| The root of Salvia miltiorrhiza Bunge is the Chinese medicinal material and widely used in the treatment of cardiovascular and cerebrovascular diseases.The secondary metabolites of S.miltiorrhiza,tanshinone,cryptotanshinone,rosmarinic acid and salvianolic acid B are the main medicinal active ingredients.Plant secondary metabolites are important substances that resist external unfavorable environment,and are also the main medicinal active ingredients,therefore,there is a contradiction between the accumulation of secondary metabolites and the requirements of their growing environmental conditions.There have a difficult problem in medicinal planting and evelopment and utilization of medicinal ingredients.Studies have shown that the WD40/MYB/bHLH transcription factor complex affects the accumulation of secondary metabolites by regulating the expression of multiple enzyme genes involved in secondary metabolic.WD40 protein plays the role of a "scaffolding" in the transcription factor complex as a backbone protein.And studies have reported that WD40 protein plays an important role in plant stress resistance,especially drought response.In this study,based on the whole genome sequence of S.miltiorrhiza,the SmWD40 gene family was screened and the key gene SmWD40-170 in response to drought stress was found.Therefore,this study intends to explore the mechanism of response to drought,and effects on the growth and secondary metabolite synthesis and accumulation by regulating SmWD40-170.The main research contents and results are shown as follows:1.Based on the complete genome database of S.miltiorrhiza,a total of 225 SmWD40 genes were identified.Through phylogenetic analysis,all SmWD40 proteins were divided into 9 subfamilies.According to the domain composition they divided into 17 subfamilies.Physiological and biochemical analysis,gene structure analysis,protein conserved motif analysis and GO annotations were performed on all members of the family using biological software.The expression patterns of all genes in different parts were analyzed by real-time quantitative PCR.In order to further study the function of SmWD40 gene,the Sm WD40 gene promoter cis-acting elements were analyzed and 110 of them had drought response elements were dehydrated at different time intervals,and a significant response gene SmWD40-170 was screened.2.Building the HBT-GFP-NOS subcellular localization vector of SmWD40-170 gene,the Arabidopsis thaliana protoplasts were isolated and purified,and the localization vector was transferred into Arabidopsis thaliana protoplasts by PEG-mediated chemical transformation.SmWD40-170 was found is localized in the nucleus and cytoplasm.3.SmWD40-170 overexpression and interference vector were constructed by Gateway technology and amiRNA technology respectively.SmWD40-170 overexpressing plants and SmWD40-170 interference plants were obtained by Agrobacterium tumefaciens-mediated transformation of S.miltiorrhiza leaf disc.Positive plants were simultaneously verified from DNA level and RNA level.The overexpressing lines were OE-3,OE-7 and OE-8,and the interference lines were i-11,i-14 and i-15.4.The stomatal opening of SmWD40-170 overexpressing and interfering strains showed that the stomata of the interference strain could not be closed normally after 3 hours of ABA and H2O2 treatment.The overexpression and interference lines were dehydrated respectively,it was found that the dehydration rate of the interference strain was higher than the control strain.At the same time,the stomatal opening after dehydration for 3h was measured,the interference strain could not be closed normally compared with the control strain.Indicated that the dehydration rate was caused by stomatal opening and closing.It is concluded that SmWD40-170 may be involved in the closure of the stomata to regulate the response of plants to arid environments.5.After transplanting the tissue culture seedlings of 60d S.miltiorrhiza overexpressing,interfering and control strains to the soil culture medium,it was found that the interference strains had smaller leaves,curled leaves and less roots than the control strains,and the whole plants grew slowly;The overexpressing lines have large leaves,developed roots and strong growth.6.The content of secondary metabolites in the expression,interference and control strains of S.miltiorrhiza with 60d in soil culture was detected.(1)The content of total phenolic acid and total flavonoids in the over-expressed and dried control lines were analyzed.The results showed that the total phenolic acid and total flavonoids in SmWD40-170 overexpressing lines were increased compared with the control,while the content of total phenolic acid and total flavonoids in Sm WD40-170 interference strains decreased significantly compared with the control.(2)Using LC-MC to detect the content of salvianolic acid B,rosmarinic acid,tanshinone ⅡA,and cryptotanshinone of the expression,interference and control strains of S.miltiorrhiza.The results showed:The content of rosmarinic acid,salvianolic acid B,tanshinone ⅡA and cryptotanshinone in SmWD40-170 overexpressing gene line was significantly increased,and the content of each component in Sm WD40-170 interference strain was significantly decreased.(3)The expression patterns of the key enzyme genes in the salvianolic acid biosynthesis pathway and the tanshinone biosynthesis pathway were analyzed in transgenic and control strains.The results showed that key enzyme genes such as SmPAL,SmC4H,Sm4CL,SmTAT,SmTAT,SmRAS and SmCYP98A14 on the salvianolic acid synthesis pathway were significantly up-regulated in the overexpressing lines and significantly down-regulated in the interference lines;In the tanshinone biosynthesis pathway The expression levels of the key enzyme genes SmDXS,SmHMGR,SmFPPS,SmGGPPS,SmCPS and SmKSL were significantly up-regulated in the overexpressing lines and significantly down-regulated in the interference lines.It is concluded that SmWD40-170 affects the synthesis and accumulation of secondary metabolites by regulating the growth of leaves and roots.In summary,this study laid the foundation for bioinformatics for the identification of members of the WD40 family of S.miltiorrhiza,and opened up new ideas for the research of SmWD40-170 in drought resistance,growth and secondary metabolism. |
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