Welcome to visit Scientia Silvae Sinicae,Today is

Scientia Silvae Sinicae ›› 2018, Vol. 54 ›› Issue (4): 38-48.doi: 10.11707/j.1001-7488.20180405

Previous Articles     Next Articles

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

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

CLC Number: