摘要
为了探讨和优化应用流式细胞术(FCM)测定药用植物的基因组大小(或称C值)的方法,分别以新鲜、幼嫩和硅胶干燥的叶片为材料,采用Otto和LB01两种细胞核提取液,利用流式细胞术对黄芩C值进行测定。结果表明,选用豌豆为内标植物时,相比LB01一步法,Otto两步法在低温提取、离心和保存条件下,碘化丙啶染色浓度为0.02 mg/mL,得到的细胞核散点图相对集中,碎片较少,容易准确取门,CV较小。不同提取材料表明,幼嫩叶片和新鲜叶片利用Otto二步法均能得到高质量的细胞核悬液,细胞核数量多且散点图集中,有利于取门和测定的C值更准确。而干燥叶片提取的细胞核得到的散点图分散,无法准确取门和测定C值。最终,系统地优化了利用流式细胞术测定药用植物黄芩基因组大小的方法,并首次成功测定黄芩1C值为0.58 pg,基因组大小约为562 Mbp。这一研究结果,将为了解黄芩的基因组值大小和利用流式细胞术测定其他药用植物C值提供参考,并将为黄芩属药用植物资源的遗传评价和保护利用等研究奠定基础。
To discuss and optimize the methods of estimating nuclear DNA content of medicinal plants by flow cytometry （FCM）, fresh leaves, younger leaves and dry leaves by silica were taken as samples and Otto＇ s buffer and LB01 buffer were used to estimate the C-value of Scutellaria baicalensis Georgi by FCM. The results showed that better results were easily and accurately gotten with relatively concentrated scatterplot, less debris, more simplicity to take gates and lower CVwhen using Otto＇s buffer with two-steps method to extract, prepare and store under low temperature with the concentration of propidium iodide （PI） at 0.02 mg/mL, compared to that by using LB01 buffer. The results of different materials demonstrated that both fresh leaves and younger leaves could get better quality suspension of nuclei by Otto＇ s and generated a larger amount of nuclei and more concentrated particles in scatterplots, which would help take gates and estimate C-value more accurately. On the contrary, younger leaves would lead to a large number of centralized nuclei, which was unable to take gates and estimate C-value accurately. At last, the method of estimating nuclear DNA content of the medicinal plant S. baicalensis was systematically optimized, and the C-value of S. baicalensis was first estimated successfullyas 1 C=0.58 pg, and the genome size was 562 Mbp by FCM. These results will provide references for recognizing the genome size of S. baicalensis and the estimation of other medicinal plants by FCM, and thus lay foundation for the genetic evaluation and conservation of medicinal plants of Seutellaga.
出处
《中国农学通报》
CSCD
2013年第25期130-135,共6页
Chinese Agricultural Science Bulletin
基金
国家自然科学基金"山茱萸种质资源遗传多样性分析及其与产地环境因子适宜性评价"(31100241)
陕西省科技计划项目"黄芩属药用植物种质资源收集和评价研究"(2011K16-02-05)
关键词
黄芩
Otto
LB01
流式细胞术
C值
Scutellazia baicalensis Georgi
Otto
LBO1
flow cytometry
C-value
