Insect Resistance Function Analysis Of PnKTI Genes In Transgenic Arabidopsis Thaliana

Protease Inhibitor(PI)is a kind of anti-nutritional substance widely existing in plants and it is considered as a natural anti-insect agent due to its inhibiting activity on proteases.Kunitz-type trypsin inhibitors(KTIs)represent one most studied serine protease inhibitor,which is one of the four major subfamilies of PI.Its most important physiological function has been suggested to protect plants from insects and pathogens and the mechanism is mainly through inhibiting the activity of digestive proteases in the midgut of insects,resulting in insects not being able to digest and absorb nutrition normally and growth and development being retarded.Populus Nigra is a perennial woody plant with 12 mechanical injury induced KTI genes(PnKTIs)identified in its genome.In order to investigate whether or not PnKTI genes have anti-herbivore functions and explore the potential underlying mechanism,this study chose four highly inducible PnKTI genes(PnKTIA6,PnKTIB1,PnKTIC1 and PnKTID2)as the target genes and introduced them into Arabidopsis thaliana by transgenic technology.After confirmation of the introduction of transgenes by real time quantitative PCR,we choose transgenic Arabidopsis lines with the highest expression levels,and fed Plutella xylostella with fresh leaves and recorded their changes from larva to adults.The results showed that the overexpression of four PnKTI genes showed inhibitory effects on the growth and development of Plutella xylostella to a varying degree,and among them PnKTIA6 had the best effect,followed by PnKTID2,PnKTIB1 and PnKTIC1 in the decreasing order.In order to further explore how the protein products of PnKTI genes inhibited the digestibility of proteases in insect guts,we isolated the total protein extracted from four types of transgenic Arabidopsis leaves and biochemically analyzed their respective inhibitory activities towards trypsin and chymotrypsin.The results showed that the PnKTIs only had inhibitory effect on trypsin,but not on chymotrypsin,and the inhibitory activities of the four gene products on trypsin were ranked in the order as PnKTIA6,PnKTID2,PnKTIB1 and PnKTIC1,which were highly consistent with the results of insect feeding test.In conclusion,this study provided more novel target candidate genes for bioengineering of insect-resistant plants and had potential instructive implications inpest control in agriculture and forestry.