Studies On Change Of Osmolytes In Lespedeza Michx Under Drought Stress And Transgenic Lespedeza Formosa With Sac B Gene

The Lespedeza Michx. is a kind of shrub with fine function of water and soil conservation and feedstuff . However, the studies on physiological responses under drought stress and enhancing the ability of drought resistance by technology of the genetic engeering were not carried out for Lespedeza Michx. In this study, regulate changes of osmoregulation material were observed in six species of Lespedeza Michx. in drought stress; On this basis, effective regeneration system of Lespedeza formosa (Vog.) Koehne was established; Sac B gene was introduced into the plants of L. formosa and the transgenic plants were analysed in molecular level. Transgenic plants can grow in medium containing more than 200 mmol/L NaCl and 5%PEGThe accumulation soluble sugar increased gradually under drought stress in Lespedeza Michx. The content of soluble protein drop gradually under drought stress. The content of heat stable protein increased while soil moisture content declined from 75-80% to 55-60%, but droped rapidly while soil moisture content declined from 55-60% to 35-40%. L. potaninii is not same as other species, its content of heat stable protein decreased gradually under drought stress. We supposed that soluble sugar play an important role in osmoregulation of The Lespedeza Michx.The establishment of effective regeneration system of L. formosa is the foundation of genetic transformation. The results indicated that there are most adventitions buds from nodosity in cotyledon in MS medium containing 6-BA 0.5mg/L, 2, 4-Dlmg/L and NAA 0.5mg/L; 6-BA play an important role in production of adventitions buds, and its optimum content is between 0.5-1 mg/L. 0.1-0.2 mg/L 6-BA is better for multiplication, NAA play an important role in rootage of buds, and its optimum content is between 0.1-0.5mg/L.L. formosa can abide high concentration of antibiotics. The growth of adventitious buds from nodosity of cotyledon is restrained when the concentration of Kanamycin is over 350mg/L. While same concentration of Kanamycin restrain intensively root development of adventitions buds. The optimum concentration of antibiotics is respectively 350mg/L +Carb500mg/L, 300mg/L+Carb500mg/L and 150mg/L+Carb300mg/L for the stage ofadventitious buds growth, multiplication and rooting of L.formosa.Nodosity in cotyledon were suitable for inoculation, propaedeutic culture were 2 d; The gene transformation of Sac B with nodosity in cotyledon of L. formosa were conducted by Agrobacterium LBA4404 (OD600=0.5); Dark culture was 2d on adventitions culture medium(AS, 100umol/L); Nodosity in cotyledon were transferred to selection culture medium including 350mg/L Kan and 500mg/L Carb.Transgenic plants were identified by technology of PCR and PCR-Southern.The results indicated that the cpy-Sac B gene had been integrated into the genome of L formosa, and five transgentic plants were obtained. The cpy-Sac B gene has expressed according to RT-PCR.Transgenic plants can accumulate more soluble sugar than the control plants. Transgenic plants can grow in medium containing up to 200 mmol/L NaCl or 5%PEG. But control plants These findings shed new light on the regulation of fructan biosynthesis in plants and the role of Fructan in reducing oxidative stress induced by osmotic stress, in addition to its accepted role as an osmolyte.