雨生红球藻原生质体制备与再生育种

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1、雨生红球藻原生质体制备与再生育种雨生红球藻原生质体制备与再生育种雨生红球藻原生质体制备与再生育种雨生红球藻原生质体制备与再生育种雨生红球藻原生质体制备与再生育种雨生红球藻原生质体制备与再生育种雨生红球藻原生质体制备与再生育种雨生红球藻原生质体制备与再生育种雨生红球藻原生质体制备与再生育种第27卷第4期2011年7月福建师范大学(自然科学版)JournalofFujianNormatUniversity(Natura1ScienceEdition)Vo1.27No.4Ju1.2011文章编号:i000—5277(2011)04—0122—05

2、雨生红球藻原生质体制备与再生育种叶涛,王明兹,黄建忠,陈必链(教育部工业微生物工程研究中心,福建师范大学生命科学学院,福建福州350108)摘要:研究了高渗溶液组成及浓度,酶的组成与质量分数,预处理方法,温度和pH等因素对雨生红球藻原生质体制备与再生的影响.结果表明:选用细胞数约为1O?mL_1的对数生长期细胞.于50mmo[?IEDTA和25mmol-LDTT缓冲液.3OC下恒温处理1h,再于质量分数2.0蜗牛酶和质量分数1.0%纤维素酶,温度3OC,pH6.7,50r?rain振荡条件下水解细胞壁.反应2h后原生质体制备率达到68.3.

3、原生质体溶液适度稀释后接入再生培养基平板,(23±1)C,1200lx光照培养.从再生藻落中获得l株高产藻株P69,其培养生物量比出发藻棘增加43.18,产物中虾青素含量提高26.9%.关键词:雨生红球藻;原生质体;制备;再生;育种中图分类号:Q949.21文献标识码:APreparationandRegenerationBreedingofProtoplastsfromHaematococcuspluvialisYETao,WANGMing-zi,HUANGJian-zhong,CHENBi—lian(EngineeringResearc

4、hCentero{IndustrialMicrobiology,MinistryofEducation,CollegeofLiSciences,FujianNormalUniversity,Fuzhou350108.China)Abstract:Effectingfactorssuchascompositionandconcentrationofhypertonicsolution,hydrolaseanditsconcentration,pretreatmentmethod,temperatureandpHduringprotoplast

5、spreparationandregenerationofHaematococcuspluvialishavebeeninvestigated.Theresultssuggestedthattheoptimumconditionswereasfollow:usingthecellswhichtotalnumberreached10?mL一,pretreatedwith50mmol?L一EDTAand25mmol?L一DTTsolutionat30Cfor1h,hydrolyzedwithmixturesolutioncontaining2.

6、09/5cellulaseand1.09/6snailenzymetoremovethecellwall,under30℃atpH6.7andwithgentlyshaking(50r?min).About68.3%cellshadbeentransformedtoprotoplasts2hlater.Dilutedtheproto—plastsandinoculatedthemontoregeneratingplates,cultivatedunder1200lxillumination,(23±1)C,toregeneratenorma

7、lceils.AmutantstrainP69hadbeenselected,anditsbiomassproductivityandastaxanthincontentincreased43.18and26.9%00thanthatoftheinitiatestrainproduced,respectively.Keywords:Haematococcuspluvialis;protoplast;preparation;regeneration;breeding雨生红球藻(Haematococcuspluvialis)是一种单细胞绿藻,其

8、细胞在生长周期中能大量合成与积累类胡萝卜素(可占干质量的2.O9/5～5.09/6),其中80%以上为虾青素及其酯类,为自然界中虾青素含量最高的生物Ⅲ.雨生红球藻虾青素开发研究在