可溶性淀粉测定方法

1、可溶性糖和可溶性淀粉：Soluble sugar was determined by the anthrone method (Li, 2000) using sucrose as the standard. Half a gram of fresh samples was placed in a 25mL cuvette added with 10 mL distilled water, allowedto sta nd at 100°C for1 h, and filtered into 25 mL volumetric flasks. Reaction mixture of m

2、L contained mL extracts, mL mixed reagent (1 g anthrone+50 mL ethyl acetate), 5mL H2SO4 (98%), mL distilled water. The mixture was heated at 100 C for 1 min and absorbance read at630nm. Soluble starch wasdetermined by the anthrone method (Li, 2000) with sucrose as the standard. The remainder of meas

3、ured soluble sugar was transferred to a 25 mLcuvette-1containing 10 mL distilled water and mL HClO4 mol L ). The cuvette was placed in a boiling water bath for 30 min, cooled to 30C, and filteredinto 25 mL volumetric flasks. Reaction mixture of mL contained mL extracts, mL mixed reagent (1 g anthron

4、e+50 mLethyl acetate), 5mLH2SO4 (98%), mL distilled water. The mixture was heated at 100C for 1 min and absorbance readat 630nm. Total non-structural carbohydrates (NSC) were the sumof soluble sugar and soluble starch.Extraction and analysis of carbohydrateCarbohydrates were analyzed essentially as

5、described by Pharr et al. (1985) and Modore et al. (1988). Briefly, 0.5 g (FW) of cucumber seedling leaves were frozen in liquid nitrogen and grounded to fine powder, then extracted three times by 5 ml 80% ethanol at 80 C each. The extracts were pooled and evaporated to dryness at 50C in vacuum. The

6、 residues were dissolved in 1 ml distilled water and were filtered through lm filter. Agilent1200HPLCsystem was used to analyze carbohydrate. Carbohydrate compounds were separated on a Waters Sugar Pak column 9 300 mm)at 50_C, using water as the mobile phase with the flow rate of ml min-1 . Stachyos

7、e, raffinose, sucrose, glucose, galactose and fructose were identified by comparison of retention times of known standards (Sigma) and quantified by a refractive index detector (G1362A RID). Total amount of the sugars was sum of the contents of stachyose, raffinose, sucrose, glucose, galactose and f

8、ructose. Starch in the extracted tissues was digested with 10 ml 30% HCI04 (v/v) for 24 h at 25°C . The react ion was termi nated by in cubati ngat 80C for 10 min. After centrifugation,the supernatant was transferredto a 25 mI vitreous bottIe and fiIIed to reguIated voIume with distiIIed water.

9、 Starch was determined spectrophotometricaIIy by reference to a gIucose standard curve.总的氨基酸测定方法：At the end of the experiment, pIants were sampIed at a singIe day within2 h to minimize the effect of diurnaI variation. PIants were cIeaned from surface saIt deposits with distiIIed water and bIotted dr

10、y. SubsequentIy, fresh weight of pIants was determined for caIcuIation of reIative growth rates (per day). Afterwards, pIants were separated into Ieaf Iaminas, roots, and rhizomes. 0nIy Iiving materiaI was used, immediateIy frozen in Iiquid N2 and stored at -20C untiI anaIysed. PIant sampIes for det

11、ermination of free amino acids and sugars were freeze-dried at-20Cin a vacuum evaporator (Christ, Germany) and puIverised (Mikro-Dismembrator; Braun Biotech, Germany). About 100 mg of powdered sampIes were extracted three times with 80%ethanoI at room temperature,centrifuged (5000 x g) and the combi

12、ned supernatants were stored at -20°C until analysed. Norleucine, as an internal standard used for recovery checks, was added to the samples during extraction. All amino acid samples were purified by ultrafiltrationthrough Ultrafree-MC Membranes(Milipore, USA), freeze-dried at-20C, and redried

13、in a 2:2:1 mixtureof methanol: Na-acetate:triethylamine at 4 C . Samples including standards were derivatized with phenylisothiocyanate to give phenylthiocarbamyl amino acids (Waters, Pico-Tag method), and freeze-dried at 4C . Samples were redissolved in phosphate buffer and run on a HPLC system (Ch

14、romasystem 500, Kontron Instruments, Germany) using a Sentry-C18 precolumn (Waters, USA) and a reverse phase Pico-Tag column x 300 mm; Waters). Separation was carried out at 46C with a gradient of acetate buffer and acetonitrile/ water. Standard mixtures of amino acids were used for identification a

15、nd quantification of the samples. The totals of free amino acids (TotAA) were calculated as the sumof all 20 detected and quantified amino acids. Their contents are given in absolute (mmol-1g DW)and relative (% TotAA) units. All sugar samples including standards were determined using a HPLC-system (

16、Dionex DX-100, USA). Samples were run on PA1-Guard precolumn and PA1 column (4 x 250 mm; CarboPac, Dionex) with 0.15M NaOH (eluent) and detected by a Pulse Amperometric Detector with a gold electrode at 24 ± 2C. Pulse setting was at 50, 600 and -600mV for 360, 120 and 420 ms, respectively. Standard mixtures of sucrose, fructose and glucose were used for identification and quantification. The totals of these three sugars was designated as TotSUG. Their contents are-1given in absolute (mmol g DW) and relative (% TotSUG) units. The osmolality in rhizomes and leaves was measured using