Study On The Defense Response And Mechanism Of Cotton Induced By Apolygus Lucorum

With the large-scale application of transgenic cotton,mirid bugs become the most important sucking pest on cotton in China,and Apolygus lucorum is the dominant species among several mirids.Based on the argument that cotton resists the mirid by promoting the synthesis of anti-insect metabolites and releasing herbivore-induced plant volatiles(HIPVs)after being attacked by the A.lucorum,this study focused on the scientific issue of the regulation mechanism by A.lucorum attack in cotton plants,using the transgenic cotton line SGK321(Bt+CpTI)which is grown on a large area in the middle and lower reaches of the Yangtze River as the material,treated the cotton 24 hours with different densities of nymphs.We demonstrated that cotton attacked by A.lucorum significantly inhibited the development of A.lucorum and attracted the natural enemy Peristenus spretus.Transcriptome and metabolome analysis were used to deeply analyze the dynamic changes of gene expression and metabolite content in transgenic cotton at different time points before and after the damage of A.lucorum.Then the secondary metabolites,HIPVs and plant hormones related to insect resistance in cotton plants were analyzed by quantitative analysis.Finally,feedback verification of plant hormones were performed.The defense response of cotton to A.lucorum were elucidated in different aspects.The main results were as follows:1.The results showed that:1)After 24 hours damage of four 3rd instar nymphs,cotton significantly attracted the P.spretus and repel the A.lucorum;the composition and content of volatiles showed that the volatiles of cotton induced by four 3rd instar nymphs are quite different from that of healthy plants.The release of(E)-4,8-dimethylnona-1,3,7-nonatriene and(E)-?-ocimene,which have been proved to have significant attracting effects on parasitoid wasps,were significantly increased.2)A.lucorum feeding on the treated cotton leaves showed slow development.The 3rd and 4th periods were significantly prolonged 1.13d and 1.08d than the control,respectively,and results in the total duration of the nymphs was prolonged(15.25d vs 12.85d).The emergence rate was significantly reduced from 66.67%in the control to 43.33%.3)By measuring the activities of detoxification enzymes in the two treatments,the enzymes of the adults fed on cotton leaves damaged by four 3rd nymphs of A.lucorum were significantly higher than that of adults feed on heathy cotton leaves,the activities of acetylcholinesterase,glutathione S-transferase and carboxylesterase were induced from 2.29±0.30,401.38±19.27 and 376.34±14.95 U/mg prot to 3.47±0.43,540.57±46.70 and 513.21±38.28 U/mg prot.The activity of mixed-functional oxidase was even as high as 49.44±7.22 U/mg prot,extremely higher than that in the control.Thus,four 3rd nymphs of A.lucorum can effectively activate the induced defense response of cotton plants.2.The results showed that:1)The number of up-regulated genes in cotton leaves gradually increased at 4h,12h and 24h after damage,which were 3847,4374 and 6236,respectively,while the down-regulated genes were only 776,1735 and 4615,respectively.2)Further analysis showed that the differential genes were mainly involved in metabolic pathways related to induced insect-resistance such as plant-pathogen interaction,plant mitogen-activated protein kinase signaling pathway,plant hormone signal transduction,?-linolenic acid metabolism,flavonoid biosynthesis,phenylpropanoid biosynthesis pathway and so on.3)The analysis also found that the expression levels of most key synthetase genes involved in the jasmonic acid(JA)biosynthesis pathway were significantly up-regulated after 4 hours of feeding,such as 13-lipoxygenase(LOX),allene oxide synthase(AOS),allene oxide cyclase(AOC),oxophytodienoate reductase(OPR),and the expression level has been maintained at a high level throughout the feeding process,4)However,the expression level of some genes of isochorismate synthase(ICS),a key enzyme in the salicylic acid(SA)biosynthesis pathway,showed a downward trend.5)The expression of most genes involved in condensed tannins were also up-regulated after 4 hours,and have been maintained at a high level.These results showed that the direct defense of cotton plants against A.lucorum is mainly induced by JA-mediated promoting the biosynthesis of condensed tannins in plants.3.The results showed that:1)The total differential metabolites between damaged and healthy cotton leaves increased gradually after 4,12 and 24 h damaged by A.lucorum nymphs,the up-regulated metabolites were 106,422,and 780,respectively,and the down-regulated metabolites was 160,299,and 588,respectively.2)By analyzing the compounds involved in the jasmonic acid biosynthesis pathway,the content of major intermediates 12,13-epoxy-9,11,15-octadecatrienoic acid(12,13(S)-EOT),12-oxophytodienoic acid(12-OPDA)and 7-isojasmonic acid-coenzyme A(7-IsoJA-CoA)were significantly induced at 24 hours after fed by A.lucorum,expect the ?-linolenic,which was significantly decreased.The content of JA and JA-Ile also showed a significant upward trend after damaged by the A.lucorum.3)The content of salicylic acid(SA)decreased after feeding by the A.lucorum,and its content was significantly lower than that of healthy plants throughout the whole feeding process until 24 hours.4)The content of abscisic acid(ABA)showed no significantly changes.5)The research on the biosynthesis pathway of condensed tannins found that the content of condensed tannins reached 26.60±1.20 mg/g at 24 h after damaged by A.lucorum,which was significantly higher than that of healthy plants.The content of phenylalanine reached the highest level at 4 hours,but decreased rapidly.The content of ?-coumaric acid also reached the highest at 4 hours.And the content also showed a downward trend after 4 hours,but there was no significant difference with that at 4h;the content of trans-cinnamic acid and dihydrokaempferol was significantly higher than that of healthy plants after feeding by A.lucorum.6)The content of gossypol showed no significant change during the whole feeding period.7)The collection and analysis of plant volatiles before and after the feeding of A.lucorum showed that with the feeding time extended,the types and contents of plant volatiles were the significantly increased,and the newly induced volatiles were mostly terpenes,including DMNT and(E)-?-ocimene,which have been.proved to have significant trapping effects on parasitic wasps.Therefore,the induced defense of cotton plants against A.lucorum is mainly mediated by JA-mediated promoting the biosynthesis of condensed tannins and the biosynthesis and release of terpenes in plants.4.The results showed that:1)Plants can significantly attract natural enemies after 48 hours post treated by 0.2 ?mol JA;the effect of plants attracting natural enemies was the best after 48 hours post treated by 2 ?mol JA and lasted until 72 hours;no effect of natural enemies was observed when the plants were applied by SA;simultaneous or successively application of the two hormones did not affect the behavior of natural enemies.2)When the A.lucorum fed on the leaves of cotton plants treated by 2 ?mol JA for 48 hours,the developmental duration of the 3rd and 4th instar nymphs were 3.32 d and 3.44 d,respectively,which was significantly longer than that of the nymphs fed on leaves of healthy plants(2.29 d and 2.19 d).The emergence rate(40.00%)was also significantly lower than that(66.67%)fed on the leaves of healthy plants.While the activities of four detoxification enzymes(acetylcholinesterase,glutathione S-transferase,mixed-functional oxidase and carboxylesterase)in the adults(3.82±0.45,645.23±49.89,491.30±57.51 and 35.11±4.16 U/mg prot)were significantly higher than those fed on leaves of healthy plants(2.29±0.30,401.38±19.27,376.34±14.95 and 25.33±1.87 U/mg prot,respectively).The developmental progress of nymphs and the activities of several detoxification enzymes in cotton plants after JA treatment were consistent with the results obtained in Chapter 2.These results validate that the JA played a dominant role in the induction of insect resistance in cotton plants and the SA could inhibit the volatile-m ediated indirect defense induced by JA.