【作者】 梁朋；

【导师】 施和平；

【作者基本信息】 华南师范大学 ， 植物学， 2003， 硕士

【摘要】 本文研究了发根农杆菌（Agrobacterium rhizogenes）ATCC15834对三裂叶野葛（Pueraria phaseoloides）叶片外植体的遗传转化以及培养基中蔗糖浓度、Ca²⁺浓度及光对三裂叶野葛毛状根生长及其异黄酮类化合物产生的影响。结果如下： 发根农杆菌（A.rhizogenes）ATCC15834感染三裂叶野葛（P.phaseoloides）叶片外植体20d后产生毛状根。毛状根能在无外源生长调节剂的MS固体或液体培养基上自主生长。PCR结果证实，发根农杆菌Ri质粒的rolC基因已在三裂叶野葛毛状根基因组中整合并得到表达。培养30d后，三裂叶野葛毛状根中的异黄酮类化合物及葛根素含量分别是非转化根的1.6和5.1倍。 较高浓度的蔗糖有利于毛状根生长，培养16d后，毛状根在含5％、4％、3％蔗糖的MS培养基中的干重增殖倍数分别为11.7、11.9、10.1；而在含2％蔗糖的培养基中的干重增殖倍数仅为5.9。与毛状根培养物的生长曲线相似，毛状根培养物中的异黄酮类化合物含量，随着毛状根培养物的生长而不断提高。其中3％蔗糖浓度有利于毛状根中异黄酮类化合物及葛根素的积累，毛状根的葛根素含量在培养12d后达到最高，约5.147mg/g DW；而培养16d后，其异黄酮类化合物的含量最高，约27.76mg/g DW。在毛状根生长的停滞期，培养基中蔗糖的消耗速度缓慢；在快速生长时期，培养基中蔗糖消耗迅速，同时毛状根中可溶性总糖含量也逐渐增高，至培养12d后达到最高；在毛状根生长的平台期（培养16d后），培养基中蔗糖几乎消耗完。 毛状根在外加Ca²⁺专一螯合剂EGTA的MS培养基中不能自主生长，而培养基中钙离子浓度为5.986、8.979mmol/L时，则明显抑制毛状根的生长及其葛根素的积累，但却有利于毛状根培养物中异黄酮类化合物的积累。 与暗培养相比，蓝光和白光都明显抑制三裂叶野葛毛状根培养物的生长，其干重分别比暗培养下降了27.04％和18.87％；但却能提高毛状根中异黄酮类化合物的含量，其中尤以白光处理的作用最明显，白光下培养的毛状根培养物中的异黄酮类化合物含量分别比蓝光、暗培养提高了17.2％和23.7％；蓝光和白光下培养的三裂叶野葛毛状根的表面呈淡绿色，而暗培养的三裂叶野葛毛状根呈淡红褐色或白色。更多还原

【Abstract】 Genetic transformation of Pueraria phaseolioides with Agrobacterium rhizogenes strain ATCC15834 and effects of sucrose concentrations, calcium and light on the growth and the production of isoflavones in hairy roots were studied. The main results were as follows:Hairy roots of P. phaseoloides could be obtained from the leaf explants 20 days after inoculation with Agrobacterium rhizogenes ATCC 15834 and could have a rapid growth on solid or liquid growth regulator-free MS medium. The transformation of hairy roots was confirmed by PCR amplification of rolC genes of Ri plasmid from A .rhizogenes. 30 days after cultivation, contents of isoflavones and puerarin in hairy root cultures were 1.6 times, 5.1 times, respectively, as much as that in control roots .High concentrations of sucrose in the medium stimulate the growth of hairy roots. The proliferation times of dry weight of hairy roots cultured for 16 days in the MS medium with 5%, 4%, 3% and 2% sucrose were 11.7,11.9, 10.1 and 5.9, respectively. Similar to the growth of hairy roots, the content of isoflavones in hairy roots was increased with the time during culturing.3% sucrose in the medium was most effective for accumulation of isoflavones and puerarin in the hairy roots. In this case, the highest content of puerarin, 5.15mg/gDW was obtained 12 days after cultivation, and the highest content of isoflavones, 27.76mg/gDW after cultivation for 16 days. Sucrose in the medium was utilized slowly at the still stage, but consumed very rapidly at fast growing stage and during this stage, the content of soluble sugar in hairy roots was increased with time and the highest sulable content was also obtained 12 days after cultivation. Sucrose in the medium was almost used up 16 days after cultivation.P. phaseolioides hairy roots couldn’t grow in the calcium-free MS liquid medium containing EGTA. Ca2+ at the concentration of 5.986 mmol/L and 8.979mmol/L dramatically inhibited the growth and puerarin accumulation of hairy root cultures, but was suitable for accumulation of isoflavones in hairy root cultures.In comparison with dark culture, white light and blue light significantly inhibited the growth of hairy root cultures, their dry weight was decreased by 18.87% and 27.04%, respectively, but could enhence the content of isoflavones in hairy roots, especially culturing in white light. The content of isoflavones in puerariaphaseolioides hairy roots cultured in white light was 1.17 and 1.23 times, respectively, as much as that in hairy roots cultured in the dark and in blue light. When cultured in the white light and blue light, the hairy roots of P.phaseoloides looked light green, but white or light red-brown in the dark.更多还原