| Tea pests have always been the major limiting factor in high quality and efficiency tea production, however, the molecular mechanism of the tea plant resistance is not clear today. While the molecular mechanism induced defense research of feeding pests on evergreen woody plant is lacking. This paper researched the differentially expressed genes in both number and type after feeding main pests (tea looper) on the tea leaves using SSH technology. On this basis, comparative studies of the express characteristics of differentially expressed fragments(TDF)related with the important defense function, as well as the expression differences in different varieties were made through fluorescence quantitative PCR. Main results are as follows:(1) A stable and reliable technical system of SSH is established, and technical support for subsequent study of differences in gene expression between different samples is provided.(2) Based on the cloning identification of induced cDNA fragments included in the forward subtractive cDNA library, and the sequencing of the fragments longer than 200bp, 280 TDF sequences were obtained. According to the length, there are 151 TDF with the length between 200bp and 400bp, 54% of the total. The number of the TDF with the length between 400bp and 600bp is 101, 36% of the total. 28 TDF is longer than 600bp, 10% of the total. Through BlastX comparison shows: the gene has high homology with known functional sequence is 157, 56% of the total, the cloning of hypothetical protein is 19, 6% of the total. There's no match for 104, 37 % of the total. And 271 sequences of TDF is submitted to GenBank(GW342615 - GW342885)。(3) The classification results of 144 induced genes'probable function show that 31 genes related to the protein metabolism, 21% of the total;18 have relationships with photosynthesis and primary metabolic functions, about 13%;11 are with the transfer function of proteins, 8% of the total; and the number of genes related to the transcription function is 12, 8% of the total;11 related to the function of signal transduction, 8% of the total;and the number of the genes related to lipid metabolism and secondary metabolic function are respectively 7 and 13, 5% and 9%;22 are related to adversity and defense functions, 15% of the total;19 are related to other metabolic pathways of plant, 13% of the total.(4) According to the reports related to plant defense mechanism of pests, there are a few may involved in the tea looper feeding: 4 TDF related with direct defense, including caffeine synthase (TDF - 55, 88), flavonols synthase (TDF - 72, 146), arginine enzyme (TDF - 2) and homocysteine enzyme inhibitors (TDF - 144), And 7 TDF have relationships with indirect defense, including the processes of biosynthesis of green leafy volatiles, aromatic volatiles and terpenes volatiles.(5) Through quantitative polymerase chain reaction (PCR) analysis of induced gene expression level, it is founded that after feeding,the raised expression of the TDF related to different defense mechanisms is different, and along with the time expression varies in different varieties. Such as the phenomenon that caffeine synthase (TDF - 55, 88) and flavonoids synthase (TDF - 72, 146) increase in different time after feeding, but caffeine synthase was increased significantly greater than flavonoids synthase in four hours after feeding. Different varieties of tea, the raised expression of the induced TDF is different, too. For example, after feeding, longjing 43, TDF - 73 raised express multiple higher than shuchazao, reach to 74.5%, then are the TDF - 55 and TDF - 139, higher than the shuchazao 57.8% and 57.1% ,TDF - 40, higher than shuchazao only 11.9%. For TDF - 72 and 105 are lower than shuchazao, respectively 53.4% and 26.9%. |
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