Abstract
In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.
Highlights
Proteinase inhibitors (PIs) have been established as plant defense agents against insects and other pests [1,2]
Since the 5′ untranslated region (5′-UTR) of the transgenes plays an important role in determining the rate of translation in plant cells [27,28,29], the failure of accumulation of SaPIN2a in leaves of transgenic plants could be attributed to low translational efficiency of SaPIN2a because of the lack of the 5′-UTR of SaPIN2a in previous binary vectors pSa7 [25] and pARTSaf [26] (Figure 1)
C, plasmid pARTSaf; Sa, total RNA (b) or purified SaPIN2a (c) from stems of S. americanum plants; wild-type plants (WT), wild-type tobacco plants; B41, transgenic plant line transformed with pBI121 vector only; P21 and P51, transgenic plant lines transformed with pARTSaf; S14 and S21, transgenic plant lines transformed with pSa7; F201 and F161, transgenic plant lines transformed with pF121
Summary
Proteinase inhibitors (PIs) have been established as plant defense agents against insects and other pests [1,2]. The adaptation of insects to PIs has resulted in the failure of some transgenic plants overexpressing PIs to resist pests [17,18,19,20]. It has been shown, that the PIs from non-host plant could effectively inhibit the gut proteinases of crop pests [16]. Larvae of the noctuids H. armigera and S. litura, which are two devastating pests causing considerable economic loss world-wide to many important crops and vegetables such as cotton, tobacco, sunflower, corn, pepper and tomato plants [23,24], were fed on leaves of the transgenic plants expressing SaPIN2a. We report the effects of SaPIN2a on trichome development of transgenic plants and on insect larval growth
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