than females. So, the mass rearing of male only will push the advancement of silk industry. The Soviet Union, Japan and China researchers have carried out some useful exploration. However, the most useful type of sexing mechanism developed for silkworm has been based on the construction of balanced lethal strains by Strunnikov. This method needs a suitable marker gene, as well as long-term screening and labor force, high cost made it difficult to promote among different species. Marec et al. proposed a genetic sexing strategy using W-linked transgene insertion in Lepidopteran. A conditional lethal gene inserted into the W chromosome, certain conditions, the female death and the males(ZZ) of this genetic sexing strains are fully wild type. The release program would be less likely to be rejected publicly on the basis of concerns about genetically modified organism. But, such genetic sexing strains had not been reported to date.Zinc-finger nucleases (ZFNs) are fusions between zinc-finger DNA binding domains and the non-specific nucleic acid enzymes FokI domain. An appropriately designed ZFN can create a double-strand break (DSB) at a single predetermined site in the genomic DNA of an organism, cells efficiently repair DSBs via the homology-directed repair (HDr) or non-homologous end joining (NHEJ) pathways. Because of the ZFNs, We can manage the genetic information by gene disruption, gene correction and targeted gene addition. Although ZFNs achieve great success in many model organisms, In silkworm, whether ZFN can achieve a highly efficiency in gene targeting needs further experimental evidence.To investigate the possibility of W-linked transgensis in silkworm, Bombyx, mori, we screened the sex ratio of 160 transgenic silkworm lines, two of which appeared sex-specific expression pattern. One transgenic line was confirmed to be female-specific expression. Southern blotting and inverse-PCR analysis showed that the transgene was inserted into the W-chromosome. We designed a ZFN reagent that target the green fluorenscent protein (GFP) gene. Direct embryo injection of ZFN-ecoding mRNA into the transgenic line pBac[Serl-the Red-sv40,3xp3EGFP]. Explore the gene targeting efficiency of GFP-ZFN mRNA in silkworm. The main results are as follows.1. Isolation of the W-linked transgensis in silkwormWe screened the sex ratio of 160 transgenic silkworm lines, the fluorescence of two lines,158 and 159, were different between males and females. The fluorescence of the offspring of two line were examined through back crossing or sibling mating, the transgene line of 158 is not the candidate of our expectation and the 159 offspring showed different between males and females. All the female offspring of the transgenic line 159 were EGFP positive, some of the male offspring showed EGFP positive, which suggested that a W chromosome linked transgene was included together with a autosomal trans gene in the transgenic line 159. To isolate the W chromosome linked transgenic line, individuals were back crossing and the offspring were investigated. Finally, an offspring line numbered 30-13 showed female-specific fashion was isolated. According to the classical Mendelian genetics, the 30-13 transgenic line must be the W-linked transgensis.2. Molecular detection of W-linked transgenic silkwormGenomic DNA of the transgenic line 30-13 was extracted. The EGFP primer were designed, genomic DNA of wild silkworm as a control, PCR analysis initially proved that the transgenic line 30-13 was the W-linked transgensis. In order to determine the copy number of the transgenic line 30-13, Southern blotting analysis was employed and the result showed that only one insertion fragment was integrated into the chromosome of silkworm. Inverse-PCR analysis showed that the transgene was inserted into the W-chromosome.3. A tentative exploration on efficiency of GFP-ZFN mRNA gene targeting in silkwormTo investigate whether the zinc finger nucleases mRNA can be efficient for gene targeting in silkworm. We designed a pair of ZFN targeting the GFP, the silkworm transgenic line pBac[Ser1-Red-sv40,3xp3EGFP] as the experimental group. The sequence of the ZFN was synthesized by a company and the mRNA of the ZFN was transcribed in vitro using the T7 mMessage mMachine transcription kit to generate a single capped transcript containing both open reading frames. Unfortunately, we had not found the knockout individuals by the embryonic injection of the ZFN mRNA.