摘要
【目的】克隆蜡梅凝集素基因并研究其抗蚜虫和抗蛞蝓活性,为植物抗虫基因工程提供理论依据和试验材料。【方法】通过随机挑选克隆测序的方法,从蜡梅花cDNA文库中克隆蜡梅凝集素基因,并构建植物表达载体,用农杆菌介导的叶盘法转化烟草,并对转基因植株进行抗蚜和抗蛞蝓试验。【结果】从蜡梅花cDNA文库中克隆到了一个凝集素基因CpLEC(GenBank登录号:DQ352145),该基因全长871 bp,ORF框长558 bp,编码185个氨基酸。该基因DNA分析表明其不含内含子。具有单子叶植物甘露糖凝集素基因家族的特征,有2个典型的甘露糖结合位点(QXDXNXVXY),和1个变异的可能结合位点(HXGXNXVXY)。Southern杂交表明,该基因在蜡梅基因组中为多拷贝。构建了35S启动子控制下的CpLEC基因的植物表达载体pBI121-CpLEC,利用根癌农杆菌介导法转化烟草获得抗卡那霉素的植株。PCR和半定量RT-PCR分析表明,CpLEC基因已经整合到植物基因组中并在转录水平上得到了有效表达。抗蚜虫鉴定表明,转基因植株及其离体叶片有效地抑制了蚜虫的群体增长率,平均抑制率分别为60%和68%。抗蛞蝓试验结果表明转基因植株有明显的抗蛞蝓活性,转基因烟草虫害指数为0.17,远低于非转基因烟草的虫害指数0.96。【结论】克隆获得了蜡梅凝集素基因CpLEC,通过转基因手段分析其抗蚜和抗蛞蝓活性,结果表明该基因对抗虫基因工程有重要的应用价值。
【Objective】The objective of the study was to clone lectin gene from Chimonanthus praecox and investigate the insect-resistance activity of CpLEC gene transgenic tobacco in order to provide a theoretical basis and experimental materials for insect-resistant genetic engineering.【Method】 The method of randomly selecting clones from the library and bi-directional sequencing was used to clone the Chimonanthus praecox lectin gene.The insecticidal activity of the CpLEC gene against the peach aphids（Myzus persicae） and limax（Philomycus bilineatus） was studied using transgenic tobacco plants expressing the CpLEC gene under the control of the constitutive CaMV35S promoter.【Result】 A full-length cDNA encoding a mannose-binding lectin was isolated from the library.The cDNA,designated as CpLEC gene,is 871 nucleotides long and has an open reading frame of 558 bp with a deduced amino acid sequence of 185 residues.Sequences analysis indicated that there was no intron within the genomic region.Conserved Bulb-type mannose-binding domain was detected within the region of 38-145aa of CpLEC gene and it contained 2 typical mannose-binding sites QXDXNXVXY and a variable binding site HXGXNXVXY.The result of Southern blot showed that CpLEC gene belonged to multi-copy gene.A plant expression vector pBI121-CpLEC was constructed in which the CpLEC gene was under the control of the cauliflower mosaic virus 35S promoter.Transgenic tobacco plants were regenerated by Agrobacterium tumefaciens mediated transformation.PCR and RT-PCR analyses confirmed that the CpLEC gene had integrated into the plant genome and was expressed at various levels in mRNA levels.The pest-resistance biological test showed that transgenic plant expressing CpLEC gene and its leaves in vitro reduced the population increase rate of the Myzus persicae,and the average inhibition rates were 60% and 68%,respectively.The transgenic plant expressing CpLEC gene also has the resistance to limax with the pest index of 0.17 which is much lower than 0.96 of non-transgenic plant.【Conclusion】 One Chimonanthus praecox lectin gene was isolated and the pest-resistance biological test showed that the CpLEC gene has applied value for plant transgenic engineering against pests.
出处
《中国农业科学》
CAS
CSCD
北大核心
2011年第2期358-368,共11页
Scientia Agricultura Sinica
基金
国家自然科学基金资助项目(31070622
30872063)
中央高校基本科研业务费专项资金资助(XDJK2009A004)
重庆市自然科学基金项目(CSTC
2006BB1336)
关键词
蜡梅凝集素基因
转基因烟草
抗性
蚜虫
蛞蝓
Chimonanthus praecox lectin gene
transgenic tobacco plants
resistance
Myzus persicae
Philomycus bilineatus
