Developement Of Insecticidal Bt Rice With Blast Resistance And The Study Of Transgenic High Lysine Rice

Rice is widely cultivated worldwide, and serves as a significant staple food for more than 50% of world population. However, rice yield suffered great damages by lepidoptern insects in most of rice-producing areas. In addition, rice is a major source of protein for animals, but the protein in rice has an incomplete amino acid profile because of a deficiency in essential amino acids for humans and livestock. With the development of plant biotechnology, the genetic engineering approach has been comprehensively applied in improvement of insect-resistance and grain nutrition.In this study, we elvaluated the insect-resistance of cry1Ac gene in 208S line and its hybrids; For the improvement of blast resistance in target line, blast resistant Pil and Pi2 genes were pyramided into 208S, and genetic background of the improved line was assayed by molecular markers in the whole rice chromosome. Furthermore, we examined the lysine (Lys) content in LYSINE-RICH PROTEIN (LRP) introduced line, PA110, and its hybrids; For evaluation of the effect on increasing Lys by LRP gene in different genetic backgrounds, the foreign LRP gene was introduced into an elite cultivar 9311 by cross and marker-assisted selection (MAS), and assayed the Lys content in corresponding lines. The results and conclusions are as followes:(1) CrylAb/lAc protein content by Elisa quantitative analysis showed a downward trend with plant grown in both photoperiod-and thermo-sensitive male sterility (PTSMS) rice,208S, and its hybrid Zheyou 3; The concentration of CrylAb/lAc protein in leaves and stems were much higher than that in endosperms and roots; The content of CrylAb/lAc protein got an identical level as donor, Huahui No.l (TT51) in all detected stages; In parental line and its hybrid, the CrylAb/lAc protein content was stably expressed, and no dominant dosage effect in the crylAb/1Ac gene was observed.(2) 208S and its hybrids by Southern blot hybridization analysis showed the same transgene copy number as donor parent, TT51, which has two copies of crylAb/lAc gene.(3) The PTSMS line 208S showed favorable agronomic and yield traits, which are suitable for PTSMS application; In addition,208S has excellent grain quality and high combination.(4) For the improvement of blast resistance in target line, blast resistant Pil and Pi2 genes were pyramided into 208S. In addition,120 markers with polymorphism between 208S and donor parent Hual517B were used for the genetic background analysis, and the recovery ratio was 96.7% through 3 times backcrossing with 208S. Furthermore, the main agronomic traits in improved line had no significant differences from backcrossed parent; the fertility analysis of pyramided line showed that it was completely sterile in Hangzhou summer, and partially fertile in Sanya winter, which illustrated the same fertility features as parent line 208S.(5) The pyramided 208S line with crylAb/lAc/Pi1/Pi2 genes showed desirable blast resistance compared with parent 208 S line; the improved line got outstanding resistance to rice leaf folder (RLF, Cnaphalocrocis medinalis Guenee) in Sanya.(6) LRP expressing pattern analysis by qRT-PCR showed that the LRP transcripts dramatically increased with the development of seed, but only a few amounts were detected in leaves from seedling to mature stage in pyramided line; these suggested that GT1-driven LRP gene was expressed in an endosperm-specific expression manner; the key genes involving in Asp metabolism were significantly influenced by the introduced foreign LRP gene; the result also indicated that the Lys enrichment in PA110 endosperm probably resulted from the reduction of amino acids catabolism; the expression of 9 genes involving in storage protein synthesis showed significant differences between PA110 and wild-type.(7) The analysis of amino acids content showed that Lys content in the brown rice and milling rice of PA110 got 30.1% and 28.0% increases compared with wild-type respectively; the total amount of all amino acids had 21.3% and 22.4% increases respectively; in PA110 leaves, the free amino acids presented an upward trend with plant growth, and showed a significant increases of all free amino acids besides Lys at mature stage compared with wild-type.(8) The introgression line with LRP gene under 9311 genetic background was developed by MAS and backcrossing breeding; the improved line 9311LRP had only 7.3% increases of Lys in seeds, while most amino acids showed a small decreases compared with parent control 9311. These indicated that the different genetic backgrounds may influence LRP gene effects on Lys fortification. In the brown rice of hybrid PA110/9311, Lys content had an obvious increases (19.6%) compared with hybrid control PA64S/9311. However, the Lys concentration only increased 2.4% compared with PA64S/9311, which suggested that LRP gene in sterile line rather than in restorer line may have better performance on Lys increase in hybrid application.In addition, the hybrid PA110/9311LRP with homozygous LRP genotype had smaller Lys increases (9.2%) than the hybrid PA110/9311 with heterozygous LRP genotype (19.6%), indicating that LRP gene under heterozygous LRP genotype may have more application values in Lys enrichment than that under homozygous LRP genotype.(9) Plant height, seed germination rate and 1000-grain weight were significantly lower than that in wild-type; furthermore, the introgression line 9311LRP also had worse performance on these agronomic traits, suggesting that LRP gene has some negative effects on grain traits. However, its hybrid PA110/9311 showed little differences from the hybrid control PA64S/9311 in most agronomic traits, especially seed germination and 1000-grain weight; PA64S/9311 LRP and PA110/9311 LRP showed above negative effects. These results indicated that the LRP expression in different genetic backgrounds illustrated distinct performance on grain traits, and using sterile line with a moderate expression of LRP gene to cross to obtain hybrids may have great values on Lys improvement in rice seeds.(10)LRP and cry1Ac genes were pyramided into 9311 line with MAS and crossing. The pyramided line showed excellent resistance to RLF and striped stem borer (SSB, Chilo suppressalis Walker), indicating that LRP gene had little influence on insect-resistance by crylAc gene. While, the Lys content in pyramided line was significantly lower than that in gene donor,9311 LRP, suggesting that LRP gene was probably interfered by cry1Ac gene on Lys fortification in seeds.