Analysis of Salt Tolerance and Insect Resistance of Transgenic Tobacco Expressing <i>AtCYSa</i> from Arabidopsis

doi:10.13523/j.cb.20180402

China Biotechnology

2018, Vol. 38

Issue (4): 8-16 DOI: 10.13523/j.cb.20180402

Analysis of Salt Tolerance and Insect Resistance of Transgenic Tobacco Expressing AtCYSa from Arabidopsis

Min YAO,Shu-hua ZHU,Fo-sheng LI,Shi-yan ZHANG,Lin TANG(

)

Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, Sichuan University, Chengdu 610064, China

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Abstract

Plant cysteine protease inhibitors play an important role in defense of biotic and abiotic stresses. The expression of the AtCYSa gene in Arabidopsis thaliana can be induced by a variety of stresses, and the over-expression of AtCYSa in Arabidopsis thaliana can enhance the ability to resist abiotic stress like salt, drought and oxidation. In order to further explore the function of AtCYSa and its application in tobacco, the plant expression vector pCAMBIA 1302-AtCYSa was constructed. Four positive transgenic tobacco lines were obtained by PCR and RT-PCR confirmation. Three transgenic tobacco lines were selected for salt stress treatment and insect resistance. In the salt stress treatment, the content of malondialdehyde in 100mmol/L NaCl and 200mmol/L NaCl treatment group was significantly lower than that in wild type. Evans blue staining and cell relative activity results showed that the cell activity of transgenic tobacco was significantly higher than that of wild type. This indicated that the expression of AtCYSa gene could protect the transgenic tobacco under salt stress. Insect activity studies showed that the total weight of the larvae in the experimental group havig a significant decrease, and the larvae mortality was significantly higher than the control. These results indicate that over-expression of the AtCYSa gene in tobacco can enhance the salt tolerance and insect resistance of transgenic tobacco.

Key words： Transgenic tobacco AtCYSa Salt tolerance Insect resistance

Received: 02 November 2017 Published: 08 May 2018

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Min YAO,Shu-hua ZHU,Fo-sheng LI,Shi-yan ZHANG,Lin TANG. Analysis of Salt Tolerance and Insect Resistance of Transgenic Tobacco Expressing AtCYSa from Arabidopsis. China Biotechnology, 2018, 38(4): 8-16.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180402 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I4/8

Fig.1 Cloning of AtCYSa gene from Arabidopsis thaliana
(a) Extraction of total RNA from Arabidopsis thaliana M: DL2000 (b) PCR of AtCYSa gene

Fig.2 The construction and identification of pCAMBIA1302-AtCYSa recombinant vector
(a) Restriction enzyme digestion of AtCYSa gene with restriction enzyme site (b) Restriction enzyme digestion of pCAMBIA1302 1~3: Plasmid; 4~5: Plasmid digestion; 6: Positive control (c) PCR detection of recombinant vector 1: Negative control; 2~4: Bacterial samples; 5: Positive control

Fig.3 Identification of AtCYSa gene in transgenic tobacoo
(a) PCR identification of AtCYSa gene in transgenic tobacoo lines (b) RT-PCR identification of AtCYSa gene

Fig.4 Assays of cystatins activity in AtCYSa transgenic tobacoo lines

Fig.5 The determination of MDA content of wild-type and AtCYSa transgenic tobacco lines under salt stress
Different number shows significant difference at 0.05 level

Fig.6 Evans blue dyeing and cell activity detection of wild-type and AtCYSa transgenic tobacco lines under 200mmol/L NaCl
(a) Evans blue dyeing of wild-type and AtCYSa transgenic tobacco lines under 200mmol/L NaCl Control: Wild type dispose with 200mmol/L NaCl (b) Cell activity detection of wild-type and AtCYSa transgenic tobacco lines under 200mmol/L NaCl, different number shows significant difference at 0.05 level

Fig.7 Insect weight changes after feeding leaf of wild-type and transgenic tobacco lines

Table 1 Insect mortality statistics after feeding leaf of wild-type and transgenic tobacco lines


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