Optimization of Extraction Conditions and Methods for Sugarcane Protoplast RNA

doi:10.3969/j.issn.1000-2561.2019.08.012

Abstract

Abstract:

In order to obtain high-yield and high-purity sugarcane protoplast RNA, this study investigated the osmotic pressure and protoplast viability on sugarcane protoplasts using the sugarcane protoplasts of ROC22 and GT28, and compared the Trizol method, improved Trizol method, improved CTAB method and kit method. The protoplast osmotic pressure was positively correlated with RNase activity, and the protoplast activity was significantly negatively correlated with RNase activity. Higher RNase activity resulted in a worse RNA extraction effect. Using 0.5 mol/L mannitol as an osmotic pressure regulator, the most active sugarcane protoplasts could be obtained. When the protoplast vigor was 70% and 90%, the RNA integrity was good and the purity was high, which met the requirements of subsequent molecular experiments. The RNA yield was the highest when the protoplast activity was 90%. Therefore, when extracting sugarcane protoplast RNA, the activity of protoplast should be at least above 70%. Using the improved Trizol method, a large number of high-quality RNA could be prepared for subsequent molecular experiments. Using this condition and method, it was ensured that a large amount of high quality sugarcane protoplast RNA was obtained. The results of the study provided methods and technical support for the molecular biology research in the somatic cell fusion breeding of sugarcane.

Key words: Saccharum officinarum, protoplast, RNA, viability, RNase

CLC Number:

S566.1

LI Suli,LIU Fangjun,LI Zhigang,LAI Peiheng,LONG Ansi. Optimization of Extraction Conditions and Methods for Sugarcane Protoplast RNA[J]. Chinese Journal of Tropical Crops, 2019, 40(8): 1537-1545.

Figures/Tables 12

Tab. 1 RT-PCR primer sequence

基因名称 Gene name	引物序列（5°-3°） Primer sequence (5°-3°)
GADPH	F：AAGGGTGGTGCCAAGAAGG R：CAAGGGGAGCAAGGCAGTT
DREB	F：GCCTGGTACATCATGCGAGT R：CCATTCTGCCTTTAGGGAGC

Fig. 1 Morphology of protoplast at different osmotic pressures A: Protoplasts in normal form, the cell membrane is flat and clear, and the shape is full; B: Protoplasts with cell membrane shrinkage, the edges of the cell membrane are wavy and vary in shape (as shown by the arrows); C: Protoplasts with increased cell debris, many fragments of protoplast rupture in the protoplast suspension (as shown by the arrows). Bar=20 μm.

Fig. 2 Protoplast viability and yield under different mannitol concentration

Fig. 3 Protoplasts with different viability A: Protoplasts with 30% viability; B: Protoplasts with 50% viability; C: Protoplasts with 70% viability; D: Protoplasts with 90% viability. Bar=20 μm, and blue cells are dead cells stained with trypan blue.

Fig. 4 Effect of protoplast viability on RNA content and RNase activity

Fig. 5 Protoplast RNA content and RNase activity under different mannitol concentration

Tab. 2 Correlation of osmotic pressure with protoplast viability, yield and RNase activity

品种 Variety	指标 Index	甘露醇浓度 Mannitol concentration	原生质体活力 Protoplast viability	RNase活性 RNase activity
ROC22	甘露醇浓度（mannitol concentration）	1
	原生质体活力（protoplast viability）	-0.023	1
	RNase活性（RNase activity）	0.926^*	-0.965^**	1
GT28	甘露醇浓度（mannitol concentration）	1
	原生质体活力（protoplast viability）	0.013	1
	RNase活性（RNase activity）	0.978^**	-0.976^**	1

Fig. 6 Electrophoresis of total RNA from protoplasts with different viability 1: Protoplast with 30% vitality; 2: Protoplast with 50% vitality; 3: Protoplast with 70% vitality; 4: Protoplast with 90% vitality.

Tab. 3 Comparison of purity, concentration and yield of total RNA from protoplasts with different viability

原生质体活力 Protoplast viability/%	D_{260 nm}/D_{280 nm}	D_{260 nm}/D_{230 nm}	RNA浓度 RNA concentration/(ng·μL^-1)	RNA产量 RNA yield/[μg·(10⁶ cell)^-1]
30	1.692	1.520	234.000	4.68±0.21^c
50	1.985	1.793	278.333	5.57±0.23^c
70	1.996	2.116	1048.333	20.97±1.21^b
90	2.001	2.014	1861.667	37.23±0.62^a

Fig. 7 Electrophoresis of total RNA from four methods 1: Trizol method; 2: Improved Trizol method; 3: Improved CTAB method; 4: TaKaRa RNA extraction kit．

Tab. 4 Comparison of purity, concentration and yield of RNA from four methods

方法 Method	D_{260 nm}/D_{280 nm}	D_{260 nm}/D_{230 nm}	RNA浓度 RNA concentration/(ng·μL^-1)	RNA产量 RNA yield/[μg·(10⁶ cells)^-1]
Trizol法Trizol method	1.915	0.987	1341.667	26.83±0.29^b
改良Trizol法 Improved Trizol method	2.045	2.268	1944.333	38.89±0.23^a
改良CTAB法 Improved CTAB method	1.862	1.428	137.667	2.75±0.17^d
TaKaRa RNA试剂盒法 TaKaRa RNA extraction kit	1.953	2.041	891	17.82±0.34^c

Fig. 8 RT-PCR amplifications of GADPH gene and DREB gene M: DL2000 DNA Marker; 1: Trizol method; 2: Improved Trizol method; 3: Improved CTAB method; 4: TaKaRa RNA extraction kit．

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