Abstract:

【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.

Key words: Chimonanthus praecox lectin gene, transgenic tobacco plants, resistance, Myzus persicae, Philomycus bilineatus