Planta

, Volume 218, Issue 4, pp 623–629 | Cite as

Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

Original Article

Abstract

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.

Keywords

Chymotrypsin Lactuca Protease Proteolysis Solanum Trypsin 

Abbreviations

CaMV

cauliflower mosaic virus

cDNA

complementary DNA

NOS

nopaline synthase

PAGE

polyacrylamide gel electrophoresis

PI

proteinase inhibitor

SaPIN2a

Solanum americanum proteinase inhibitor IIa

SDS

sodium dodecyl sulphate

T-DNA

transferred DNA

Notes

Acknowledgments

This work was supported by funds from The University of Hong Kong (to M.-L.C.). Z.-F.X. received a postgraduate studentship from The University of Hong Kong.

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Department of BotanyThe University of Hong KongHong KongChina
  2. 2.Key Laboratory of Gene Engineering of the Ministry of EducationZhongshan (Sun Yat-sen) UniversityGuangzhou 510275China

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