提取钝裂银莲花叶片中的基因组DNA,用来克隆其Actin基因序列片段,为研究其生理功能及其他基因在钝裂银莲花中的表达和调控奠定基础.根据GenBank中登录的植物肌动蛋白基因同源核苷酸保守序列设计简并引物.以钝裂银莲花叶片总DNA为模板,利用RT-PCR技术获得了3个Actin基因片段.序列分析表明,3个Actin基因片段长为780bp,含有一段内含子,第一段外显子编码128个氮基酸,具有2个Actin基因的功能位点;与其他植物同源序列进行分析表明,其核苷酸序列同源性在81％-90％之间,氨基酸序列同源性在95％-100％之间.第二段外显子编码84个氨基酸,与其他植物同源性序列进行分析表明其氨基酸序列同源性在94％-99％之间.本研究中得到的3个基因序列是Actin基因的同源片段,分别命名为AOACT1,AOACT2,AOACT3.

以3种花色(黄色、浅黄色和白色)钝裂银莲花为研究对象,分别采用TTC染色、I2-KI染色、无机酸测定和蔗糖萌发4种方法,测定了钝裂银莲花(Anemone obtusiloba)的花粉活力,同时比较了不种方法对花粉活力的测定效果,并了解了花粉活力在一天内的动态变化情况.结果表明:(1)钝裂银莲花花粉可育率很高,达80％左右.花粉直径为28.75～33.75μm.(2)TTc浓度影响花粉活力的测定,当浓度为0.5％时,所测花粉活力的效果较好.(3)I2-KI法的染色效果不理想,染色率仅为8.33％～22.2％,且3种花色花粉活力测定数据间差异不显著.(4)3种花色钝裂银莲花花粉活力有差异,黄色最高,浅黄色次之,白色最低.TTC染色法是简单快速测定钝裂银莲花花粉活力的最适方法.一天内花粉活力在12:00～ 14:00最大.

Eleven species of Anemone obtusiloba collected from 4 areas of Gansu [Hezuo(HZ),Luqu(LQ),Awancang(AWC),and Azi(AZ)] were analysed by inter single sequence repeats(ISSR) to elucidate the inherit diversity level and structure.Seven hundred and one bands were amplified by 13 primers and 392 bands were polymorphic,a polymorphic locus percentage(P) of 55.92%.The Shannon index(I) was 0.372 and Nei's gene diversity(H) was 0.250.Based on Nei's Gst value and analysis of molecular variance(AMOVA),showed that species inheritance of differentiation of A.obtusiloba existed mainly.In inter-populations: I was 0.183 and H was 0.114 at the population level.These data indicated that there were high inherent diversities in the population of A.obtusiloba.Using UPGMA,three populations were clustered: HZ,LQ and AWC,and AZ.Further analysis showed that there was no correlation between population genetic diversity level and altitude,water,extractable phosphorous or longitude and latitude,but there was a significantly negative correlation with precipitation.

The genome DNA extracted by the improved method of CTAB from Anemone obtusiloba collected from Hezuo region in Gansu was used as mode to optimize the ISSR reaction conditions by orthogonal experiment and single factor experiment. This study showed that the optimal conditions of ISSR reaction for A. obtusiloba were 20 μL reaction system, and it contained 10× Buffer 2 μL, Mg2＋ 2.5 mmol . L -1 Taq enzyme 2 U, Genome DNA 2 mg. L-1, dNTPs 0.3 mmol- L-1 and ISSR primer 0.4 mmol .L -1. The process of PCR were 94 ℃ for 5 min, 35 cycles （94 ℃ 30 s,51 ℃ 45 s,72 ℃ 90 s）, and 72℃ for 7 min, and the consult based on primer UBC 807.