【作者】 贾彩凤；

【导师】 李艾莲；

【作者基本信息】 中国协和医科大学 ， 生药学， 2008， 博士

【摘要】 金荞麦（Fagopyrum dibotrys（D.Don）Hara）,为蓼科（Polygonaceae）荞麦属（Fagopyrum）多年生草本植物,其根茎具有清热解毒、消肿止痛、治疗肺癌等功效。由于过度盲目采挖以及生态环境污染等,野生金荞麦资源遭到毁灭性破坏,1999年列入《国家重点保护野生植物名录（第一批）》中。进行优良品种选育,创造新的种质资源成为金荞麦资源可持续发展的关键。辐射处理具有较高的突变频率,诱发植物的体细胞发生变异,经无性繁殖遗传给后代,后代性状稳定快,育种周期缩短,成为获得新种质资源的有效途径之一。本研究首次对北京地区引种的江苏产地金荞麦（绿茎）的根茎进行⁶⁰Co-γ射线辐射,辐射剂量分别为0、5、10、15、20和30Gy,剂量率2.005Gy/min。在M₂代辐射剂量分别为5、10、15和20Gy的处理中,获得红茎突变株,并在本生长季进行扦插繁殖。从中选择生长健壮的绿茎和红茎植株,对不同发育时期（苗期、生长期和开花期）的农艺性状、光合特性、生理指标变化及有效成分含量差异等方面进行分析评价,研究辐射处理对金荞麦生长发育的影响,并利用AFLP分子标记进行植株的遗传多态性分析。研究结果概括如下:1农艺性状分析辐射效应对不同类型金荞麦的生物学效应具有很大差异,绿茎植株的开花期较对照提前,且单株根茎干重提高;红茎植株的开花期则相对滞后且单株根茎干重降低。辐射除对两类金荞麦（绿茎、红茎）的株高具有明显抑制作用外,各时期的一级分枝数、主茎节数、叶柄长和叶面积等性状影响差异不显著。2光合特性研究绿茎植株不同发育时期的叶绿素a、叶绿素b及总叶绿素含量均高于对照,红茎植株各时期的叶绿素含量随辐射剂量增强而逐渐减少;两类会荞麦的类胡萝卜素含量分别与各自的叶绿素含量变化趋势相反。辐射对绿茎植株的叶绿素a/b无明显影响,红茎植株的叶绿素a/b增加。两类金荞麦的净光合速率P_n、气孔导度G_s和水分利用效率WUE随辐射剂量增强而逐渐降低,蒸腾速率T_r值则逐渐升高。胞间CO₂浓度C_i随辐射剂量增强而逐渐降低。这表明辐射处理对金荞麦光合作用的限制主要是由气孔限制因素引起的。两类金荞麦苗期和生长期的初始荧光F_o、原初光能转换效率F_v/F_m、光化学量子效率φPSⅡ、电子传递速率ETR和光化学猝灭系数qP逐渐降低,而开花期呈升高趋势。各时期非光化学猝灭系数NPQ显著提高,通过热耗散作用保护光合机构。同一辐射剂量内,绿茎植株各时期的叶绿素含量均高于红茎植株,类胡萝卜素仅开花期较高。绿茎植株苗期和生长期的P_n、、F_o、F_v/F_m和qP均高于红茎植株,开花期各参数值较低。NPQ在各时期均表现为红茎植株较高于绿茎植株。3生理指标测定细胞膜透性、过氧化物酶活性和类黄酮含量等是衡量植物在辐射胁迫条件下抗性强弱的重要生理指标。辐射处理导致金荞麦的质膜透性加大,膜脂质过氧化,MDA含量增加。SOD、CAT、APX、GR酶活性和类黄酮含量均显著高于对照,保护膜系统减少活性氧危害。除SOD、POD、CAT和花青素含量在个别时期随辐射剂量增强而逐渐降低外,其它指标均随辐射剂量增强而逐渐升高。同一辐射剂量内,SOD、类黄酮、质膜透性各时期均表现绿茎植株较高,GR变化趋势相反;POD、APX酶活性苗期和生长期时绿茎植株较高,CAT变化趋势相反。MDA、花青素含量表现苗期和开花期绿茎植株较高,生长期各指标含量降低。4有效成分含量研究辐射处理对金荞麦的有效成分含量具有显著影响,绿茎植株除15Gy与对照无差异外,其它处理与对照之间的（-）-表儿茶素含量均达到极显著差异（p＜0.01）。红茎植株除5Gy外,其它处理的（-）-表儿茶素含量与对照均达到极显著差异（p＜0.01）。本试验中,作为研究对象的辐射处理植株的（-）-表儿茶素含量均高于对照,其中红茎植株15Gy的（-）-表儿茶素含量最高达到0.1027%,绿茎植株20Gy的（-）-表儿茶素含量最高为0.0841%。这表明利用辐射处理对金荞麦进行（-）-表儿茶素含量的改良是可行的。5 AFLP分子鉴定利用AFLP技术能在早期从分子水平上对金荞麦辐射后代进行鉴定和选择,提高育种效率。8对引物组合共扩增出944条清晰可辨的带,其中700条为多态性带,平均多态性位点为73.99%。用UPGMA法进行聚类分析,相似系数在0.74～0.87之间。辐射后金荞麦植株的基因组变异程度与辐射剂量成正相关,即随辐射剂量增强,植株的变异程度逐渐增加。总之,金荞麦通过降低光合色素含量、提高抗氧化酶活性及生理物质含量等方式调节并适应辐射处理的伤害。辐射处理不仅提高金荞麦的有效成分含量,还能增加种质的遗传多态性。辐射诱变技术的应用,为金荞麦的新品种选育工作开拓崭新的领域,对于提高中药材的产量和质量具有重要意义。更多还原

【Abstract】 Buckwheat（Fagopyrum dibotrys（D.Don.） Hara.）,an erect perennial herb of Polygonaceae,whose rhizome is a traditional folk medicine for treatment of lung abscess, dysentery,rheumatism and tumefaction.It had been one of the "List of National Key Conservative Wild Plants in China（the first group） "because of destruction by blind harvest and environment pollution.γray had been one of the most efficient ways to create mutants in plants,with the advantages of convenient operation,short cycle and high somatic mutation.Buckwheat rhizomes of Beijing introduced from Jiangsu of green stem were radiated byγray with dosage of 0,5,10,15,20 and 30Gy at rate of 2.005Gy/min.Red stem mutants were induced in 5,10,15 and 20Gy of M₂ genenration.Agronomic traits, photosynthesis and chlorophyll fluorescence,antioxidative enzymes and（-） epicatechin of selected green and red plants were analyzed in the paper,and AFLP technology was applied for genetic diversity.Results showed as followed:（1） Radiation enhanced the flowering and root yield of green stem buckwheat,while delayed those of red mutant.In addition to the obvious inhibition of plant height,there were no significant effect on the other agronomic traits,such as number of branches, number of main stem,petiole length and leaf area during all period.All traits of green plant were larger than red mutant except for petiole length under the same radiation dose.（2） Chl a,Chl b and Chl a+b of green buckwheat were higher compared with control,while those of red mutant lowered,both decreasing with elevating dose ofγray.Car content of both green and red buckwheat was contrasted to Chl content,respectively.There was no significant effect on Chl a/b of green buckwheat,but that of red mutant increased byγradiation.P_n,Gs and WUE of both buckwheat reduced while Tr increased byγray,and Ci decreased withγray increasing,which showed that leaf photoinhibition was the result of stomatal limitations.Fluorescence parameters,such as F_o,F_v/F_m,ΦPS2,ETR and qP declined significantly as compared with control due to leaf photoinhibition during seedling and growth stage, while enhanced in flowing.NPQ increased significantly to protect PS2 by enhancing thermal dissipation during all period.Chlorophyll content of green buckwheat was higher than red mutant during all period, carotenoids just higher in flowering period under the same radiation dose.P_n,F_o,F_v/F_m and qP of green buckwheat were higher than those in red mutant in seedling and growth stage, while lower in flowering period.NPQ of red mutant were higher than green one on the whole period.（3） Membrane permeability,antioxidative enzyme and flavonoids are the significant index resistant to radiation.Membrane permeability was accelerated byγradiation,and MDA content increased as the result of membrane oxidation.The physiological index,such as SOD,CAT,APX,GR and flavonoids were significantly higher than those in control. Except that SOD,POD,CAT and anthocyanin content decreased in some period,others increased gradually with rising dose ofγray.Membrane permeability,SOD and flavonoids of red mutant were higher than those in green during all period under the same radiation dose,that opposited to GR.POD and APX of green buckwheat were higher than those of red mutant in seedling and growth period, while decreased in flowering period;that were contrary to CAT.MDA and anthocyanin of green buckwheat were higher than those of red mutant in seedling and flowering period, while decreased in growth period.（4） Active ingredient of buckwheat was affected significantly by radiation treatment.There was significant difference between that of green buckwheat and control except for 15Gy （p＜0.01）,and between red mutant and control except for 5Gy（p＜0.01）.（-）-epicatechin content of plant radiated byγray was higher compared with control,with high value of 0.0841%in 20Gy of green buckwheat and 0.1027%in 15Gy of red mutant.It was feasible to improve（-）-epicatechin content of buckwheat byγradiation.（5） AFLP technology was applied in mutant identification and selection to improve the breeding efficiency at the early stage.944 pieces of clear bands were amplified by 8 pairs of primers combinations,of which 700 were polymorphic with an average polymorphism site of 73.99%.UPGMA showed the resemble index from 0.74 to 0.87.Genome variation of buckwheat was positive related to radiation dose.In conclusion,buckwheat adapted to radiation injury through lower chlorophyll content,higher antioxidative enzyme and active ingredient content.（-） epicatechin content and genetic diversity increased byγradiation.The application ofγradiation opened new field for medicinal plants variety selection,and had great significance in yield and quality improvement.更多还原