Development Of Two-toxin Bt Rice And Effect Analysis Of Backcross On Transgenic Rice

The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins that have specific toxicity to lepidopteran insects, and has been applied as a microbial insecticide to the control of crop pests for several decades. With the development of plant genetic engineering, Bt genes encoding insecticidal crystal protein have been introduced into many crop species for pest controlling. Transgenic Bt crops were first commercialized in 1996 and placed a large selection pressure on the target insect populations, so Bt crops face a risk that insect could evolve resistance to them rapidly. Several insect resistance management (IRM) tactics, including gene stacking, have been proposed to delay the development of pest resistance to Bt crops.Rice is one of the most important food crops, and the staple food for more than half of the people in the world. Insect damage is a great threat to rice production worldwide, causing serious loss every year. For controlling the lepidopteran stemborers and leaffolders, many Bt genes have been introduced into rice, and transgenic lines with resistance were obtained successfully. Agrobacterium-mediated transformation is the most common method of developing Bt rice, but its transgenic procedure causes somaclonal variation continually and influences the agronomic performance of transgenic rice plants.In this study, we pyramided two Bt genes to develop two-toxin rice by sexual crossing between four single-gene Bt lines possessing crylAb, crylAc, cry1C and cry2A respectively, and evaluated their insect-resistance and agronomic traits. Meanwhile, we continuously backcrossed three single-gene Bt lines possessing cry1Ac, crylC and cry2A respectively for three times, using their original variety as the recurrent parent, and investigated the agronomic traits of BCF3(n-1,2,3) homozygous lines at last. The main results in this study are as follows:1. The segregation ratios of Bt gene in the F2 generation were detected through PCR analysis, and it was confirmed that inheritance of Bt gene still follows Mendelian segregation in the pyramided progeny. Homozygous rice lines of two pyramided Bt genes were obtained in the F3 generation.2. The results of enzyme-linked immunosorbent assay (ELISA) indicated that the expression of crylAb, cry1Ac and cry2A was comparatively stable after gene stacking, and most pyramided lines contained a comparable Bt protein level to their single-gene parents. The expression of cry1C decreased in two-gene lines. The levels of crylAb, cry1C and cry2A expressed in different tissues in a decreasing order was leaf, endosperm, stem and root, while the decreasing order of cry1Ac was leaf, stem, root and endosperm.3. In the laboratory assay, all transgenic rice lines caused 100% mortality of the first-instar yellow stem borer (Scirpophaga incertulas), and the mortalities of second-instar larvae were higher than 75%. When using striped stem borer (Chilo suppressalis) as test insect, the combinations of lAb+1C, 1Ac+1C and 1Ac+2A caused mortality higher than 80%. Two-gene lines of 1Ac+1C and 1Ac+2A caused apparently higher mortality than corresponding single-gene parents, so the synergistic effects were observed. In addition, larvae of striped stem borer showed higher tolerance to Bt protein compared to yellow stem borer, and the second-instar larvae were less susceptive than the first-instar larvae.4. In the field evaluation, original varieties Minghui 63 (MH63) and Shanyou 63 (SY63) were damaged seriously. All transgenic rice lines and their hybrids exhibited excellent efficacy against stemborers, with few deadhearts. Single-gene line 2A and its hybrid were damaged slightly by leaffolders, while all the other transgenic lines and hybrids had no fold leaves.5. Both single and two-gene rice lines showed some variations in morphological and yield traits, but most two-gene lines had a comparable yield per plant with MH63. Bt hybrids showed relatively fewer variations in agronomic traits, and none of the two-gene hybrids had significantly lower yields than SY63.6. BCnF3 homozygous rice lines were obtained by a germination assay after continuous backcrossing and selfing, and proved to remain resistant to lepidopteran pests. 7. The agronomic traits of three single-gene lines were recovered to a large extent, and the yield per plant of BC3F3 homozygous lines was comparable with that of MH63.8. The transgene insert sites in single-gene parental lines possessing cry1C and cry2A were analyzed detailedly. Results of Southern hybridization indicated that cry1C and cry2A had two and single copy at the single site respectively. The flanking sequences of cry1C and cry2A were cloned by inverse PCR, and located on chromosome 11 and 12 respectively.