可溶性淀粉測定方法

1、可溶性糖和可溶性淀粉：Soluble sugar was determ ined by the anthrone method (Li, 2000) usingsucrose 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 100C for1 h, and filtered in to 25 mL volumetric flasks. Reacti on mixture of mL co

2、ntained mL extracts, mL mixed reage nt (1 g anthron e+50 mL ethyl acetate), 5mL H2SO4 (98%), mL distilled water. The mixture was heated at 100 C for 1 min and absorbanee read at630nm. Soluble starch wasdetermined by the anthrone method (Li, 2000) with sucrose as the standard. The rema in der of meas

3、ured soluble sugar was tran sferred to a 25 mLcuvette containing 10 mL distilled water and mL HCIO4 mol L ). The cuvette was placed in a boiling water bath for 30 min, cooled to 30C , and filteredinto 25 mL volumetric flasks. React ion mixture of mL contained mL extracts, mL mixed reage nt (1 g anth

4、ron e+50 mLethyl acetate), 5mLH2SO4 (98%), mL distilled water. The mixture was heated at 100C for 1 min and absorba nee read at 630nm. Total non-structuralcarbohydrates (NSC) were the sumof solublesugar and soluble starch.Extracti on and an alysis of carbohydrateCarbohydrates were analyzed essential

5、ly as 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% etha nol at 80 C each. The extracts were pooled and evaporated to dryness at 50C in vacu

6、um. The residues were dissolved in 1 ml distilled water and were filtered through lm filter. Agile nt1200HPLCSystem 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. S

7、tachyose, raffinose, sucrose, glucose, galactose and fructose were ide ntified by comparis on of retention times of known standards (Sigma) and quantified by a refractive in dex detector (G1362A RID). Total amount of the sugars was sum of the contents of stachyose, raffinose,sucrose, glucose, galact

8、oseand fructose. Starch in the extracted tissues was digested with 10 ml 30% HClO4 (v/v) for 24 h at 25C . The react ion was termi nated by in cubati ngat 80C for 10 min. After centrifugation,the supernatant was transferredto a 25 ml vitreous bottle and filled to regulated volume with distilled wate

9、r. Starch was determ ined spectrophotometrically by refere nee to a glucose sta ndard curve.總的氨基酸測定方法：At the end of the experime nt, pla nts were sampled at a sin gle day with in2 h to minimize the effect of diurnal variation.Plants were cleaned fromsurface salt deposits with distilled water and blo

10、tted dry. Subsequently, fresh weight of plants was determined for calculation of relative growth rates (per day). Afterwards, pla nts were separated into leaf lami nas, roots, and rhizomes. Only living material was used, immediately frozen in liquid N2 and stored at- 20C until analysed. Plant sample

11、s fordeterm in ati onof free amino acids and sugars were freeze-dried at - 20 Cin a vacuum evaporator (Christ,Germa ny)and pulverised(Mikro-Dismembrator; Braun Biotech, Germa ny). About 100 mg of powdered samples were extracted three times with 80%ethanol at room temperature,centrifuged (5000 x g) a

12、nd the combined supernatants were stored at - 20C 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 ultrafiltrati onthrough Ultrafree-MC Membra nes(Milipore, USA), freeze-dried at- 20C , a

13、nd redried in a 2:2:1 mixtureof methanol: Na-acetate:triethylamine at 4 C . Samples including sta ndards were derivatized with phe nylisothiocya nate to give phe nylthiocarbamylamino acids (Waters, Pico-Tag method), andfreeze-dried at 4C . Samples were redissolved in phosphate buffer and run on a HP

14、LC system (Chromasystem 500, Kontron In strume nts, Germa ny) 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 acet on itrile/water. Sta ndard mixtures of ami no acidswere used fo

15、r identificationand quantificationof the samples. The totalsof 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 determ ined using

16、 a HPLC-system (Di onex DX-100, USA). Samples were run on PA1-Guard precolumn and PA1 column (4 x 250 mm; CarboPac, Dionex) with 0.15M NaOH (elue nt) and detected by a Pulse Amperometric Detector with a gold electrode at 24 土 2C . Pulse setting was at 50, 600 and - 600mV for 360, 120 and 420 ms, res

17、pectively. Sta ndard mixtures of sucrose, fructose and glucose were used for identificationand quantification.Thetotals of these three sugars was designated as TotSUG. Their contents are given in absolute (mmol g DW) and relative (% TotSUG) units. The osmolalityin rhizomes and leaves was measured using