Signal Transduction Of Endophytic Fungus On The Synthesis And Accumulation Of Alkaloid In Sophora Alopecuroides L.

Endophytic fungal elicitor as a specific chemical signal,which can induce plant cells to produce a series of physiological and biochemical effects about signal molecules,and thus to selectively induce expression of specific genes of plants,the activity of key enzymes of metabolic activation process,eventually leading to changes in the accumulation of certain products in secondary metabolic pathways.Howerver,signal transduction and other molecular mechanisms which about Endogenous fungal elicitors promote the synthesis of secondary metabolites in medicinal plant cells is not yet clear.In this study,the tissue culture seedlings of Sophora alopecuroides L.were taken as the experimental materials.4 species of endophytic fungi concentrates of S.alopecuroides L.were prepared as the fungal elicitors to explore the effect on the accumulation of Oxymatrine(OMA).Determination of defense enzymes activity in tissue culture seedlings of S.alopecuroides L.induced by endophytic fungal elicitors,explored the physiological defense mechanism of endophytic fungi promoting the biosynthesis and accumulation of alkaloids.Under the action of endogenous fungal elicitors,analyse the content changes of the main signal molecules and OMA in the tissue culture seedlings of S.alopecuroides L.,investigated the effect of endophytic fungal elicitors from S.alopecuroides L.on key signaling molecules and alkaloids in the host.Real-time fluorescence quantitative PCR(qRT-PCR)technology was used to establish a method of detecting the gene expression of Lysine decarboxylase(LDC)一 a key enzyme for the synthesis of alkaloid in S.alopecuroides L..We preliminary discussed the effect of endophytic fungal elicitors on gene expression of key enzyme in alkaloid synthesis in the host,and analyse the relationship between LDC gene expression and the accumulation of OMA and signal molecules in the host,which revealed the mechanism of biosynthesis and accumulation of alkaloids in S.alopecuroides L.inducted by endophytic fungi.The main conclusions were as follows:1.4 species of endophytic fungi from S.alopecuroides L.,and their concentrated filtrates were prepared as the fungal elicitors.To investigate the effects of 4 different endophytic fungal elicitors on the biosynthesis of OMA and physiological defense reaction in tissue culture of S.alopecuroides L..The results showed that:4 species of endophytic fungi concentrates of S.alopecuroides L.could improve the content of OMA in tissue culture seedlings of S.alopecuroides L..Among them,the endophytic fungi XKGKDF40 was most effective,during 0-15 days of induction,OMA content continued to increase and remained higher than the control,reaching a maximum of 3.616 mg/g on the 15th day,2.09 times the 15th day of the control group.4 species of endophytic fungal elicitors could promote the activity of PAL which was one of the key enzymes on the accumulation of alkaloids.Also they could promote the activity of antioxidant enzymes,such as POD,APX and CAT.2.Under the influence of endophytic fungal inducers XKZKDF11 and XKYKDF40,analysed the changes of nitric oxide(NO),salicylic acid(SA),and jasmonic acid(JA)and the main active ingredient OMA in the host plant under different induction time.And explored the relationship between signal molecules induced by endogenous fungal elicitors and changes in OMA content,PAL,APX,and CAT activities.The results showed that:XKGKDF11 and XKYKDF40 endophytic fungal elicitors induced the increase of host signaling molecule NO bursts first,followed by SA and JA.The time of them reaching their maximum were detected at 3th day,9th day and 12th day after induction,respectively.During the induction process,3 kinds of signal molecules were involved in the regulation of PAL,APX,and CAT activity,and then the host OMA content peaked.We can speculate that NO,SA,and JA jointly mediated the endophytic fungal elicitors XKZKDF1l and XKYKDF40 to promote the synthesis of OMA.It was also found that the burst of NO was one of the earliest responses to the treatment of endophytic fungal elicitors in the tissue culture of S.alopecuroides L..The signal molecules SA and JA had antagonistic or complementary effects on the synthesis of OMA by the endophytic fungal elicitors.3.Target gene primers and reference gene primers were designed according to LDC and Lectin gene sequences of S.alopecuroids L..By relative quantification methods,optimizing the qRT-PCR reaction system and reaction conditions,to establish the detection method for LDC gene expression of key enzymes in alkaloid synthesis of S.alopecuroides L..The results showed that:The results of qRT-PCR were better when the cDNA content in the system was 200 ng/μL and the annealing temperature was 61 ℃.The standard curve of the target gene QLDC and the reference gene Lectin was constructed,in which the cycle threshold and template concentration showed a good linear relationship,the amplification efficiency is above 99%,and the sensitivity is 25 times that of semi-quantitative PCR.The endophytic fungi XKYKDF40 concentrated filtrate was prepared as the fungal elicitor,and it was found that the expression level of LDC gene in the host was consistently increased with the OMA content.Expression of host LDC gene reached the peak on the 15th day,which was 260.95 times that of the 0 day after induction treatment.4.Based on the establishment of stable quantitative RT-PCR detection of the expression system of Lactuca sativus LDC gene,analyzed the effect of endophytic fungal elicitor XKZKDF11 on the expression of key enzymes in alkaloid synthesis,explored the relationship between LDC gene expression and host OMA content and signaling molecules.The results showed that:The fungal elicitor XKZKDF1I significantly promoted the expression of LDC gene in the host and was higher than the control group at the same time in each induction period.During 0-12 days of induction,LDC gene expression and OMA content creased synergistically.On the 12th day of induction,the expression level of LDC gene reached the peak,which was 394.41 times of the 0 d of induction and 21.26 times of that of the control group of the same period.Combining with the changes of signal molecules in S.alopecuroids L.,it was found that during the induction process,the trend of changes in the expression levels of JA and LDC gene was basically synchronized.It is speculated that JA participates in the induction of LDC gene expression by the endophytic fungal elicitor XKZKDF11.