Regurgitant Derived From The Tea Geometrid Ectropis Obliqua Suppresses Wound-indeced Polyphenol Oxidases Activity In Tea Plants And Genes Clone Of Related Proteins

Polyphenol oxidases (PPOs) have been reported to play an important role in protecting plants fromherbivore attack. However, little is known about their role in tea plants. The tea geometrid Ectropisobliqua Prout (Lepideptera: Geometride) is a devastating leaf-feeding insect pest, and can causeconsiderable damage when broken out. However, next to nothing is known about whether they cancounteract PPOs’ defense trait. Previous results are shown that jasmonic acid (JA) treatment resulted inincreases in PPO activity and the effect of JA is dose dependent. E. obliqua caterpillars grew anddeveloped more slowly on JA-treated tea plants than on control plants, and larval weight gainsdepended on the dosage of JA. Artificial diet complemented with PPOs reduced the growth and survivalrate of E. obliqua caterpillars, and there was a negative relationship between PPO level and the larvalgrowth and survival rate. In the present study, I first proved the defensive trait of tea PPOs to E. obliqualarvae. Secondly, we measured the activity of PPOs in tea leaves that had been treated by E. obliquainfestation, wounding plus E. obliqua regurgitant or wounding plus distilled water to understand theresponse of E. obliqua. Try to clarify the co-evolution relationship between tea plants and E. obliquafrom one side. At last, I cloned two genes related to salivary secretion form larval salivary gland of teageometrid, and constructed recombinant plasmids of prokaryotic expression of the two genes. Here wehave prepared for there over-expression and biological functional verification in the future. The mainresults are as follow:(1) Tea extract, catechins and artificial diet were used as substrates to prove mushrooms PPO cancatalyze polyphenols in these substrates to quinones. Tea extract grinded with PVPP could catalyzeartificial diet and catechins as well. These results show that mushroom PPO has a similar catalyticfunction to tea PPO. The previous result of artificial diet complemented with PPOs reduced the growthand survival rate of E. obliqua caterpillars is the supplementary proof of the defensive function of teaPPOs.(2) Unlike mechanical wounding, which is an effective inducer of tea plant PPO activity, woundingplus the herbivore regurgitant or herbivore infestation suppressed the wound-induced PPO activities,especially at4d after treatment. These results suggest that E. obliqua larvae have evolved to elude thetea plant’s defense by inhibiting the production of PPOs.(3) Two genes related to salivary secretion were cloned from salivary gland, which were slectedfrom transcriptome of tea geometrid larvae salivary glands. EoSP1has an ORF of858bp, coding285amino acids. And EoNL2has an ORF of1041bp, coding346amino acids. Deduced amino acidssequences were analyzed using NCBI-blast and evolutionary analysis, and the results showed that thesetwo genes were the target genes. Then recombinant plasmids EoSP1/pET28a and EoNL2/pET28a areconstructed using pET28a（+）,which is a prokaryotic expression vector.