| To illustrate the plant secondary metabolism is a principal and basic work which is helpful for utilizing plant secondary metabolites to improve medicine production and push the research work about natural products.Compared with the primary metabolism,the secondary metabolism is very difficult to be recognized,but at the same time it is also an important research area with great potential.The research work about biosynthesis pathway is regarded as that in the area of mechanisms in the study of plant secondary metabolism,and it is also the core of the secondary metabolism.Thus,it is of great significance to study the biosynthesis pathway of plant flavonoids,not only for revealing the chemical composition of different plant in the formation,transformation and the relationship of different chemicals,but also in the production of flavonoids using modern biotechnique methods.Kudzu[Pueraria lobata(Willd) Ohwi.]originating in China is a perennial leguminous plant and is also an important wild plant resource.The main components of biological activity are puerarin,daidzin and daidzein.Isoflavonoid Content(dry weight) is high up to 7-8 percent,is several times that of soybean isoflavonoids(0.1 to 0.2 percent dry weight),which is helpful for studying on the separation and detection of secondary metabolism.The establishment of kudzu cell suspension culture and kudzu callus provides a good research basis and the research system for secondary metabolism,and can be used as the good materials for illustrating the flavonoid biosynthesis pathway in plants.In this paper,kudzu cell suspension cultures and callus cultures are selected as the materials,the correlation was examined between the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase and the accumulation of isoflavonoids in kudzu in vitro,as well as the correlation of the isoflavone biosynthesis and the accumulation of the possible precursor compounds such as prephenate, p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu in vitro is examined in the experiment about the PAL specific inhibitor and the 4CL specific inhibitor,which provide the enzymological evidences for verifying the novel possible pathway of isoflavone biosynthesis,and confirming the existence of the precursor compounds such as prephenate, p-hydroxyphenylpyruvate,p-hydroxyphenyllactate and p-coumaric acid during the production of kudzu isoflavonoids.The main results are as following:With the increase of the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase in the life cycle of kudzu cell culture,the accumulation of isoflavonoids increased gradually,and was examined several days later of the increase of enzyme activities.The correlations between the activities of prephenate dehydrogenase and p-hydroxyphenylpyruvate reductase and the accumulation of kudzu isoflavonoids are high,and the coefficients are 0.8812 and 0.8046,respectively.The assay for the PAL activity and the TTC viability in kudzu callus were examined, the results showed that the optimum concentration of L-Phe is 2mM,the optimum pH value is 8.8,the optimum temperature is 60℃,and the reaction products of PAL could maintain stability within 2.5h.The optimum TTC concentration for kudzu viability is 0.6% (w/v),the optimum pH value is 7.5,and the appropriate time range is12～16h.In the experiments using different concentrations of PAL-specific inhibitor(AOA) and 4CL-specific inhibitor(MDCA),the changes of the fresh weight,dry weight,the PAL activity,kudzu viability and the isoflavonoid content in kudzu callus were examined,the results showed that the optimal concentrations of AOA and MDCA were 10μM and 25μM,respectively.Three different treatments were performed with the AOA concentration of 10μM,and were as followings:①kudzu callus sample after 0 day without AOA treatment,②kudzu callus sample after 24 day without AOA treatment,and③kudzu callus sample after 24 day with 10μM AOA treatment.Samples were collected for the analysis of the isoflavonoid contents.The results showed that the isoflavonoid content in the third sample was 6.141 mg.g-1 higher than that in the first sample,and showed the significant difference (P=0.05).The isoflavonoid content in the second sample was 18.197 mg.g-1 higher than that in the third sample,and also showed the significant difference(P=0.01).The above data suggested that the other pathway of isoflavone biosynthesis might exist in kudzu callus.At the same time,the difference between the isoflavonoid content(③-①) and the isoflavonoid content(②-③) reflected the contributions of two different pathways to the isoflavone biosynthesis in kudzu callus.The main pathway of flavonoids biosynthesis in kudzu callus was from phenylalanine,through cinnamate,p-coumaric acid and p-coumarate:coenzyme A to chalcone,and the novel possible pathway of isoflavone biosynthesis may be the auxiliary pathway in kudzu callus.Under the condition of 10μM AOA treatment,the contents of prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu callus were higher than the control,and showed the significant difference(P=0.01).The results show that AOA treatment inhibited the PAL activity,but the specific mechanism is unknown.The isoflavonoid content in kudzu callus sample with 25μM MDCA treatment after 24 days was 2.217 mg.g-1 higher than that in the sample after 0 day,but showed no significant difference.At the same time,the isoflavonoid content in kudzu callus sample after 24 days without MDCA treatment was 22.251mg.g-1 higher than that after 24 days with 25μM MDCA treatment,and showed the significant difference(P=0.01).From which 4CL was the key enzyme of isoflavonoid biosynthesis,and specific inhibitor MDCA seriously blocked the isoflavone biosynthesis in kudzu Callus.Furthermore,the content of prephenate in kudzu callus with 25μM MDCA treatment was much higher than that in control,but showed no significant difference,and the contents of p-hydroxyphenylpyruvate and p-hydroxyphenyllactate were higher than control,and showed the significant difference(P=0.01).The results also showed that MDCA blocked the reaction from p-coumaric acid to p-coumarate:coenzyme A,and blocked the pathway of isoflavonoid biosynthesi,and reduced the accumulation of isoflavonoids,which further increased the content of prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate in kudzu callus through feedback inhibition.The comparing results also further confirmed prephenate,p-hydroxyphenylpyruvate and p-hydroxyphenyllactate might be the precursors of isoflavone biosynthesis.
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