Expression Analysis Of The Rubber Synthetic Gene In Suspension Cells Of Taraxacumkok-saghyz Rodin

As one of the four major industrial raw materials,natural rubber（NR）plays an important role in the industrialization and economic development of the world and is an important strategic material in China.Hevea brasiliensis,Taraxacum koksaghyz Rodin and Parthenium hysterophorus L.are collectively referred to as the world’s three major rubber-producing plants.Currently,98%of the world’s natural rubber comes from rubber tree.The regulation of rubber biosynthesis is a major theoretical issue in the production of natural rubber,and it is also a hot spot in scientific research.Laticifers is a secretory tissue in the bark of rubber tree.Its main function is to synthesize and store the natural rubber.In the bark of the rubber tree,the vascular cambium differentiated and formed the secondary laticifers which play an essensial role in synthesis of natural rubber,the differentiation of laticifers is one of the most important the oretical issues in the production of NR with which positive correlation to the number of the secondary laticifers.It is an important index for rubber tree yield breeding.We have identified the key signaling molecule that promotes the differentiation of laticifers is Jasmine,and jasmonic acid signaling pathway is involved in regulating the biosynthesis of NR.Taraxacum koksaghyz Rodin,also known as Russian dandelion.Native to Kazakhstan and China’s Tianshan region.It is a perennial herbaceous plant of the genus Chicory of the family Chicory.The natural rubber produced is cis-isoprene,and its structure and properties are comparable to those of rubber produced by the rubber tree.Moreover,from the point of view of the physiological process of producing rubber,there is a similarity.Therefore,Taraxacum koksaghyz Rodin is considered to be an ideal model plant for studying the mechanism of rubber production,and it has high scientific application value and significance.At present,due to the low genetic transformation efficiency of rubber trees and the long function period of gene identification,we use Taraxacum koksaghyz Rodin suspension cells as materials.The jasmonic acid signaling pathway of the COR response and the expression profile of genes related to the rubber synthesis pathway were identified by transcriptome sequencing and quantitative real-time PCR（qRT-PCR）.At the same time,the genetic transformation system of Taraxacum koksaghyz Rodin suspension cells was established,and the key component genes HbMYC2 and HbMYC3 of the rubber tree jasmonic acid signaling pathway were successfully transformed into Taraxacum koksaghyz Rodin suspension cells.The research results will provide a reference for the study of the mechanism of rubber synthesis using Taraxacum koksaghyz Rodin.The main results are as follows:First,we obtained 88.000 unigene from the analysis of transcriptome which base on the genetic transformation suspension cell sample treatment by COR.Although the expression multiple of 28 unigene were picked randomly and analysed by qRT-PCR,the expression trend of most unigene were co-related to transcriptome.Under the criterion of q-value<= 0.05,Fold-change（FC）>=2,according to the transcriptome,among the process of JA bio-synthesis,there were 4 members of LOX gene family and 1 member of AOC gene family were up-regulated compared to control.In JA-signal pathway,one of JAZ gene（contig₅248）was strictly up-regulated,with the log2FC value of 5.94.In MYC gene family,2 of them were down-regulated and one of which was probed in 8 h sample,and the rest of 12 genes were up-regulated.According to the heat-map of unigene related to JA bio-synthesis and JA-signal pathway,most gene were up-regulated induced by COR,so COR activated JA-signal pathway.Also,COR activated the MVA pathway which in the upper reaches of the NR bio-synthesis in suspension cell of Taraxacumkok-saghyz Rodin.COR induced several genes in the MVA pathway expressed,including ACAT、HMGS、HMGR、MVK gene family,3 members of ACAT gene family,1 member of HMGS gene family,8 members of HMGR gene family,1 members of MVK gene family were up-regulated among them.At the beginning of NR bio-synthesis,1 unigene of GPS gene family was up-regulated and expressed in 8 h,2 unigene of FPS gene family were up-regulated.We analysed the expression pattern of several genes correlated to the NR bio-synthesis by qRT-PCR in suspension cell of Taraxacumkok-saghyz Rodin with treatment of COR.It indicated that COR induced IDI1,FPS,SRPP2,HMGR,SRPP3,SRPP4 gene up-regulatedstrictly,but hardly has effects on expression of GGPPS,CPT1,SRPP1.In addition,COR was produced by a pathogenic mutant of Clove Pseudomonas and modulated the growth of plant,so we analysed other plant hormone signal pathway in suspension cell of Taraxacumkok-saghyz Rodin,including SA,ETH,IAA,GA3 which are induced related unigene up-regulated mainly and ABA,cytokinin,brassinolide which hardly changed the expression pattern.Second,the susceptibility of suspension cell to the ticarcillin of antimicrobial agents and to the Hygromycin B have been measured,the expression plasmid pCAMBIA1302 transformed into suspension cell by Agrobacterium mediated-transformation methodand the genetic transformation rate tested by Green-fluorescent-protein（GFP）and Hygromycin B.It indicates that suspension cell were insensitive to Ticarcillin in the density of 0-300 mg/L and the most suitable density of Hygromycin B for screening resistant callus was 10 mg/L.The testing of resistant callus by Hygromycin B,PCR and Fluorescence confocal microscope indicated that the construct agrobacterium which in the density of 0.6 in the OD600 blend suspension cell in the rate of 1:1.2（V/V,suspension cell:agrobacterium）for 30 minutes and co-culturing for 10 d,the we could harvest the positive genetic transformed resistant callus in the screen media.Finally,we constructed and analysed the positive genetic transformed callus.HbMYC2 and HbMYC3 gene was clone to the over-expression vector pHB,respectively,and then transformed into suspension cell via Agrobacterium mediated-transformation method,the positive HbMYC2,HbMYC3 over-expression callus was cultivated in screen media and detected by PCR.Compared to control（suspension cell）,in the positive HbMYC2,HbMYC3 over-expression callus,FPS,CPT1,SRPP4 gene not change the expression pattern,HMGR,IDI1,GGPPS,SRPP1,SRPP1,SRPP2,SRPP3 gene was strictly down-regulated which opposite to the expression pattern of suspension cell induced by COR,the reason need further research.