Establishment Of Plant Regeneration System And Genetic Transformation Of Creeping Bentgrass; Cloning And Sequence Analysis Of Na～+/H～+ Antiporter Gene From Creeping Bentgrass

In recent years, people pay more attention to the habitation and the business of lawn has become a rising industry. Turf can be used to protect and transfigure environment, decontaminate atmosphere, preserve soil and water, reduce noise pollution, it has become an indispensable thing in our daily life. Salinization is the major abiological factor of confining plant growth, so it is very important to breed salt-tolerance species. From the 1970s, genetic engineering technology has been widely used as an efficient method in plant breeding and can solve many difficulties existed in conventional breeding process.Genes with resistant function could be introduced into embryogenic calluses of turfgrasses, and transgenic plants with modified resistance could be regenerated. Rsearches about the damage of salinization and mechanism of salt-tolerance has been carried out for a long time, many salt-tolerance genes have been cloned, and a lot of transgenic plants have been produced.Creeping bentgrass (Agrostis stolonifera L.) is sod-forming perennial plant, it is a kind of cool-season turfgrasses native to Eurasia. Due to its resistances to waterlogging, uniformal growth, slender and closed leaves, creeping bentgrass has been widely used in building of city square, golf course and bowling alley. However, creeping bentgrass lacks the ability of resistance to other stresses and has low staying quality, so there is an increasing demand for new cultivars with desirable traits.1. Regeneration system and genetic transformation.Embryogenic calluses were the most ideal and common receptor of transformation. Up to now, many research results about regeneration system of different gramineous turfgrass explants have been documented. But the regeneration system of Viper, Penn-A-4 and Putter were not reported. The effects of somatic embryogenesis and the hormone concentration on rengeneration of these species also have not been analyzed. In this work, we try to establish the regeneration system of these three genotypes and observe their embryogenesis process, to lay experiment foundation for improvement of their quality.Mature seeds of creeping bentgrass were used as explants, the different stages of somatic embryogenesis were observed and analyzed. The concentration of 2,4-D had remarkable influence on callus induction, somatic embryogenesis and plantlet regeneration of creeping bentgrass. It has shown that 2.0mg/L 2,4-D, 0.1mg/L 6-BA was optimal for callus induction, and the frequency of plant regeneration would decline along with the increase of the 2,4-D concentration. The concentration of 6-BA supplemented in regeneration medium was 1.0mg/L, and the frequency of plant regeneration was above 90% in most case. Supplement of myoinositol with proper concentration in the medium could promote the growth of regenerants and produce strong plants. Somatic embryogenesis of creeping bentgrass included the stages of globular-shaped, heart-shaped, torpedo-shaped and cotyledon-shaped embryoid.In this study, embryogenic calluses of creeping bentgrass were treated with 0.6mol/L and 0.4mol/L mannitol respectively. The calluses precultured 5h and 12h were used as target materials of transformation. The Na~+/H~+ antiporter gene AtNHXl was transferred into creeping bentgrass via particle bombardment and Agrobacterium-mediated methods. The Agrobacterium strain was LBA4404 containing pHZX1. AtNHX1 gene under the control of cauliflower mosaic virus 35S promoter and the terminator region contained the polyadenylation signal of the nopaline synthetase gene was subcloned into pBI121 by replacing the GUS-coding region. The selective marker is npt II gene. The calluses were selected with 100mg/L kanamycin, and finally 6 (biolistic method) and 3 (Agrobacterium method) green plants were regenerated respectively. In PCR experiments, only 1 (biolistic method) and 2 (Agrobacterium method) of them were confirmed to be transgenic plants.Na~+/H.+ antiporter plays an important role in plant salt tolerance. In plant, the tonoplast Na~+/H~+ antiporter is responsible for the compartmentalization of Na~+ from cytoplasm into vacuole.Plant Na~+/H~+ antiporter genes were highly conservative, degenerate primers could be designed according to the conservative sequence of known Na~+/H~+ antiporter genes, and homologous gene could be cloned from other species. In the present work, total RNA of bentgrass was isolated and the first strands of cDNA were synthesized at first, a middle sequence approximately 300bp in size of creeping bentgrass Na~+/H~+ antiporter cDNA was amplified through gradient PCR technology with degenerate primers. After cloning, sequencing and homologous comparison with Blast method, the middle sequence was discovered to have very high homology with known Na~+/H~+ antiporter genes, and the amino acid sequence identity can reach as high as 88%. We can initially determine this sequence as the middle fragment of Na~+/H~+ antiporter gene of bentgrass.We will design special primers according to this middle fragment, and isolate the entire cDNA sequence using 3'-RACE and 5'-RACE. Then expression analysis and function identification will be carried out.