Effects Of Stress On Effective Component And Stress-tolerant Gene Of Isatis Indigotica Fort.

Isatis indigotica Fort, is a main plant source and raw material of indigowoad root and leaves, which are very important Chinese traditional medicine. Indirubin, the major active constituent of indigowoad root and leaves, is a kind of secondary metabolites of Isatis indigotica Fort, and it is also a kind of indole alkaloid. Presently, many researchers consider that the amount of secondary metabolites in plant will increase while the plant suffers from adverse environmental stress. Therefore, the general pattern of production and accumulation in plant of the indirubin, the kind of secondary metabolites of Isatis indigotica Fort., may also work this way. The existence of this phenomenon is related to the physiological and biochemical reaction in vivo when Isatis indigotica Fort. suffers from stress. Furthermore, it is also related to the molecular mechanism of resistance and adaption to stress of Isatis indigotica Fort. There must be some molecular foundations accounting for that phenomenon above.The exploration of this field is very helpful to get a clear picture about the habit and stress-resistance of Isatis indigotica Fort. Furthermore, it can also instruct people to cultivate Isatis indigotica Fort. more scientifically and rationally, thus the raw material of indigowoad root of higher quantity and better quality will be produced. Meanwhile, the study and discussion of this field can provide some theoretic bases for future research on the secondary metabolism and molecular mechanism of stress-tolerance of plant better.In the first part of this research, the seedlings of Isatis indigotica Fort. were used as experimental materials to analyze the change of the amount of indirubin while they were under different stresses. In the second part of this research, a new stress-tolerant related gene, named IiLEA, was cloned from Isatis indigotica Fort. and the mode of expression of IiLEA in seedlings of Isatis indigotica Fort. treated with different stresses was analyzed by Northern blotting. The main experimental methods and conclusions of this thesis are as follows:1. The seedlings of Isatis indigotica Fort. cultivated for 30 days in aseptic environment was treated with 250mmol/LNaCl, natural drought method, 20mmol/L ABA, 4℃ low temperature, respectively. The content of indirubin was extracted from Isatis indigotica Fort. seedlings at the indicated times and analyzed by HPLC method.The results showed that the content of indirubin could increased up to 59.05%~ . 161.67% in salt, drought and ABA treatment during 12h. And the content of indirubin increased up to 141.4% under low temperature stress for 24h. The conclusion implied that the content of indirubin in the Isatis indigotica Fort, seedlings could accumulate by appropriate stress and ABA treatment efficiently. The 250mmol/L NaCl stress treatment could get the most effect.2.693bp cDNA encoding late-embryogenesis-abundant(LEA) protein fragment was cloned from the seedlings of Isatis indigotica Fort.by method of RT-PCR on the basis of the homologous genes of Arabidopsis thaliana, Brassica napus, Chorispora bungeana etc. in GenBank. Sequensing analysis shows that there is 88.3% identity in nucleotide sequence and 83.3% identity in amino acid sequence to that of Brassica napus. The Open Reading Frame (ORF) of this sequence comprised of 648 base pairs encodes LEA protein with 215 amino acids. The molecular weight and isoelectric point of this protein is 23kDa and 8.07. There are 7 repeating 11-mer amino acid motif, the most important character of group 3 LEA protein, in amino acid sequence of this protein.3.The seedlings of Isatis indigotica Fort, cultivated for 30 days in aseptic environment was treated with 250mmol/L NaCl, natural drought method respectively. The total RNA was extracted from it and the expression pattern of its IiLEA gene in vivo at the different stress treatment time was analyzed by Northern blotting method. The results showed that the IiLEA gene started to express in vivo when seedlings of Isatis indigotica Fort, had been treated with 250mmol/LNaCl for 3 hours, and the longer time of stress treatment was, the.higher IiLEA gene expressed. When the seedlings of Isatis indigotica Fort.wexe treated with 250mmol/L NaCl for 12 hours, the expression level of IiLEA gene became lower. The expression pattern of IiLEA gene of Isatis indigotica Fort, seedlings which was treated with drought stress was the same as those treated with salt stress. This conclusion implied that IiLEA gene could be induced by appropriate stress while it did not express in vivo as the seedlings of Isatis indigotica Fort, growing normally.