【作者】 邱婧；

【导师】 林毅；

【作者基本信息】 安徽农业大学 ， 遗传学， 2008， 硕士

【摘要】 霍山石斛是安徽省的名贵中药材,国家重点保护的名贵药用植物,为药用石斛中的珍品。在霍山石斛组织培养过程中,经过多代培养的试管苗是否仍然保持原有品种的遗传性状,关系到这一名贵中药材的产业化。本试验以霍山石斛为实验材料,分别以霍山石斛成熟种子、茎段和愈伤组织为试管苗的初始材料进行继代培养,在分子水平上利用RAPD、ISSR、DALP、SRAP标记进行遗传稳定性分析,旨在找出能够使霍山石斛稳定遗传的组织培养快繁方法,从而推动霍山石斛这一名贵中草药的产业化。主要研究结果如下:1.试验采用改良的CTAB法提取霍山石斛继代试管苗基因组DNA可用于各种分子标记方法分析,并且具有适用、成本低、简单、省时等优点。2.通过对反应条件的优化试验,建立了各种分子标记方法的较适宜体系。其中,SRAP是一种新发展起来的分子标记技术,具有稳定可靠、扩增效果好、可重复性强的优点。优化的反应体系为:25μL的反应体系中,模板DNA量30ng、2.5mmol/L Mg²⁺浓度、0.8μmaol/L的上下游引物、0.2mmol/L的dNTPs以及Taq酶1U。扩增程序为:94℃预变性5min,94℃变性1min,35℃复性1min,72℃延伸1min,5个循环,94℃变性1min,50℃复性1min,72℃延伸1min,35个循环,最后72℃延伸7min。3.利用RAPD分子标记方法对不同继代次数（1～4代）的霍山石斛试管苗进行分析,结果表明:引物S22对茎段诱导培养的试管苗检测时,分别在第3、4代试管苗约1200bp处检测出谱带的减弱;此外,引物S22和S25对愈伤组织诱导的试管苗检测时,分别在第4代试管苗约1200bp和500bp处检测出谱带的减弱。4.利用ISSR分子标记方法对不同继代次数（1～4代）的霍山石斛试管苗进行分析,结果表明:对愈伤组织诱导的继代培养试管苗检测至第4代时,引物UBC-820在大小约为2250bp处的谱带带型变弱,引物UBC-870在大小为1500bp处的谱带带型变弱;引物UBC-807对茎段诱导和愈伤组织诱导的继代试管苗检测时,大小为750bp的谱带都明显减弱。5.利用DALP分子标记方法对不同继代次数（1～4代）的霍山石斛试管苗进行分析,结果表明:引物组合DALP234-DALPR对愈伤组织继代试管苗检测时,第4代的各条谱带明显变弱,大小分别为2250、1500、1000、750bp;另一引物组合DALP241-DALPR对愈伤组织继代试管苗检测时,从第2代开始,大小约为3000bp的谱带带型逐渐减弱。6.利用SRAP分子标记方法对不同继代次数（1～4代）的霍山石斛试管苗进行分析,结果表明:对愈伤组织诱导的继代培养试管苗检测时,引物组合m8-em5在大小约为400bp处的各代谱带带型变弱。4种分子标记所反映的霍山石斛继代试管苗遗传稳定性基本相同。以成熟种子为初始材料的继代培养,其试管苗在所检测的范围内均未发生变异,具有高度的遗传稳定性;以茎段和愈伤组织为材料的继代培养,其试管苗在检测范围内,均能检测到谱带带型的减弱,但均未探查到任何变异位点。根据这些结果,3种继代方式比较之下,以茎段、愈伤组织为初始材料的继代培养,在继代过程中存在一定程度的变异,其中愈伤组织变异程度略大于茎段,以成熟种子为初始材料的继代培养是更能稳定遗传的试管苗快繁方式。更多还原

【Abstract】 Dendrobium huoshanense is a kind of precious traditional Chinese medicine in Anhui Province,and is protected by government for its medicinal value.Whether D.huoshanense seedling after subculturing several times can maintain genetic stability concerns its industrialization.The genetic stability of D.huoshanense repeatedly subcultured in vitro for one to four times with three different original materials of mature seed,stem segment,and callus,was examined by using RAPD,ISSR,DALP and SRAP,and a rapid propagation way which can maintain D.huoshanense genetic stability is hoped to find out,thus promoting its industrialization.1.In the study,the genomic DNA of D.huoshanense repeatedly subcultured in vitro extracted by advanced CTAB method could be fully used for all kinds of molecular marker method,furthermore,the method explored was applicable,economical,simple and rapid.2.To gain a suitable system for various molecular marker method,the experimental profile and parameters were optimized.SRAP is a newly developed molecular marker method,which is stable,reliable,effective and reproductive.The optimized reaction system was as follows:25μL reaction system containing 30ng template,2.5mmol/L Mg²⁺,0.8μmol/L primers,0.2mmol/L dNTPs and Taq polymerase 1U.The optimal amplification program was as follows:5min at 94℃,followed by 94℃for 1min,35℃for 1min,72℃for 1min,5 cycles,1min at 94℃,50℃for 1min,72℃for 1min,35 cycles,and final extension time of 72℃for 7min.3.D.huoshanense seedling repeatedly subcultured in vitro for one to four times was analyzed by RAPD,and the results showed that some weakening bands at approximately 1000bp were found within the third and the fourth descend,when D.huoshanense repeatedly subcultured in vitro with materials of stem segments were detected by primer S22;Besides,some weakening bands were found at 1000bp and 500bp within the fourth descend,when D.huoshanense repeatedly subcultured in vitro with materials of callus were detected by primer S22 and S25.4.D.huoshanense seedling repeatedly subcultured in vitro for one to four times was analyzed by ISSR,the results indicated that weakening bands of primer UBC-820 were found at approximately 2250bp,primer UBC-870 at approximately 1500bp,within the four descends of D.huoshanense repeatedly subcultured in vitro with materials of callus;Some bands weakened clearly at approximately 750bp,when D.huoshanense repeatedly subcultured in vitro with materials of callus and stem segments were detected by primer UBC-807.5.D.huoshanense seedling repeatedly subcultured in vitro for one to four times was analyzed through DALP,the results indicated that every band of the fourth descend weakened clearly,which were 2250,1500,1000,750bp;Some bands at approximately 3000bp gradually weakened from the second descend,when D.huoshanense repeatedly subcultured in vitro with materials of callus were detected by primer DALP241-DALPR.6.D.huoshanense seedling repeatedly subcultured in vitro for one to four times was analysis through SRAP,the results showed that weakening bands of primer m8-em5 were found at 800bp within the four descends of D.huoshanense repeatedly subcultured in vitro with materials of callus.The four molecular marker methods showed D.huoshanense seedlings repeatedly subcultured in vitro maintained genetic stability basically the same.No variation was detected within the descends of D.huoshanense repeatedly subcultured in vitro with materials of mature seed,maintaining highly genetic stability;Some bands weakening were detected,but no variation spot was found within the descends of D.huoshanense repeatedly subcultured in vitro with materials of callus and stem segments.With the comparison of the three subculturing methods,it indicated that some variations were found within the descends of D.huoshanense repeatedly subcultured in vitro with materials of callus and stem segments,and callus varied more than stem segments.Repeatedly subculturing with the materials of mature seed was better rapid propagation way to maintain the genetic stability of D.huoshanense.更多还原