新疆耐盐植物藜的甜菜碱醛脱氢酶基因(CaBADH)的克隆、盐胁迫表达分析及植物表达载体的构建

Cloning of Betaine Aldehyde Dehydrogenase Gene(CaBADH)from Chenopodium album L. and Expression Analysis of Salt Stress and Construction of Plant Expression Vector

【目的】为开展植物耐盐基因工程提供候选基因。【方法】研究以新疆耐盐植物藜为材料,利用同源克隆技术从藜中克隆到BADH基因,并通过RT-PCR方法对其在不同盐胁迫下的表达进行了初步分析,随后将该基因构建至高效植物表达载体pCN2300上。【结果】(1)经测序分析并与藜科其他植物进行同源性比对显示,得到的序列为藜的BADH基因,开放阅读框长度为1503bp,编码500个氨基酸;(2)以BADH基因核心序列设计引物对此基因在盐胁迫下的表达进行了RT-RCR分析,发现其本底表达量较高,以100mmol/LNaCl处理2、5、12和24h后其表达量没有明显增加趋势,而以50mmol/LNaCl或KCl长期胁迫后其表达量则比对照和100mmol/L时的表达量高;(3)经双酶切鉴定,已成功将CaBADH基因构建到植物表达载体pCN2300,得到重组质粒pCN2300-CaBADH。【结论】研究为进一步从生理和分子水平阐明藜的耐盐机制提供了一定参考。并为通过转基因技术获得耐盐作物新品种打下基础。

[Objective]To provide candidate genes for plant genetic engineering in improving salt tolerance,[Method] in present study,Chenopodium album L.,a salt-tolerant species distributed in Xinjiang was used as material,BADH gene was cloned through homology cloning technique,and a preliminary analysis was performed for expression of BADH under different salt stresses by RT-PCR,then a plant expression vector pCN2300-CaBADH was constructed.[Result]Results showed that：（1） After being sequenced and multiple alignment with other several Chenopodiaceae species,it indicated that CaBADH was successfully cloned,and its nucleotide sequence was 1 503 bp,500 amino acid deduced by code;（2） RT-PCR analysis with primers of BADH gene core sequence under salt stress showed that CaBADH exhibit relatively high basal level in Chenopodium album,and there was no significant increase of transcript level after 2,5,12,24 h stress under 100 mmol/L 1 NaCl;However,a higher expression level was observed under 60 d stress of 50 mmol/L NaCl or KCl treatment,rather than that of 100 mmol/L NaCl or KCl compared with the control.（3） By double digestion verifying,CaBADH gene was correctly inserted into the plant expression vector pCN2300,and recombinant plasmid of pCN2300-CaBADH was obtained.[Conclusion]The present study will provide evidence for further exploring salt tolerant mechanisms of C.album in physiological and molecular levels,and also help scientists in developing new salt-tolerant varieties through transgenic technology.