| The defense reaction induced by insect feeding is gradually formed in the process of plants evolution.The systematic defense reaction,which happens not only in the local position of insect feeding but also occurring in other tissues or organs that are not damaged,plays an important role in environmental adaptation of plants.The research of the mechanisms of plant systematic defense reaction is one of the hotspots in the fields of plant physiology and ecology,and the research on shoot-to-root responsiveness(StR)which is induced in leaves by pests feeding has become a focus in recent years.Different plant species have different molecular mechanism of StR and therefore an in-depth exploring of the complex StR mechanism it is very necessary.Tea(Camellia sinensis)is a very popular and health nonalcoholic beverage worldwide.Tea geometrid(Ectropis oblique),whose larvae can cause severe damage by feeding on tea leaves,is one of the most common pests of tea in China.However,the research about molecular mechanism of StR in tea plants is still in infancy.In this research,the transcriptome sequencing(RNA-seq)and bioinformatics methods were applied and comparative analysis of the differentially expressed genes was performed to explore their expression patterns.Subsequently,qRT-PCR was performed to verify the differentially expressed genes,and the changes of JA,SA,ABA contents before and after tea geometrid feeding were compared.These results are helpful to clarify the molecular mechanism and systemic transfer characteristics of signaling molecules inducing the defense responses from leaves to roots after tea leaves are fed by Ectropis oblique.This research enriches and improves our understanding of the defense mechanism of tea plants,and provides theoretical support for the insect-resistant tea variety breeding,resistance genes exploring and pest integrate controlling.The main results are as following: 1.After quality control of the raw data which was produced by a Illumina platform,12.9,13.7,12.8,13.3 Gb clean data were obtained from CKL,CKR,EL and ER(three biological replicates per library),respectively.We used de novo assembly to obtain reference transcriptome,and generated 102,845 unigenes in total.The overall length of them was 74,486,413 nt,and the average length was 724 nt.The N50 was 1153 nt.In the analysis of gene expression annotation,63,588(92.3 %),41,126(59.7 %),37,179(54.0 %),23,813(34.6 %),46,027(66.8 %)unigenes were annotated to NR,Swiss-Prot,KEGG,COG and GO databases,separately.The total number of annotated unigenes was 68,907.2.There are 5,418 differential genes induced in leaves(3,769 were upregulated genes and 1,649 were down-regulate genes)and 9,902 in roots(7,050 were upregulated genes and 2,852 were down-regulate genes)compared with before feeding by Ectropis oblique.Further analyses showed that there were 9,568 and 5,084 differential genes specially induced in roots and leaves,respectively.In addition,there were 334 differential genes(288 were upregulated genes and 46 were down-regulate genes)induced in roots and leaves simultaneously.3.The bioinformatic analysis showed that the differentially expressed genes,which were induced in roots and leaves,were related to calcium signals,hormonal signals,signal transduction,reactive oxygen species metabolism,transcription factors and defense reaction after feeding by Ectropis oblique.The FPKM values showed that the expression levels of caffeine,phenylpropanoid and HIPVs biosynthesis pathways in leaves were much higher compared with those in roots.However,the expression level of CML(related to calcium signals,12.3 fold),CDPK(related to signal transduction,4.0 fold),and APX and SOD(related to ROS,4.1 and 4.6 fold)were much higher in roots than in leaves,respectively.The expression levels of these genes show no significant difference in leaves before and after feeding by Ectropis oblique.4.The results of qRT-PCR showed that the expression level of LOX3 achieved highest at 6 h and 9 h in leaves and roots after Ectropis oblique feeding,and was about 72-and 9.9-fold changes compared to controls,respectively.The peak expression of SAMT was achieved at 6 h in leaves and roots after Ectropis oblique feeding,and was about 2.4-and 1.3-fold changes than controls,respectively.The time of peak expression of L-ASO,which related to ROS synthesis pathway,was consistent with SAMT.The fold changes of leaves and roots were 1.2 and 1.1 compared to control,respectively,and its expression was down-regulated at the other time.The expression of the defense genes,such as PI,achieved the peak at 12 h in leaves by pests feeding,and was about 5.3-fold changes compared to control.However,the peak expression of PI in roots was appeared at 6 h,and was about 1.2-fold changes compared to control.5.LC-MS results showed that the content of JA in leaves was higher than in control plants during 3-24 h after Ectropis oblique feeding.The maximum content was appeared at 9 h,and about 19.7-fold changes compared to control.However,the JA content in roots was higher than control during 3-9 h after Ectropis oblique feeding.The maximum content was appeared at 3 h,and about 6.0-fold changes compared to control.The characteristic of JA-ile was similar to that of JA.The maximum content was appeared at 3 h in leaves and roots after Ectropis oblique feeding.However,the SA content in leaves and roots were lower compared with control at each time point after Ectropis oblique feeding.The maximum content of ABA was appeared at 24 h and 6 h in leaves and roots after Ectropis oblique feeding,respectively.The ABA content was higher than control in roots at each time point,expect at 9 h after Ectropis oblique feeding.In general,this study can help us to understand the overall defense reaction of tea plants in response to the Ectropis oblique feeding,and establish the effective strategy of preventing insect feeding and reducing the pesticide application. |
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