Temperature Difference On Syringin And Coniferin Contain In Dendrobium Catenatum And Related Gene Expression

Dendrobium catenatum is a valuable Chinese herb in the Orchidaceae,which is the first of the nine Chinese herb.Dendrobium catenatum contains polysaccharides,flavonoids,alkaloids,coniferin,syringin and other phenolic medicinal ingredients.It is one of the effective way to realize the sustainable utilization of wild Dendrobium catenatum resources to use Protocorm-like body(PLB)instead of seedling.Temperature is an important factor affecting matter accumulation in plants,and the temperature difference between day and night has significant effects on photosynthesis,physiological characteristics,flower bud differentiation,matter accumulation and yield and quality of crops.In this paper,Dendrobium catenatum PLB were used as materials to optimize PLB proliferation of Dendrobium catenatum.A method for simultaneous determination of coniferin and syringin in Dendrobium catenatum was established.DcF5 H,DcCOMT,DcCAD and DcCAD promoter sequences were cloned.The quantitative expression of DcF5 H,DcCOMT,DcCAD,coniferin and syringin in different temperature difference treatment were analyzed.The main research results are as follows:1 Proliferation optimization of the PLB of Dendrobium catenatumThe effects of carbon source,amino acid and different culturemethods on the proliferation of Dendrobium candidum were studied,PLB as material,1/2 MS+50 g/L mashed potato as basic culture medium.The results showed that glucose promoted the growth of PLB and delayed the differentiation of PLB,At 30 d,the proliferation coefficient was 3.75;The addition of amino acids inhibit the proliferation rate of the PLB.The proliferation rate of PLB in solid medium was significantly lower than that in liquid medium.At 30 d,the proliferation coefficient of suspension culture was twice than that of solid culture.Under 25/21 ? temperature treatment,Dendrobium catenatum PLB proliferation state is best,the proliferation of PLB mass is small,the color green,and less differentiation;Most suitable for Dendrobium catenatum PLB proliferation medium is 1/2 MS+50 g/L mashed potatoes+25 g/L glucose suspension culture,at the same time set 4 ? temperature difference between day and night.2 Simultaneously determination of coniferin and syringin in Dendrobium catenatum by UPLCA method was developed for simultaneous determination of coniferin and syringin in Dendrobium catenatum by ultra-high performance liquid chromatography(UPLC).To provide technical support for further study on the influence of diurnal temperature difference in Dendrobium catenatum.the chromatographic separation was carried on an ACQUITY UPLC BEH C18(2.1 mm×100 mm,1.7 ?m)ingradient elution with a mobile phase of 1% acetic acid aqueous solution(A)/methanol(C),flow rate: 0.20 mL/min,column temperature 35 ?;The injection volume is 5.0 ?L.The gradient elution condition was initially 0% A;0~3 min,80% A;3~8 min,80% A;8~10 min,95% A.The quantitative wavelength was 283 nm.The two substances were completely separated within 10 minutes.The extraction method of an orthogonal test was designed,and the results showed that the sample extracted with pure water,extraction ratio 1 : 40,and the extraction time1 h(vibrate every half an hour)had the highest extraction rate of the two substances.The proposed method was applied to the determination of four samples of Dendrobium catenatum PLB in temperature difference treatment.The results showed that the most conducive to the coniferin is25/21 ? accumulated temperature difference,15 d,coniferin content up to 204.78 mg/kg,1.33 times that of control.The most conducive to syringin is 25/17 ? accumulated temperature difference,10 d,syringin content up to 27.04 mg/kg,2.02 times that of control.Moderate temperature difference appears to promote the effect,while big temperature difference lowers the effect.3 DcF5 H,DcCOMT and DcCAD gene cloning and bioinformatics analysis of Dendrobium catenatumThis study use Dendrobium catenatum PLB as material,PCR technology were used to clone DcF5 H,DcCOMT,DcCAD.resultsshowed that the ORF of DcF5 H gene 1 536 bp,prediction encoding 511 amino acids,subcellular localization prediction its positioning in the cellular matrix,DcF5 H protein contain cytochrome P450 protein on the conservative domain structure,It belongs to the oxidase family of proteins.DcCOMT gene ORF long 1 086 bp prediction encoding 361 amino acids,subcellular localization prediction results show that DcCOMT protein play a role mainly in the cytoplasm,DcCOMT contains seven conservative domain structure,is a kind of regulating dimers protein methylation reaction.DcCAD gene ORF long 641 bp,prediction encoding 216 amino acids,subcellular localization prediction results show that DcCAD protein mainly play a role in the mitochondria,found it contain a typical structure of alcohol dehydrogenase conservative domains and NAD(P)combined with conservative domain structure,presumably the protein play a role of reduction,has characteristic of reductase.We investigate the codon usage bias of DcCOMT.The results showed that the ENC,GC and GC3 s site were 49,0.452 and 0.396 respectively for DcCOMT,which suggests that the codon bias level of DcCOMT in Dendrobium catenatum research was low,biased towards the synonymous codons with A or U on the 3rd site.mutation pressure is the main force in the formation of DcCOMT codon preference.The relationship between DcCOMT gene of Dendrobium catenatum andPhalaenopsis bellina is close,and their GC1 s,GC2s,GC3 s,GC,CAI and ENC are similar,indicating that the closer the species evolutionary relationship is,the more similar their codon preference is.In addition,the yeast expression system was more suitable for the heterologous expression of DcCOMT gene of Dendrobium catenatum.The model plant Nicotiana tabacum and Solanum lycopersicum could be used as genetic transformation receptors.Dendrobium catenatum DcCAD gene promoter amplified to get the gene promoter sequences 2 003 bp,DcCAD promoter analysis showed that the gene transcription starting area is located in-147--197,transcription start site is located in the first-156 bp G,The promoter contains cis-elements such as light,hormone,growth and metabolic regulatory elements and circadian.It may be closely related to response to stress,metabolism process of Dendrobium catenatum.4 Quantitative expression and correlation analysis of DcF5 H,DcCOMT and DcCAD genes of Dendrobium catenatum by qPCRExpression pattern of DcF5 H,DcCOMT,DcCAD gene under25/25 ?,25/21 ?,25/17 ?,25/13 ? diurnal temperature difference was analyzed,the result showed that: The expressions of DcF5 H and DcCOMT genes were significantly negatively correlated with the contents of syringin and coniferin,presumably DcF5 H DcCOMT similar regulation mode,and the two genes negative regulation of the syringinand coniferin.The expression level of DcCAD gene was significantly positively correlated with the content of syringin,suggesting that DcCAD and DcF5 H,DcCOMT had opposite regulation modes.indicating that DcF5 H,DcCOMT was negative control the metabolism of coniferin and syringin.DcCAD expression increased the content of syringin and coniferin in Dendrobium catenatum.