Salt-tolerance And Disease-resistance Breeding Of Wheat Transgene

Common wheat (Triticum asetivum L.) is one of the most important crops for food supply in the world. It is a mainly planted crop in the northern area of China except for northeast province. But its output has being affected severely, because of attack of insect, bacteria and fungi, as well as the entironment deterioration duo to soil salinization and desertization. It is a most effective method to breed new abiotic stress and disease tolerant wheat species. Beside the traditional way of breeding, the great progress in plant gene engineering, in particular the monocotyledon plant gene transformation by Agrobacterium, along with the more and more new functional genes cloned, great ardour has being given to breed wheat with high quality, salt- tolerance and dry-tolerance and antidisease by genetic engineering. In this thesis, we studied the transgenic method mediated by A. tumefaciens, as well as explored the effect of vacuolar Na~+/H~+ antiporter gene to salt tolerance and antifugal a-thionin DB4 gene to powdery mildew of wheat.1. Impact ofNa~+/H~+ antiporter gene AtNHX1 on salt tolerance of common wheatWheat productivity is severely affected by soil salinity mainly due to Na~+ toxicity to plant cells. To improve the yield performance of wheat in saline soils, we have generated transgenic wheat expressing a vacuolar Na~+/H~+ antiporter gene AtNHXl from Arabidopsis thaliana, to enhance the capacity of reducing cytosolic Na~+ by sequestering Na~+ in the vacuole of plant. 1.1 Young embryos and embryogenic calli infectionThe strain of A .tumefaciens GV3101, AGL1, EHA105 carrying npt â…¡gene and AtNHXl gene drove by promoter 35S in vector pROK2 was used to transfer wheat c.v. Yanyou 361, Yan 103 and Hesheng 3. Young embryos, younger calli and long-term cultured embryogenic calliderived from young embryos were infected and co-cultured with A.tumefaciens. The transformed explants were selected by paromycin 50-150mg/L and 26 resistant plants generated from 92 calli were identified by PCR amplification with the primers of AtNHXl genes. The positive frequency of PCR was about 1.3(YanlO3) and 2.9%(Hesheng 3) respectively, and one copy or two copies integration in the wheat genome was confirmed by Southern hybridization. Chromsome in situ hybridization also found fragments integrated to the position closed to telomere. Transgenic lines had different degree of AtNHXl gene expression confirmed by RT-PCR, and there .was no proportional relation between the copies of foreign gene and their expression quantities.The salt-tolerance of transgenic lines were analyzed from flowing physiochemical index:(D Survival rates of transgenic wheat were higher than control wheat after they were treated by 170mM and 300mM NaCl for 5 days. Under 170mM NaCl concentration, survival rate of transgenic line B53 is twice that of control wheat, and under 300mM NaCl solution, all control wheat were died, while 38% transgenic line B53 escaped.(D In the different salt solution with 0, lOOmM, 200mM and 300mM NaCl, germination rates of transgenic wheat were also higher than control. Under 300mM NaCl concentration, germination rates of line B53 were 50%, while control wheat were only 38%.(3) The AtNHXl transgenic wheat lines exhibited improved biomass production such as, shoot fresh and dry weight, length and number of tillers, root fresh and dry weight and length, at the vegetative growth stage and germination rates in severe saline conditions. The B59(Null) without AtNHXl from segregated from transgenic line have lower biomass than transgenic wheat as the control wheat. So salt-tolerance improving was brought by AtNHXl, not due to variation of tissue culture.? A field trial revealed that the transgenic wheat lines produced higher grain yields and heavier and larger grains in the field of saline soils with 0.5% and 0.7% NaCl.(5) The transgenic lines accumulated a lower level of Na+ and a higher level of K+ in the leaves than non-transgenic plants under saline conditions (100 mM and 150 mM NaCl). These results indicate that the salt tolerance of wheat and grain yield in saline soils can be improved by enhancing the level of the vacuolar Na+/H+ antiporter.1.2 Shoot meristem infectionShoot meristem of wheat Hesheng 3 was uncovered and infected by A. tumefaciens AGL1 harbored with AtHNXl gene promoter. DNA was extracted from plant with new leaves for PCR and Southern blot analysis. Eighteen transgenic plants obtained from 100 infected shoot, and the transformation efficient is higher in this test than in the calli infection. The yields of partial transgenic lines are almost 170% of control plants. This reveals that the expression frequency ofAtNHXl is higher when it was driven by promoter Ubiquitin than 35S.2. Na+/Ff antiporter gene cloned from Festuca arundinaceaAccording to above results, it is necessary to research the action of vacuolar Na+/H* antiporter gene of monocot. salt plant to wheat salt resistant improvement, so as to breed transgenic wheat with stronger salt resistance. The translated protein sequence has 97.04% homogous to wheat antiporter, while has a low homogous to that of A. thaliana. It provides a better choice of gene source for the salt-tolerant breeding of wheat and other monocot crops through gene engineering.The acting mechanism of Na+/H* antiporter gene to wheat breeding is discussed.3. Impact ofa-thionin gene DB4 on powdery mildew resistance of common wheatAntifugal a-thionin DB4 gene, drove by specific ribulose bisphosphate carbxylase/oxygenase (rubisco) activase promoter (RACP) of green tissue or osmotic protein promoter, along with npt II gene was transformed to wheat c.v. Hesheng 3 and Jinan 177 mediated by A. tumefaciens. Plants with npt II resistance were confirmed haying foreign genes of both npt II and DB4 in wheat genome by PCR and Southern blotting analysis. Part of transgenic progenies lightened and delayed to appear powdery mildew.In combination 1 with 3, we compare and discuss the affected factor on wheat transformation via A. tumefaciens.