| In this paper, we take semi-parasite plant Thesium chinense (Santalaceae) as experimental materials, focusing on the research of haustorium formation which is a key process of parasite. We aim at separating and identifying chemical signals during haustorium development, and selecting several haustorium development-related genes. These researches are important to reveal the mechanism of haustorium formation and construction of parasitism between parasite and host plants. It is of great theorietical and practical significance that elucidate the parasitic mechanism to solve and develop the production of T. chinense.The concrete research content is as follows:1. To separate and identify chemical signals which inducing T. chinense haustorium formation, we cultured Prunella vulgaris acted as host plant, we used the method of GC-MS to mensurate the components of T. chinense root’s secretion collected with Amberlite XAD-4 resin. A total of 53 compounds had been detected, including hydrocarbons, esters, organic acids, ketones, alcohols, nitrogen containing compounds, phenolic, Aldehyde and quinine. Despite its low concertration in phenol quinines, 2,4-di-tert-butylphenol and 2,5-di-tert-butyl-1,4-benzoquinone were critical compounds for its phenyl structure, especially the latter owned a core structure of 1,4-benzoquinone, as similar with DMBQ (a haustorium induced factor commonly reported), the occurrence of electron transfer in phenol, hydroquinone and semiquinone state gave stimulus signals in haustorium formation.2. Treated with different concentrations of DMBQ as exogenous signals to induce haustorium formation in T. chinense, results showed that DMBQ worked effectivly on inducing haustoria, but induction effects vary widely in different concentrations. Low concentration solution (1μM,0.1μM) showed stronger ability to induce haustoria formation than high concentration solution (1000μM,100μM). When treated with 1μM DMBQ after 60 d, nearly 20 haustoria appeared per seedling, but when treated with 1000 μM DMBQ, T. chinense seedlings grew slow without roots elongation and scarcely possible detected haustoria formation. The ratio of haustoria number and roots weight reflected inducing ability and 1 μM DMBQ showed perfect with a ratio of 110.52 when treated to induce haustoria.3. To optimize a simple and effective method for total RNA extraction from root of T. chinense which rich in polysaccharides and phenol, the experiment compared and analyzed the quality of total RNA extracted by TRIzol, CTAB-LiCl, SDS/phenol and improved TRIzol methods from root of T. chinense through electropherogram, UV spectrophotometry and RT-PCR verification. Results indicate that the total RNA extracted by improved TRIzol method were clear and no dispersion with a ratio of OD260/OD280 was 1.943, the integrity of the RNA was well, and there was no obvious contamination with DNA and other impurities, was suitable for RT-PCR test. In conclusion, the improved TRIzol method is efficient for total RNA extraction from root of T. chinense.4. RNA extracted from the normal roots (without haustoria) was used as a driver and haustorium was used as a tester to conduct suppression subtractive hybridization. After two rounds of PCR reactions, The amplified, differentially expressed cDNA fragments were cloned into a T-Vector and then transformed into E. coli for SSH library construction.395 randomly selected clones from the subtracted libraries were sequenced and 276 ESTs obtained after removing ailed sequencing, poor quality sequences with poly(A)+results and redundant sequences. The total length of these retained ESTs were 132,926 bp, from minimum 157 bp to maximum 1,059 bp in length with an average length of 481.62 bp.5. According to BlastN and BlastX searches with credible expectation values (E-value<10-6),230 ESTs showed significant homology to previously identified genes in GenBank. Among these sequences,199 ESTs obtained GO annotation which consisted of molecular function, biological process and cellular component. Based on 2nd level of GO annotation, catalytic activity and binding activity (DNA, RNA, protein, anions) took up a large proportion of 40.7% and 39.1% in molecular function; metabolic process and cellular process owned a percentage of 22.2%and 22.5% in biological process; cellular component meant where these genes functioned, and cell had a highest percentage of 32.9%, followed by organelle (23.9%) and membrane (18.0%).6. To investigate whether the differentially expressed candidate genes were expressed during haustorium development, relative expression of these genes in haustoria was determined at the following stages:haustorium-underdeveloped stage (normal roots of T. chinense without induction), haustorium initiation stage (the stage that swellings appeared in exodermic of roots), haustorium maturation stage (vessels appeared in this stage) and haustorium penetration stage (haustoria connected roots of parasite and host plants). Results showed that relative expression level of TcPME was about 4-fold higher at haustorium penetration stage compared to other stages. Meanwhile, the relative expression level of TcAux/IAA was highest at haustorium initiation stage and decreased at the subsequent two stages. TcPrx and TcPAL showed a similar expression pattern during haustorium formation, and their relative expression gradually increased to a degree of 17.28-fold and 9.63-fold higher at haustorium penetration stage than haustorium-underdeveloped stage, respectively. qRT-PCR data also revealed that relative expressions of the four ESTs up-regulated in the samples induced by hosts compared with the control. For the control, relative expressions showed more gently, maximum transcripts of TcPME, TcAux/IAA, TcPrx and TcPAL were 1.36-fold (6 d),1.40-fold (12d),1.77-fold (20d) and 1.59-fold (12d), respectively. While maximum transcripts on treatment group were 2.09-fold,3.18-fold,6.03-fold and 2.88-fold, respectively. Three ESTs relative expressions were at its maximum at 20 d except TcAux/IAA (12 d). |
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