The Expression Profile Of Ebf Produced By Transgenic Wheat Plants Affects Aphid And Predator Behavior

In China, the dominant species is Sitobion avenae Fabricius(Hemiptera: Aphididae) in most wheat growing areas. At present, chemical control of S. avenae is the major approach. With the development of biotechnology, transgenic crops engineered for resistance to insect pests have important economic and ecological effects on reducing use of insecticides and environmental pollution. E-β-farnesene(EBF) is the main component of the aphid alarming pheromone, a volatile sesquiterpene, which has the function either as a direct repellent to aphid or act as a kairomone for attraction of natural enemies of aphids. EBF is also emited by some plant taxa. Therefore, plant-derived EBF synthase(EBFS) gene could be cloned and transferred into wheat genome through transgenic technology to make wheat synthesize and release EBF to repel aphids and attract natural enemies, which maybe a new strategy for wheat aphid control. Aiming to provide a technical evaluation standard for aphid-resistant effect of transgenic wheat, this article will conduct comprehensive evaluation to transgenic wheat strains from the target gene transcription, the expression, aphid repelling effect and behavior of predatory natural enemies. The main results are listed as follows:The detection of EBFS gene expression profiles: the EBFS gene in wheat lines(designed by No 1-12) were detected by PCR. Firstly, we got 10(No 1-7 and 10-12) positive strains from PCR detection. Further, expression of EBF synthase gene was dectected using real time PCR for positive strains on leaves at three leaf stage, tillering stage, double ridge stage jointing stage, filling stage, and on wheat ear at heading stage, blossom stage, filling stage, milky ripe stage. Results showed EBFS expression profile was different from the different growth stage and on the different plant parts. EBFS were expressed on leaf and wheat ear of all the positive strains. EBFS expression was different from different growth stage of different strains. EBFS expression of majority strains were slightly higher on the leaf of jointing stage; EBFS expression of majority strains was slightly higher on wheat panicle at heading stage; EBFS expression of majority strains was not significantly different between leaf and panicle at grouting stage.The detection of EBF release: volatiles of jointing stage of transgenic wheat were analyzed using two-dimensional gas chromatography/time-of-flight mass spectrometry(GC×GC-TOFMS). Results showed EBF were detected only in four strains which were No1, No4, No5 and No6, and quantity of EBF were about 4 ng·d-1·g-1, 80 ng·d-1·g-1, 20 ng·d-1·g-1,and 10 ng·d-1·g-1, respectively. Quantity of EBF was higher in No4, and EBF was not detected in other strains.Determination of olfactory behavior of aphids and predatory natural enemies to transgenic wheat: aphid: S. avenae olfactory behavioral responses to transgenic wheat which could release EBF was detected using Y-tube olfactometer and a species of ladybird, Harmonia axyridis behavioral responses to transgenic wheat was detected using four-arm olfactometer. Odors of four strains of transgenic wheat releasing EBF were as the odor source, respectively. Results showed strain of No.4 and No.5 had significant repelling effect on winged aphid(N=86, P=0.0059; N=81, P=0.0413); No.4 strain had significant attraction of Harmonia axyridis, because time ladybird staying in the treatment arm was longer than the control arm(N=60, P<0.0001) and the number of times entering treatment arm was more than the control arm(N=60, P=0.0132).Technical specifications of aphid-resistant evaluation of transgenic wheat were established initially in this study:1) Transgenic wheat positive lines were identified rapidly using PCR;2) EBFS gene expression profiles of transgenic wheat were detected using real-time PCR;3) EBF release of transgenic wheat were detected using GC×GC-TOFMS;4) Aphid-resistant effect were evaluated by means of tests on aphids and their natural enemies olfactory behavioral responses to transgenic wheat.After the above four step identification, the results showed that No.4 strains of transgenic wheat at jointing stage had the higher EBF release and could repell aphid(S. avenae) and attract their predator(H. axyridis). The results provide experimental basis for screening suitable transgenic wheat strains for aphid control and the upstream of transgenic wheat breeding.