Salt-tolerance Breeding Of Tall Fescue Via Gene Engineering As Well As Cloning, Expressing And Functional Analysis Of Stress Relative Genes Of NHX/TaPOD/TaPOX In Salt-tolerance Plants

Salt stress has a harmful effect on plant growth and seed production, and thereby causes increasing damages to crop productivity worldwide. One possible method, by utilizing the area of saline-alkali land, is to cultivate salt-tolerant plant. Tall fescue (Festuca arundinacea) is an important perennial cool-season grass, and it is widely used for both forage and turf purposes. This grass is a hexaploid outcrossing species with a high degree of selfincompatibility, which makes conventional breeding quite difficult, including breeding for salt tolerance. Development of its salt stress tolerance by genetic engineering is of momentous significance to maintain perennial ever green turf and save water resource. This will also make enlargement of establishment area, and especially can improve ecological environments in western regions of China.To generate salt-tolerant turf and forage, supported by the National Basic Research Program of China (Project No.: 2006CB100100) and the National Transgenic Fund of China (Project No.: 2003/5250322), we had transformed tall fescue (Festuca arundinacea) with AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana. In this thesis, we report the systematical studies on factors affecting plant regeneration and Agrobacterium-mediated transformation of embryogenic calli from tall fescue, as well as the analysis of inheritance and salt-tolerant phenotypes in independent transgenic lines.Furthermore, full-length cDNA of Na⁺/H⁺ antiporter and peroxidase involved in salt stress were cloned from Agropyron elongatum and salt-tolerant somatic hybrid wheat cultivar SR3. We also primarily investigated the effect of these genes on plant stress tolerance.The main results are as follows:1) Impact of AtNHX1 on salt tolerance of tall fescuePlant regeneration system from embryonic calli of eight tall fescue cultivars was established. The four types of eight embryo-derived calli were transformed with two Agrobactrium tumefaciens strains AGL1 and GK3101. AGL1 harboring intion-AtNHX1 expression vector pROK2U containing ubiqutin promoter and npt II marker gene. GV3101 harboring intron-AtNHX1 expression vector pROK2 containing 35S promoter and npt II gene. After infection and co-culture with AGL1 or GV3101, the embyogenic calli were selected with 50-150 mg/L paromomycine. The resistant plants regenerated from the resistant calli were selected further with 10-20mg/L kanamycin. Genome DNA of the resistant plants was checked with specific primers and probe from AtNHX1 gene. The results of PCR assay and Southern blot analysis indicated that exogenous AtNHX1 gene have been transferred into different cultivars of Festuca arundinacea. The result shows different transformation frequencies among the four cultivars.Inheritance of transgenic T₁ and T₂ lines were identified by PCR. Southern blot hybridization showed the uniform loci of the inserted genes among To, plants and some of T₁ and T₂ lines. Most of the transformants carried single transgene copies. RT-PCR shows different expression amount of the trangens in T₂ plants.These transgenic lines showed no phenotypic changes or yield reduction. Germination rate and biomass production under saline conditions used for indicators of salt tolerance, and we found that transgenic plants were more resistant to a 200 mM NaCl solution than control plants. In conclusion, we demonstrated single copy inheritance of AtNHX1 in most of the T₁ and T₂ lines of transformed tall fescue, with a close 1:1 segregation ratio. These transgenic progenies have been approved to release in a given area by Chinese Agriculture Department (No. 2006-T048). 2) Isolation and characterization of full-length cDNA of NHX from Agropyron elongatumFull-length cDNA of TeNHX1 and TeNHX2 genes have been cloned from salt tolerance Agropyron elongatum using the RACE (rapid amplification of cDNA ends) method. Sequence measurement and analysis showed that the nucleotides and deduced amino acid sequences of the fragments were similar to the other NHX genes reported. Moreover, there was also a similar LFFIYLLPPI domain with these genes. Through functional expression in yeast AXT3, we found that the different Na⁺/H⁺ antiports provided different level of salt tolerance with divisive concentrations of NaCl. RT-PCR analysis indicated that the expression of NHX genes had tissue specificity, consistent with the function of Na⁺ compartmentalize.3) Isolation and characterization of the full-length cDNA of the salt-tolerant hybrid wheat line SR3 involved in salt stress.A new somatic hybrid introgression line SR3 from common wheat JN177 and Agropyron elongatum with higher salt-tolerance than the parent wheat has been generated in our lab, which passed Shandong provincial regional yield trial for new salt-enduring wheat cultivar.The full-length cDNA of TaPOD and TaPOX genes have been cloned from salt-tolerant hybrid wheat line SR3 with the DDRT-PCR and 5' RACE (rapid amplification of cDNA ends), according to the information of cDNA-Chip in our lab. The sequences of the deduced amino acids were analyzed, and the gene expressions of SR3 and parent wheat under saline stress were investigated. RT-PCR, Northern bloting and cDNA-Chip revealed that the transcription of wheat peroxidase gene was up- regulated in salt-tolerant line SR3 and down-regulated in JN177 under saline stress. A possible stress-responding mechanism of this gene has been discussed.In combination with the cDNA-Chip analysis, it is suggested that there are two kinds of peroxidase involved in salt stress and drought stress respectively in the genome of SR3, these genes might responsible for the tolerance of stress. All these genes are compellent in the genetic engineering of turf and forage improvement.The TaPOD gene was introduced into SR3 by Agrobacterium tumerfaciens mediated transformation of shoot apical meristem. PCR and Southern blotting confirm the inserts of aim genes to SR3 genomes. The enzyme activity analysis indicated that transgenic T₁ plants could overexpress foreign POD, which exhibited higher expression level of peroxidase activity than the control.