【作者】 杜金友；

【导师】 陈晓阳；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2005， 博士

【摘要】 胡枝子属（Lespedeza Michx.）,是优良的水土保持和饲料型豆科灌木。有关胡枝子干旱生理、遗传改良等方面研究还处于空白阶段。本研究通过干旱胁迫实验,测定了胡枝子6个种的渗透调节物质的变化,在此基础上,选择美丽胡枝子（L.formosa（Vog.）Koehne）为材料,研究了再生系统和遗传转化体系,将抗逆基因Sac B导入美丽胡枝子中,并通过分子检测以及功能验证,获得了转基因植株。 干旱胁迫可以导致胡枝子体内可溶性蛋白含量的持续下降,热稳定蛋白含量在一个短暂的升高后,迅速下降。可溶性糖含量随着土壤相对含水量的下降逐步升高。从本试验结果来看,可溶性糖在胡枝子渗透胁迫的调节方面可能起到了重要作用。 以美丽胡枝子为材料,研究6-BA、2.4-D以及NAA等激素条件下不定芽的分化、增殖和生根情况。实验结果表明,6-BA 0.5mg/L+2.4-D1mg/L+NAA 0.5mg/L获得的有效不定芽最多,美丽胡枝子子叶节分化不定芽受6-BA影响较显著,其比较适宜的浓度应在0.5～1mg/L之间。获得高质量的增殖不定芽的适宜6-BA浓度应在0.1～0.2 mg/L之间。NAA浓度对不定芽生根有极显著影响,生根的最适NAA浓度在0.1～0.5mg/L之间。由此,建立了美丽胡枝子的再生系统。 美丽胡枝子对抗生素耐性较强。当卡那霉素的浓度大于350mg/L,才可以抑制子叶节不定芽的分化。但是高浓度的卡那霉素（Kan）对美丽胡枝子不定芽生根具有较强烈的抑制作用,羧苄青霉素（Carb）对子叶节不定芽分化及茎段增殖的抑制作用更弱。据此确定了抗生素筛选培养浓度:不定芽分化培养为Kan350 mg/L+Carb500mg/L;继代培养为Kan300mg/L+Carb500mg/L;从第二次继代后,羧苄青霉素的浓度可以降到300mg/L;生根培养为150mg/L+Carb300mg/L。通过实验,确立了美丽胡枝子的遗传转化体系:（1）以生长健壮的美丽胡枝子无菌苗子叶节为材料,预培养2d;（2）将子叶节在OD₆₀₀值为0.5左右的农杆菌菌液中浸染30-35min;（3）吸干菌液后,在含100uM乙酰丁香酮的MS₀培养基中,黑暗共培养2天;（4）转至含有350mg/L Kan和500mg/L Carb的分化培养基中进行筛选。 据PCR检测结果,转基因植株与载体上原基因的PCR结果在大小上是一致的;PCR-Southem检测结果显示,转基因美丽胡枝子扩增出的片段与探针能够杂交,插入美丽胡枝子基因组中的外源片段在核苷酸序列方面也与原基因一致;从RT-PCR检测结果看出,插入的cpy-Sac B基因已经正常表达。所有这些实验验证了目的基因已经整合到了美丽胡枝子的基因组中。 通过模拟胁迫验证了转Sac B基因美丽胡枝子的功能。转基因植株的可溶性糖含量比未转化苗有较大幅度提高,可以在NaCl 200mmol/L以及PEG 5%中生长,而未转化苗停止生长并黄化及有部分植株落叶。说明转化材料携带的Sac B基因能够表更多还原

【Abstract】 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.更多还原