Establishment Of High Efficiency Foreign Gene Transformation System Of Chlamydomonas Reinhardtii And Studing On Gene Transformation Of Dicrateria Inornata

We optimized conditions for gene transformation of Chlamydomonas reinhardtii and established high-efficiency transforming method for Chlamydomonas reinhardtii. We studied on the sensitivities of 8 species of marine microalgae to 8 types selectable markers usually used in land plant genetic engineering. The selectable markers of Dicrateria inornataPlatymonas genetic engineering were determined, the method of transformation of Dicrateria inornata was also determined. The research contents and results in details as follows:I .Electorporation of chlamydomonas reinhardtii was used to optimize conditions for introduction of BLE gene. The conditions that were varied included capacitance, electrical field strength, temperature and osmolarity. Following optimization ,high-efficiency is obtained under the condition of 3.OX 125 j.?F capacitor, 20mM ,nannitol and 20mM sorbitol in 0C+(20?5) ~C temperature We determined the sensitivities of Chlamydomonas reinhardUi to zeocin, the chlarnvdomonas growth can be inhibited in I I.?g/ml zeocin liquid medium and on 10 ii g/ml zeocin solid medium. The transformants have survived for 8 months on no-zeocin medium.2.The sensitivities of Dicrateria inornata~ Platymonas SP, Pyramidomonas SP~ Goniotrichum alsidii, Chlorella SP-. Porphyridium cruentum. Nitz.cchiaClosterium, Porphyridium SP. to selectable markers usually used in land plant genetic engineering :hygromycin chloramphenicol geneticinstretomycin ~cefotaxime. methotrextate. phosphinotricin zeocin was studied. The results showed that Dicrateria inorna!a and Platy onas are more sensitive to chloramphenicoh geneticin~ zeocin than other algae, in 340 iig/ml chIoramphenicol or l00 u g/ml geneticin' l00 ll g/mI zeocin liquidmedium can inhibit the growth of Dicrateria inornata,in 340 u g/mlchIoramphenicol or 600 P g/ml geneticin, 200 ll g/ml zeocin 1iquid mediumcan inhibit the growth of Plalymonas. Hygromycin' chIoramphenicol'geneticin. stretomycin 1 cefotaxime \ methotrextate' phosphinotricin' zeocincan't inhibit the growth of Pyramidomonas SP. Goniotrichum alsidii.Chlorella SP. PorPhyridium cruenIum, Nitzschia CIoslerium. PorPhyridiumSP. .ChIoramphenicoI' geneticin. zeocin are suitabIe seIectabIe agents forDicraleria inornaIa and Platy1nonas genetic engineering. But we need seIectthe transformants as soon as possible, it take ChloramphenicoI 1 geneticin'zeocin Iong time to inhibit the gro''th of Dicrateria inornata. Platymonas.it needed to optimize conditions for condition of selection.3. We studied the sensitivities of DicraIeria inornata to zeocin in l0%normal seawater concentration5. L D5, of l 06cel Is/m I DicraIeria inornala tozeocin was determined. Transformed the whole DicrateriQ inornata cells byelectroporation. used the BLE gene as the seIectable gene,compareddifferent eIectrical field strenRth '' ith the effect on the survivaI rate ofDicrateria inornata celIs, the suitable transformation condition wasdetermined. On the l000 V/cm. 800 V/cm. 600 V/cm' 500 V/cm eIectricaIfield strength, the survival rates are higher (45%~66%), on the 500 V/cmeIectricaI fieId strength, the SLIrvival rate is the highest. The transformantstreated by 800 V/cm electricaI t1eld strength can survive l5 days on 10 Pg/mI zeocin liquid medi um.