Preparation Of Spheroplasts From Arthrospira And RAPD Analysis Of Spirulina And Arthrospira

Protoplasts (spheroplasts) are the appropriate materials of physiological research and genetic breeding. For the genetic engineering of Arthrospira, the preparation of protoplasts is an indispensable means too. In this paper, we discussed the preparation methods of spheroplasts from Arthrospira platensis FACHB341. Though the experiment, we improved the existing preparation methods of spheroplasts from Arthrospira, including the optimal methods of removing sheath, the optimal pH value of buffer solution, the optimal osmosis stabilizer, the optimal combination of enzymes, the concentration of lysozyme and the optimal time of enzyme hydrolysis. From experimental results, we found, for preparation methods of spheroplasts from Arthrospira, the ultrasonic wave, the ionic treatment and the enzyme hydrolysis was entirely necessary. The following is the best combined mode: When Arthrospira platensis FACHB341 got to the log phase, we put the culture media in the 50mL centrifuge tube, and treated it by ultrasonic wave with 3~5W energy, 1.0s pulse on and 0.5s pulse off, this step lasted for 2min. Then the algae filament was collected by centrifuge(3000rpm, 10min), and was washed in Zarrouk culture media contained 1.0 - 1.5mol/L NaCl for 5min. The algae filament was collected again by centrifuge(3000rpm, 10min), and was washed in sterile Zarrouk culture media for 2~ 3 times, then was collected by centrifuge (3000rpm, lOmin) and was transferred into the enzyme solution(0.2mol/L phosphoric acid buffer solution pH 7.2, 0.8mol/L KCL, 0.5% lysozyme). The enzyme solution was placed in the 28℃~30℃ water bath , and was shaked slowly for 5~7h, then the spheroplasts were detected by hypotonic explosion, were counted repeatedly every 1.5h after Evan's dyeing. Therefore, the yield of spheroplasts could be numerated. By this method, we got 40% active spheroplasts from Arthrospira platensis. For the genetic transformation and thegenetic engineering study of Arthrospira, our research establishes experimental foundation.In this paper, we studied the following nine strains of the algae by the method of RAPD: Arthrospira platensis FACHB341, FACHB438, FACHB439, FACHB793, FACHBJP1, OUQDS6, OUQDS8, Arthrospira maxima OUQDSM and Spirulina FACHB351. To get a steady RAPD reaction system, we optimized the primary factors that influenced the stability of reaction system at first. The results were as follows: 25uL reaction volume, 50ng template DNA, 2.5mmol/L Mg2+, 2umol/L primer concentration, luL lOmMdNTP, 1U Taq-polymerase. The reaction programme: Pre-denaturation (94°C, lOmin), denaturation (94°C, lmin), annealing (36°C lmin), extension (72 °C 2min), 40 cycles, extension (72 °C lOmin). According to the electrophoresis bands, we-got the comparability of samples by the formula S=2Nxy/ (Nx+Ny) based on Nei-Li principle, the corresponding genetic distance also could be obtained by D=l-S, then we analyzed it by using Phylip software, and constructed the clustering analysis map. The results were as follows:(1) After filtering 60 primers, we found 15 random primers with better repeatability, clear electrophoresis bands and high polymorphism. Using these 15 primers, we amplified 140 electrophoresis bands from nine strains, every primer amplified 9.3 electrophoresis bands in average. In these bands, there were 139 polymorphism bands, and the proportion exceeded 99%.(2) From the genetic distance among these strains, we found that the genetic distance between FACHB351 and OUQDS8 was the farthest (0.1925), and the genetic distance between FACHBJP1 and FACHB793 was the nearest (0.0793). As a whole, the genetic distances of nine strains of the algae were rather near. This result explained that their genetic background was rather near too, and it was also accordant with their status.(3) From the clustering map, the nine strains gathered into three species firstly, FACHB351 was the first species, FACHB341 was the second species, and the other seven strains gathered into the third species. The third species also can be classified into two branches, the first branch was FACHB439, FACHB438 and other five strainsgathered into a branch, thereinto, the genetic distances between FACHB793 and FACHBJP1, OUQDS8 and OUQDSM were the nearest, these two strains belong to a branch severally.Above results were consistent with some characteristics of nine strains from physiology, biochemistry and molecular biology, but there were still some discrepancies compared to the classical classification based on the microshape of algal filament, therefore, it has very important academic and practical significance that establishes a reasonable and effective Spirulina classifying method and effective means of new strain at the molecular level as quickly as possible.