Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a.
SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R1 progeny. SaPIN2a mRNA was detected in both the R0 and R1 transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.
Xu
Zeng-Fu
ZF
Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Teng
Whei-Lan
WL
Chye
Mee-Len
ML
D001483:Base Sequence; D002918:Chymotrypsin / Q000378:metabolism*; D017931:DNA Primers; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D007700:Kinetics; D018545:Lactuca / Q000201:enzymology*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology*; D011480:Protease Inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012231:Rhizobium / Q000235:genetics; D014158:Transcription, Genetic; D014361:Trypsin Inhibitors / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; C445075:PIN2 protein, Solanum americanum; D010940:Plant Proteins; D011480:Protease Inhibitors; D012333:RNA, Messenger; D014361:Trypsin Inhibitors; D002918:Chymotrypsin