Resistance Evaluation And Mechanism Of Camellia Sinensis Response To Ectropis Obliqua

As major leaf-eating insect in tea garden,Ectropis obliqua Prout has many generations,fast reproduction and easy to break into disaster,which seriously affects the normal growth and development of tea plants,the yield and quality of tea leaves.To effectively control E.obliqua and breed the insect-resistent tea plant for solving fundamentally this problem.In this study,according to observing the phenotype of tea plant leaves and the growth and development of E.obliqua feed different tea plant leaves,we selected insect-resistant ‘Shangmeizhou’(RG)and insect-susceptible ‘Pingyang Tezhaocha’(SG)for transcriptome sequencing and analysis of metabolites,and clarified the molecular mechanism of tea plant resistant to E.obliqua,and excavated resistance-related genes and key secondary metabolites,and analyzed resistancerelated gene families in bioinformatics and expression patterns,and cloned key resistance genes and the genetic transformation of tobacco.The main results of this dissertation are as follows:1.According to paraffin section,the internal structure of leaves of 12 tea plants(‘Shangmeizhou’(MZ),‘Biyun’(BY),‘Longjing Changye’(LC),‘Jiulongcha’(GL),‘Lanshan Kucha’(LS),‘Longjing 43’(LJ),‘Baihaozao’(BH),‘Huixiangcha’(HX),‘Dafang Gongcha’(DF),‘Fuyun 6’(FY),‘Zhenong 113’(ZN),‘Pingyang Tezaocha’(PY))was observed.The leaves of 6 tea plants(MZ,BH,HX,DF,GL,ZN)have two to three layers cells of closely arranged palisade tissue,and PY,FY,LC,LJ,LS,BY have two layers cells of loosely arranged palisade tissue.Twelve tea leaves were fed to E.obliqua larvae,and the feeding amount of MZ,BY,DF and HX was significantly less than PY,and the weight gain of the larvae was positively correlated with the feeding amount.The larvae fed on MZ leaf had the lightest weight and the smallest volume.The larvae fed on PY leaf was the largest and the best growth.The number of larval pupae of GL,PY was higher,while that of HX,BY and MZ was lower.In the feeding preference test,first instar larvae were highly tend to MZ and LJ;The second instar larvae had a greater feeding preference to MZ and FY;Third-instar larvae had a tendency to LS,GL and HX,but a little tendency to MZ;The larval selection of MZ decreases with the increase of instar,while the feeding selection of PY does not change much with the increase of instar.The PI of PY was higher,than that of MZ.MZ was selected as E.obliqua resistant tea plant(RG)and PY as E.obliqua susceptible tea plant(SG)for further research.2.The key metabolites of RG and SG were analyzed,75 and 74 differentially expressed metabolites(DEMs)were identified in RG and SG,respectively.These DEMs included flavonoids,amino acids and their derivatives,fatty acids,nucleic acids and their derivatives.In both cultivars,metabolites involved in the flavonoid pathway are highly enriched during TG attack when compared with control plants.On closer analysis,it was found that the type of flavonoid products was totally different.For example,epicatechin(EC),gallocatechin(GC)and catechin(C),and leucocyanidin were uniquely increased their level in RG,whilst kaempferol,delphinidin,quercetin,proanthocyanidin,and procyanidin were significantly increased their level in SG.Jasmonic acid(JA),which have been known as the most important phytohormone linked to plant defense against herbivores,was highly induced in RG,while only the hydroxylated derivatives of JA was identified as DEMs in SG.Interestingly,the level of fructose was significantly repressed in RG,while it was significantly induced in SG during TG attack.We also observed that SG accumulated a significantly higher level of SA and theanine under either control or TG feeding condition.In the differential metabolic pathway,RG and SG during TG attack were mainly involved in flavonoid biosynthesis and biosynthesis of phenylpropanoids.Therefore,flavonoid metabolism pathway and phenylpropanoids metabolism pathway plays an important role in plant insect-resistant reaction.3.There was a difference between the volatile species and release amounts of control treatment and pest treatment in RG and SG.The volatile species and release amounts in RG and SG become larger.Terpene compounds such as β-Myrcene,E-3,7-dimethyl-1,3,6-Octatriene,3,7-dimethyl-1,6-Octadien-3-ol,α-Cyclopentene,α-Farnesene,Squalene,(2Z)-2-Pentenyl acetate,4-Methyl tetradecane and fatty acid compound such as(E)-2-Hexenal,(E)-3-Hexen-1-ol,(Z)-2-Hexen-1-ol was significantly increased.Compared with SG,the volatiles of RG during TG attack,such as β-Myrcene,E-3,7-dimethyl-1,3,6-Octatriene,3,7-dimethyl-1,6-Octadien-3-ol,α-Cyclopentene,α-Farnesene,Squalene,(2Z)-2-Pentenyl acetate,4-Methyl tetradecane,(E)-2-Hexenal,(E)-3-Hexen-1-ol,(Z)-2-Hexen-1-ol,were increased.Furthermore,the release amount of Squalene only increased in RG after insect pests.4.RG and SG after TG attacked were prepared and then paired-end sequenced.342,961 non-redundant unigenes with an average length of 594 bp were achieved,and a total of 156,440(45.61%)unigenes were annotated in at least one database.14,260 DEGs were obtained,of which 7,587 were up-regulated and 6,673 were down-regulated.KEGG pathway analysis revealed that carbohydrateand amino acid metabolism related pathways were more activated in SG.Compared with SG,all the genes involved in jasmonic acid syntheses such as FAD7A-1,LOX2.1,and JMT in RG were shown a higher expression level.Genes encoding PAL and LDOX,which involved in a rate-limiting step of phenolic and anthocyanin biosynthesis,respectively,were significantly induced in RG.In addition,most of the terpene synthases genes(TPS03、TPS04 and TPS21),which catalyze key steps in the formation of terpenoids,were also more induced in RG.7,587 up-regulated genes were mainly related to lipid modification,cell surface receptor signaling pathway,xylem development,and transport,especially involved in phosphorylation and metabolic process.997 genes were successfully mapped,and among which 103 TFs(59)or PKs(44)were found to be interacting with 894 nodes and 24,294 edges.PPI network analysis revealed that six candidate TFs interacting with 61 other genes,and thirtythree candidate PKs which were belonging to LRR-RLK,Ser/Thr kinase,CDPK,and MAPK family with 271 other genes.5.From the transcriptome of all tissues of tea plant,a total of 149 NBS protein sequences were obtained through filtering.These NBS protein sequences were further divided into seven types: CC-NBS-LRR(52),TIR-NBS-LRR(3),CC-NBS(25),TIR-NBS(3),NBS(20),NBSLRR(40)和 RPW8-NBS(6).According to analysis of bioinformatics and gene expression level,Cs4100(CsNBS-1)and Cs187031(CsNBS-2)were selected for full-length cloning and genetic transformation analysis.6.The sequences full length of CsNBS-1 and CsNBS-2 are 3696 bp and 3718 bp,respectively,belonging to CNL type.The ORF region are 2748 bp and 2706 bp respectively,and the full length of amino acids are 915 aa and 901 aa respectively.The genetic transformation through agrobacterium-mediated transformation,a total of 41 positive plants of kanamycin resistance were obtained.