Analysis of Physio-biochemical Characteristics of T2 α-tubulin SoTUA Transgenic Sugarcane

doi:10.3969/j.issn.1000-2561.2020.04.008

Abstract

Abstract:

Six SoTUA transgenic lines (T1, T2, T3, T4, T5, T6) were selected from the positive SoTUA transgenic sugarcane lines of T₂ generation after PCR amplification and sequencing. The transgenic lines and control (wild type, WT) were treated with low temperature stress (4 ℃), and the level of malondialdehyde (MDA) and osmotic adjustment substances (soluble sugar, soluble protein) and activities of peroxidase (POD) and superoxide dismutase SOD were determined, and RT-qPCR analysis was done for SoTUA gene expression in sugarcane leaves at 0, 5 and 10 days of the treatment. The relative expression of α-tubulin transcription factor in the SoTUA transgenic sugarcane and WT was up-regulated, getting higher with the cold stress time under low temperature treatment at 4 ℃. The relative expression level was always higher in the SoTUA transgenic lines than that in WT at 5 and 10 days of the treatment. At the 10th day, the relative expression level of the transgenic line T1 was significantly higher than that of other transgenic strains (P<0.05). The content of MDA increased gradually under cold stress, but it was found significantly lower in the transgenic sugarcane than that in WT by 41.77% (P<0.05). The trend of POD activity varied among different lines, but it was higher in the SoTUA transgenic lines than in WT (P<0.05). The SOD activity of transgenic lines T1, T2, T4 and T6 increased first and then decreased while SOD activity of WT and transgenic lines T3 and T5 increased gradually. The soluble sugar content increased gradually and the trend of soluble protein varied in different strains. The expression of SoTUA gene was positively correlated with soluble sugar. Using the membership function comprehensive analysis method, the ranking result for cold resistance was obtained as T3 > T2 > T1 > T5 > T6 > T4 > WT. Up-regulated expression of SoTUA gene in the transgenic sugarcane could improve cold resistance, and the genetic stability is good.

Key words: sugarcane, SoTUA gene, cold resistance, physio-biochemical characteristic, genetic stability

CLC Number:

S556.1

CHEN Jiaoyun,KHAN Qaisar,WEI Jianglu,TANG Lihua,DONG Dengfeng,LI Yangrui. Analysis of Physio-biochemical Characteristics of T₂ α-tubulin SoTUA Transgenic Sugarcane[J]. Chinese Journal of Tropical Crops, 2020, 41(4): 685-693.

Figures/Tables 12

Tab. 1 Primer sequences for PCR amplification

基因名称 Gene name	正向引物(5°-3°) Forward primer (5°-3°)	反向引物(5°-3°) Reverse primer (5°-3°)
SoTUA	CCGTGGTGATGTTGTTCCTA	GGGTGGCTGATAGTTGATGC
GADPH	AAGGGTGGTGCCAAGAAGG	CAAGGGGAGCAAGGCAGTT

Fig. 1 Electrophoresis detection for sugarcane total DNA WT: Wild type sugarcane; 1-6: Transgenic sugarcane lines T1, T2, T3, T4, T5, T6.

Fig. 2 PCR identification for SoTUA transgenic sugarcane M: DL2000 DNA Marker; 1: Recombinant plasmid pCAMBIA3300-SoTUA-Bar (positive control); 2: WT (negative control); 3: H2O;4-9: Transgenic sugarcane lines T1, T2, T3, T4, T5 and T6.

Fig. 3 Electrophoresis detection for sugarcane total RNA WT: Wild type; 1-6: Transgenic sugarcane lines T1, T2, T3, T4, T5, T6.

Fig. 4 Relative expression of SoTUA gene in sugarcane leaves under low-temperature treatment Different lowercase letters mean significant difference at 0.05 level.

Fig. 5 Changes of soluble sugar content in leaf of sugarcane under low temperature stress Different lowercase letters mean significant difference at 0.05 level.

Fig. 6 Changes of soluble protein content in leaf of sugarcane under low temperature stress Different lowercase letters mean significant difference at 0.05 level.

Fig. 7 Changes of MDA content in leaf of sugarcane under low temperature stress Different lowercase letters mean significant difference at 0.05 level.

Fig. 8 Changes of POD activity in leaf of sugarcane under low temperature stress Different lowercase letters mean significant difference at 0.05 level.

Fig. 9 Changes of SOD activity in leaf of sugarcane under low temperature stress Different lowercase letters mean significant difference at 0.05 level.

Tab. 2 Correlation coefficients between various indicators in sugarcane leaves

指标 Index	SOD活性 SOD activity	可溶性蛋白含量 Soluble protein content	可溶性糖含量 Soluble sugar content	MDA 含量 MDA content	POD活性 POD activity
可溶性蛋白含量	-0.833^**
可溶性糖含量	-0.108	-0.286
丙二醛的含量	0.285	-0.407	0.137
过氧化物酶活性	-0.367	0.545^*	-0.480^*	-0.479^*
SoTUA基因表达量	0.135	-0.383	0.612^**	0.437^*	-0.603^**

Tab. 3 Comprehensive evaluation of cold-resistance of sugarcane lines

株系 Line	SOD活性 SOD activity	可溶性蛋白含量 Soluble protein content	可溶性糖含量 Soluble sugar content	POD活性 POD activity	MDA含量 MDA content	综合评价值 Total score	排名 Rank
WT	21.26	5.36	4.22	5.66	4.19	40.70	7
T1	11.63	16.42	4.89	12.12	16.48	61.54	3
T2	4.61	13.72	18.79	9.24	17.05	63.41	2
T3	9.38	21.63	12.13	2.50	18.21	63.85	1
T4	21.56	7.61	2.81	8.70	14.95	55.63	6
T5	23.05	6.30	4.55	13.02	13.46	60.38	4
T6	10.12	20.06	3.92	7.56	16.62	58.29	5

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Analysis of Physio-biochemical Characteristics of T₂ α-tubulin SoTUA Transgenic Sugarcane

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