REGULAR ARTICLES

Cell Research (2003); 13(4):301-308

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

Jian Hong YAO¹, Xiu Yun ZHAO¹, Zhi Hua LIAO¹, Juan LIN¹, Zhong Hai CHEN¹, Fei CHEN¹, Jun SONG¹, Xiao Fen SUN¹, Ke Xuan TANG^1,2,*

¹State Key Laboratory of Genetic Engineering, School of Life Sciences, Morgan-Tan International Center for Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R and D Center, Fudan University, Shanghai 200433, China
²Fudan-SJTU-Nottingham Plant Biotechnology R and D Center, Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200030, China

Received Nov-7-2002 Revised June-18-2003 Accepted June-23-2003

Correspondence: Prof. Ke Xuan Tang 0086-21-65642772 (phone) 0086-21-65648376 (fax) kxtang1@yahoo.com or kxtang1@sohu.com

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The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

Key words: araceae, cDNA cloning, lectin, Pinellia ternata, RACE-PCR.