Cloning and molecular characterization of a novel lectin gene from Pinellia ternata.
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.
Yao
Jian Hong
JH
State 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.
Zhao
Xiu Yun
XY
Liao
Zhi Hua
ZH
Lin
Juan
J
Chen
Zhong Hai
ZH
Chen
Fei
F
Song
Jun
J
Sun
Xiao Fen
XF
Tang
Ke Xuan
KX
D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D030021:Pinellia / Q000235:genetics; D037121:Plant Lectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
C047938:Pinellia ternata lectin; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins