异源表达<i>AtCYSa</i>基因烟草的耐盐和抗虫特性分析

doi:10.13523/j.cb.20180402

中国生物工程杂志

2018, Vol. 38

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

研究报告

异源表达AtCYSa基因烟草的耐盐和抗虫特性分析

姚民,朱淑华,李佛生,张士彦,唐琳(

)

四川大学生命科学学院生物资源与生态环境教育部重点实验室成都 610064

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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摘要：

植物半胱氨酸蛋白酶抑制剂在植物防御生物与非生物胁迫过程中发挥着重要的作用。拟南芥中的半胱氨酸蛋白酶抑制剂AtCYSa基因的表达能够受到多种胁迫的诱导,且在拟南芥中过量表达AtCYSa基因可以增强转基因拟南芥抵御盐、干旱和氧化等非生物胁迫的能力。为了进一步探究AtCYSa基因的功能以及在烟草中的应用,构建了植物表达载体pCAMBIA 1302-AtCYSa。通过PCR以及RT-PCR验证共获得4株阳性转基因烟草,选择其中3株转基因烟草进行盐胁迫处理和抗虫活性实验。在盐胁迫处理中,100mmol/L NaCl和200mmol/L NaCl处理组的丙二醛含量显著低于野生型对照组。伊文思蓝染色和细胞相对活性结果表明,转基因烟草的细胞活性比野生型烟草明显偏高。这说明在盐胁迫处理下,AtCYSa基因的表达能够起到保护转基因烟草的作用。抗虫活性研究发现,实验组的幼虫总重均呈明显的下降趋势,且幼虫死亡率显著高于对照组。这些结果表明,AtCYSa基因在烟草中的过量表达能够增强转基因烟草的耐盐以及抗虫的能力。

关键词： 转基因烟草; AtCYSa; 耐盐; 抗虫

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

收稿日期: 2017-11-02 出版日期: 2018-05-08

ZTFLH:

Q789

基金资助: 国家自然科学和技术重大专项(2016ZX08010001-010);国家自然科学基金(31270360)

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引用本文:

姚民,朱淑华,李佛生,张士彦,唐琳. 异源表达AtCYSa基因烟草的耐盐和抗虫特性分析[J]. 中国生物工程杂志, 2018, 38(4): 8-16.

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.

链接本文:

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

图1 拟南芥AtCYSa基因的克隆

图2 pCAMBIA1302-AtCYSa表达载体的构建与鉴定

图3 AtCYSa转基因烟草的鉴定

图4 AtCYSa转基因烟草cystatins活性检测

图5 盐胁迫下野生型烟草与AtCYSa转基因烟草MDA含量的测定

图6 野生型烟草与AtCYSa转基因烟草在200mmol/L NaCl处理下的伊文思蓝染色和细胞活性检测

图7 喂食野生型及转基因烟草叶片后黄粉虫体重的变化情况

表1 喂食野生型及转基因烟草叶片后黄粉虫死亡率统计

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