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林业科学 ›› 2018, Vol. 54 ›› Issue (4): 38-48.doi: 10.11707/j.1001-7488.20180405

• 论文与研究报告 • 上一篇    下一篇

杉木叶片原生质体分离及RNA提取体系的建立

唐佳妮, 林二培, 黄华宏, 童再康, 楼雄珍   

  1. 浙江农林大学 亚热带森林培育国家重点实验室 杭州 311300
  • 收稿日期:2017-05-02 修回日期:2017-05-26 出版日期:2018-04-25 发布日期:2018-05-28
  • 基金资助:
    浙江省农业(林木)新品种选育重大科技专项(2016C02056-5);浙江省省院合作林业科技项目(2016SY16);国家自然科学基金项目(31300565)。

Isolation and Total RNA Extraction of Leaf Protoplasts in Chinese Fir

Tang Jiani, Lin Erpei, Huang Huahong, Tong Zaikang, Lou Xiongzhen   

  1. The State Key Laboratory of Subtropical Silviculture Zhejiang A & F University Hangzhou 311300
  • Received:2017-05-02 Revised:2017-05-26 Online:2018-04-25 Published:2018-05-28

摘要: [目的]以杉木组培苗幼嫩叶片为材料,分析不同因素对杉木原生质体分离的影响,建立杉木叶片原生质体分离体系,以期为杉木基因功能验证提供有效的技术平台;比较不同RNA提取方法,筛选获得杉木原生质体总RNA提取的有效方法,为今后利用杉木原生质体开展分子生物学研究提供技术支持,也可为其他林木原生质体的总RNA提取研究提供参考。[方法]选取杉木组培苗最上端未分轮的幼嫩叶片为材料,通过分析5种不同酶组合、真空处理时间(0、5、10、15、20 min)、渗透压(0.3、0.4、0.5、0.6 mol·L-1甘露醇)、BSA浓度(0.1%、0.2%、0.3%、0.4%)及酶解时间(1、2、3、4、5 h)5个条件,进行杉木叶片原生质体的分离。采用改良Trizol法、改良Trizol+10 μg糖原法、CTAB-LiCl法、CTAB-LiCl+10 μg糖原法、改良CTAB-异丙醇法、改良CTAB-异丙醇+10 μg糖原法、天根RNA提取试剂盒法共7种方法提取杉木原生质体RNA,通过杉木内参基因和2个特异基因进行RNA质量验证,比较不同方法的提取效果。[结果]在1.5%纤维素酶R-10、1%离析酶R-10、0.2%果胶酶Y-23、0.5 mol·L-1甘露醇以及0.3% BSA的混合酶液中,真空处理10 min,酶解2 h,可分离出高活力的纯净杉木原生质体,其产量可达到7.27×106个·g-1,活力可达到94%。7种方法皆能提取出杉木叶片原生质体的总RNA,但不同方法间存在明显差异,其中改良Trizol法、改良Trizol+10 μg糖原法、改良CTAB-LiCl法、改良CTAB-LiCl+10 μg糖原法提取的RNA有不同程度的降解现象,其余方法所提取的RNA完整性较好,且OD260/280和OD260/230值均在1.8~2.0范围内;在RNA得率方面,改良CTAB-异丙醇+10 μg糖原法可高效提取杉木原生质体RNA,106个细胞RNA产量平均可达到6.89 μg,分别是改良CTAB-异丙醇法和天根RNA提取试剂盒法的1.73倍和1.58倍;综合考虑,改良CTAB-异丙醇+10 μg糖原法提取杉木原生质体RNA的效果最好。[结论]以杉木组培苗幼嫩叶片为材料,分离条件为纤维素酶R-10浓度1.5%、离析酶R-10浓度1%、果胶酶Y-23浓度0.2%、甘露醇浓度0.5 mol·L-1、BSA浓度0.3%,真空处理10 min,酶解2 h,可大量获得高质量的杉木原生质体;利用改良CTAB-异丙醇+10 μg糖原法,可高效提取杉木原生质体RNA,106个细胞RNA产量平均可达到6.89 μg。研究结果为杉木重要性状关键基因的功能研究及其在育种中的应用提供了重要基础。

关键词: 杉木, 原生质体, RNA, CTAB法, 糖原

Abstract: [Objective] To establish protoplast isolation system for Chinese fir(Cunninghamia lanceolata) leaves, the effects of different factors on protoplast preparation were analyzed by using young leaves of Chinese fir plantlets from tissue culture. Meanwhile, the most effective method for extraction of total RNA from Chinese fir protoplast was chosen through comparison of different RNA extraction methods.[Method] The young leaves of Chinese fir plantlets were selected as explants, and the effects of major factors, such as five enzyme combinations, vacuum treatment time (0, 5, 10, 15, 20 min), osmotic pressure (0.3, 0.4, 0.5, 0.6 mol·L-1 mannitol), BSA concentration (0.1%, 0.2%, 0.3%, 0.4%) and enzymolysis time (1, 2, 3, 4, 5 h) were investigated to construct optimum protoplasts isolation system for Chinese fir leaves. Seven RNA extraction methods, such as improved Trizol method, improved Trizol+10 μg glycogen method, CTAB-LiCl method, CTAB-LiCl+10 μg glycogen method, improved CTAB-isopropanol method, improved CTAB-isopropanol+10 μg glycogen method and TIANGEN RNA extraction kit were applied to compare the efficiency of RNA extraction from Chinese fir protoplasts.The quality and yield were detected by reference genes and two specific genes.[Result] The optimum condition for protoplast isolation from Chinese fir leaves included a mixed enzyme solution of 1.5% cellulase R-10, 0.2% pectolyase Y-23, 1% macerozy R-10, 0.5 mol·L-1 mannitol and 0.3%BSA, vacuum treatment for 10 min, and digestion for 2 h. High activity and pure protoplasts could be prepared from Chinese fir leaves by using the optimum condition. The protoplast yield could reach to 7.26×106 per gram with 94% viability rate. All seven methods could extract RNA from protoplasts. However there were obvious differences between different methods. RNA degradation was found in the improved Trizol method, improved Trizol+10 μg glycogen method, CTAB-LiCl method and CTAB-LiCl+10 μg glycogen method.While, the other three methods obtained good RNA quality, of which the values of OD260/280 and OD260/230 were in the normal range of 1.8-2.0. Additionally, the yield of RNA was the highest in improved CTAB-isopropanol+10 μg glycogen method, up to 6.89 μg per 106 protoplasts, which was 1.73 times and 1.58 times of the improved CTAB-isopropanol and TIANGEN RNA extraction kit, respectively. In conclusion, the modified CTAB-isopropanol+10 μg glycogen method was most efficient for RNA extraction from Chinese fir protoplasts.[Conclusion] In this study, by using the young leaves of Chinese fir plantlets, a protoplast isolation technique was established with the condition of 1.5% cellulase R-10, 0.2% pectolyase Y-23, 1% macerozy R-10, 0.5 mol·L-1 mannitol and 0.3%BSA, 10 min vacuum treatment, and 2 h enzymolysis. Meanwhile, the improved CTAB-isopropanol+10 μg glycogen method could be used to extract total RNA from Chinese fir protoplasts with the average yield of 6.89 μg per 106 protoplasts. All the results of this study provided an important basis for the functional analysis of key genes and their application in breeding of Chinese fir.

Key words: Cunninghamia lanceolata, protoplast, RNA, CTAB method, glycogen

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