Function Analysis Of Defence-Related Genes Of Tea Plant (Camellia Sinensis)and Identification And Application Of Its Synomones Induced By Tea Green Leafhopper

Tea plant(Camellia sinensis) is an important cash crop which originates in southwestern China, and now has become one of the three largest non alcoholic beverages in the world. Tea plant suffers from many damages caused by phytophagous insects during the process of growth. As a water soaking drink, it is restrained to use chemical pesticides to control insects. With higher and higher demand of hygienic quality and safety of tea, it is necessary to develop pollution-free and organic tea production. Tea plant releases volatiles when attacked by herbivorous insects, and these volatiles serve as behavior-suggested chemical signals providing valuable information among tea plants, herbivores and their natural enemies. It shows great advantages to study and utilize these volatiles obtained by means of extraction, artifical synthesis and release from transgenic plant to regulate insect behavior and defense insect damage. The results are as follows:(1) The biosynthetic pathway of jasmonates is one of the main pathways induced by herbivores, and plays a dominant role in the plant defence process induced by herbivores. Allene oxide cyclase (AOC) is an essential enzyme in jasmonates biosynthetic pathway. AOC catalyzes the stereospecific cyclization of unstable allene oxide to (9S,13S)-12oxo-(10,15Z)-phytodienoic acid (OPDA). Here we cloned a cDNA from C. sinensis, named as CsAOC (Genbank:HQ889679), by RACE, showing highly homologous to the AOCs of other species. The full-length cDNA of CsAOC was916bp with5:and3’UTR, poly(A) tail and a738bp open reading frame (ORF). The comparison between the full-length cDNA and genomic DNA of CsAOC revealed that the genomic DNA contained two introns, one was94bp and the other one was402bp. The putative protein includes245amino acids with a molecular weight of26.5kDa and pI of9.0. It had no signal peptide, but contained an N-terminal cTP. The deduced CsAOC amino acid sequence had high homology with other plant AOCs via multiple alignments. Secondary structural prediction of CsAOC protein was performed using the SOMPA program. CsAOC protein was composed of21.22%a-helix,25.31%extended strand,6.12%p-turm and47.35%random coil. The E. coli expression vector for CsAOC which was constructed for the first time showed that the size of the protein produced through the prokaryotic expression is equal to the experiment have expected. The recombinant protein existed in the form of an inclusion body. RT-QPCR analysis revealed that the expression of CsAOC could be regulated by various stresses and elicitors. MeJA had the most prominent effects on stimulating the expression of CsAOC. The gene was up-regulated following SA and wounding treatments and was also induced in response to feeding by the tea geometrid and tea green leafhopper. The results showed that it can regulate AOC expression and jasmonate biosynthesis.(2) In phytocoenose, the terpenoids serve as behavior-suggested chemical signals having an effect on the relationship of plants and plant-insect. So the biosynthetic pathway of the terpenoids plays an important role in plant defence reaction.3-hydroxy-3-methyl coenzyme A reductase (HMGR) is just the first rate-limiting enzyme and is an important regulating point of the metabolism of terpenoids in the cytoplasm. A full-length cDNA encoding3-hydroxy-3-methyl coenzyme A reductase, named as CsHMGR (Genbank: JN584161), was cloned. The full-length CsHMGR was2051bp with5’-UTR, a1773bp ORE,3’-UTR and poly (A) tail. The comparison between the full-length cDNA and genomic DNA of CsAOC revealed that the genomic DNA contained two introns, one was136bp and the other one was108bp. The putative protein includes590amino acids with a molecular weight of63.38kDa and pi of8.82. The deduced CsHMGR amino acid sequence had high homology with other plant AOCs via multiple alignments. Secondary structure of CsHMGR protein was composed of44.92%α-helix,14.41%extended strand,5.59%β-turm and35.08%random coil. It had two NADP (H) binding sites and two HMG-CoA binding sites. The E. coli expression vector for CsHMGR showed that the size of the protein produced through the prokaryotic expression is equal to the experiment that have expected. The recombinant protein existed in the form of an inclusion body.(3) In order to effectually utilize plant synomones to attract mymarids parasitizing the eggs of tea green leafhoppers, Empoasca vitis, the fresh infact tea shoots (ITS), the leafhopper-damaged tea shoots (LDTS), infact tea tender stems (ITTS) and ovipositing-injured tea tender stems (OITTS) acted as odor sources respectively, whose volatiles were collected via custom-made push-pull aeration system with Super Q being absorbent, eluted by organic solvent, concentrated under a flow of nitrogen. The compounds in the volatiles were qualitatively analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that new component was detected, such as (E, E)-a-farnesene. The content of (E,E)-α-farnesene was larger from LDTS and OITTS. In tea plantations, the attraction of (E,E)-a-farnesene to mymarids Stethynium empoascae Subba Rao and Schizophragma parvula Ogloblin was strongest. In the various tea plants growing areas during summer and autumn, a batch of synomones were used to trap the two species of mymarids, with (E, E)-a-farnesene being most attractive. The jasmine yellow boards with mixture of (E, E)-a-farnesene, benzaldehyde and (E)-2-hexenal could efficaciously attract the two species of mymarids together and direct them to parasitize the eggs of tea green leafhoppers. The investigation supplies a successful example that the tea plant synomones can be used in attracting the mymarids to suppress tea green leafhoppers.(4) Generally speaking, there are more richness and abundance of natural enemies in July and October than other months in tea plantations, which are fit for the test of the attraction of synomones to natural enemies and comparison between various synomones, so as to screen out the synomones of synomones formula with the strongest attraction. Based on the results of behavioural bioassay of our research group, the various synomones and their mixture were loaded onto rubber nob and manufacture into lures, which were appended on the yellow jasmine sticky boards. The set were called traps, which were used to detect the attraction of synomones and their mixture to natural enemies, and screen out the optimal component and the formule. The results showed that the difference in the richness, abundance and indice of diversity of the natural enemies trapped by various synomones. The hexane and Z-3-hexenol attracted less the richness and the abundance than other synomones, with higher indices of diversity. The damages by the leafhopper activate the relative defence genes, and emit the volatile synomones, which attract mymarids together, parasitize the eggs of the leafhoppers, and suppress the population of the leafhopper. The results were referred to the attration for natural enemies.