Studies On Molecular Genetics Of Germplasm And Morphological Reformation In Spirulina

In this study, genetic diversity of various strains of Spirulina platensis is analyzed with RAPD molecular marker and antibiotics marker, the results are compared with their morphologic characters, aimed at constructing a more reasonable classification criterion and knowing more about the genetic background of Spirulina. Additionally, it is found that under the cultivated condition the linear filament can retransite to the normal curved one which is similar to original spiral filament. By comparing the biological characteristics of original normal filament, linear filament and the curved filament retransited from linear filament, certain evidence of the morphologic variation regulated by a special transposon are detected on the level of protein and DNA, which will help us to discover the mechanisms of this morphologic variation on molecular genetics level and solve the problem in production of Spirulina in large scale. The main results are as follows:1. Studies on the Spirulina morphology and its RAPD molecular marker analysisMorphologic characters of filaments and cell about seven strains of Spirulina platensis (Sp-D, Sp-Z, Sp-S, Sp-B, Sp-C, Sp-Fl, Sp-F2) are statistically analyzed. The results show that they have some morphologic difference.A preparation method of genome DNA in Spirulina with high quality and high extraction rate for RAPD analysis is studied. The high quality genome DNA was obtained from the pelitomorphic Spirulina about 10% water content after lyophilized, isolated with CTAB buffer and two-time-precipitation method. And the genome DNA of seven strains of Spirulina platensis as mentioned above are analyzed with 106 random primers using random amplified polymorphic DNA (RAPD) technique. With the 106 random primers, 23 primers revealed their obvious polymorphisms and generated a total of 151 fragments, 120 of which were polymorphic. Calculated with Nei's formula and the unweighted pair-group method with arithmetic averages (UPGMA), the Cluster analysis results demonstrated that the classification of seven strains of Spirulina platensis with RAPD markers is consistent with some of their physiological and biochemical charactersas well as some molecular biological characteristics. So, RAPD marker can be used as an important profile in the classification of Spirulina at DNA level. A more scientific and reasonable criterion for the Spirulina classification can be established with RAPD technique and morphological characteristics. '2. Studies on the antibiotic markers in SpirulinaThe tolerance of seven strains of normal filaments (Sp-D, Sp-J, Sp-Z, Sp-S, Sp-B, Sp-C, Sp-Fl), a super-long strain induced by irradiation[Sp-Z(SL)], two strains of linear filaments variated from normal filaments [Sp-D(L) and Sp-J(L)] of Spirulina platensis to 5 kinds of antibiotics (Chloromycetin, Ampicillin, Kanamycin, Lincomycin, Streptomycin) is analyzed and compared statistically. The results indicated that: (1) Tolerance of ten strains to Kanamycin, Chloromycetin and Streptomycin are consistent with each other. All of them are highly tolerant to Kanamycin, but comparatively sensitive to Streptomycin, of which the median inhibited concentration (IC50) is between 56.4 u g/ml and 76.0 u g/ml, and the most sensitive to Chloromycetin, of which the median inhibited concentration (IC50) is between 13.1 u g/ml and 21.5 u g/ml. (2) The tolerance of ten strains to Ampicillin and Lincomycin are quite different. Among them, Sp-Z and Sp-S are remarkably tolerant to Ampicillin, next to the two is Sp-C. Though the growth of Sp-D, Sp-D(L) and Sp-Z(SL) is distinctly inhibited by Ampicillin, they are still alive in the cultures; whereas Sp-J, Sp-J(L), Sp-B and Sp-Fl are lethal in this condition. It means that they are most sensitive to Ampicillin. Meanwhile, the results show that Sp-D, Sp-J, Sp-Z and Sp-C are relatively tolerant to Lincomycin, but the other 6 strains of meterials are not. (3) Sp-D, Sp-J and Sp-Z differ with their morphological varieties or mutant Sp-D(L), Sp-J(L) and Sp-Z(SL) about the tolerance to Li...