【作者】 王涛；

【导师】 柳参奎；

【作者基本信息】 东北林业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 碱茅(Puccinellia chinampoensis)是一种抗旱、耐盐碱(NaHCO3,Na2CO3)性极强的盐碱地特种植物,它与水稻、小麦一样都属禾本科,但在长期的进化及对盐碱适应过程中,形成了与其它禾本科植物相比极为特殊的抗逆机制,而且具有极强的抗盐碱能力。通过植物组织培养技术建立碱茅再生体系,进一步筛选出高效转基因体系,为以后碱茅的遗传转化研究实验做了准备。本研究以碱茅为试验材料,全面而系统地探讨了影响碱茅种子愈伤诱导的一些主要因素,并通过农杆菌EHA105介导下对GUS基因遗传转化。试验的研究结果为：在碱茅种子愈伤组织的诱导过程中,不同灭菌剂及灭菌时间对种子灭菌效果和诱导情况差异明显,使用70%酒精3min+10%次氯酸钠30min的灭菌处理效果较好。在MS培养基上添加不同浓度的2,4-D对碱茅种子诱导产生愈伤组织的效率不同,最适2,4-D浓度为4 mg/L。选择以改造培养基I(N6大量元素、MS微量元素、两倍MS铁盐、五倍MS有机物,其中肌醇的用量不变)作为碱茅种子愈伤组织诱导的基本培养基。不同光照培养条件对碱茅种子诱导产生愈伤组织的效率不同,在短光照(12/12h)、培养温度为24-260C时,碱茅种子愈伤组织诱导的时间短,诱导率较好。在碱茅种胚愈伤组织的继代过程中,比较三种培养基对愈伤组织继代的影响,实验结果表明选择以改造培养基S(MS大量元素、四倍MS微量元素、MS铁盐、N6有机物)作为愈伤组织继代的基本培养基,在2,4-D浓度为4mg/L时,添加浓度为1mg/L的ABA可以改善愈伤组织质量,增加更多的胚性愈伤组织形成。在碱茅种胚愈伤组织芽的分化过程中,以MS作为分化基本培养基,不同的激素配比添加对愈伤组织分化作用效果有显著差异。分化率最高的是附加6-BA 0.4mg/L+IAA0.04mg/L的培养基,即MS培养基+6-BA 0.4mg/L+IAA 0.04mg/L+CH 600mg/L+脯氨酸500mg/L+30%蔗糖+8%琼脂。分化培养基中加入山梨醇和麦芽糖代替蔗糖可以有效抑制愈伤组织褐化,而且可以提高不定芽分化率。在诱导培养基,继代培养基和芽分化培养基中都添加500 mg/L脯氨酸可以有效的防止愈伤组织褐化,进而提高分化率。确定碱茅愈伤组织分化期的卡那霉素筛选压是建立遗传转化体系的必要条件,我们采用了最佳筛选浓度为50mg/L。侵染后选用抽滤除菌方式是筛选过程中抑菌效果最佳的。用含有GUS基因的农杆菌EHA105介导转化碱茅愈伤组织,经过卡那霉素筛选的愈伤组织瞬时表达率达到了47.14%。更多还原

【Abstract】 Puccinellia chinampoensis is a special gramineous plant like rice and wheat, which can survive well in the saline and alkali environment with severe drought and salt stress. The difference from rice or wheat is Puccinellia chinampoensis has evolved a series of resistance mechanism via years of adaptation in the saline and alkali soil. In this study, transformation system of Puccinellia chinampoensis has been constructed and optimized, following with optimized regeneration system, through plant tissue culture technique. The whole constructed system will be pre-determinative for the further studies as gene interference.In this study, some crucial factors which affected the embryo culturing using Puccinellia chinampoensis as the main plant material were discussed comprehensively and systemically. And the genetic transformation of put-LS2 mediated by Agrobacterium tumefaciens was followingly conducted using the above callus. The main results obtained in this study contains as follows.In the callus induction process of Puccinellia embryo, different sterilant and sterilized time to seed embryo showed different effects and induced situation obviously. In this study, the best combination was 70% ethanol for 3min and 10% sodium hypochlotous for 30min. The rate of callus induction was different in the medium containing different concentrations of 2,4-D and the optimized was 4 mg/L. The altered medium I (N6max+MSmin+2×MSFe+5×MSvitamin (inositol no change)) was chosen to be the basic medium to induce the callus. The time of cultivation in light also have effect on the callus induction. The shorter of the light time, the higher rate of the callus induction would get.In the callus subculture of Puccinellia chinampoensis seeds,different mediums showed different impacts. The result showed that the modified medium S(MSmax+4×MSmin+MSFe+ N6vitamin) was chosen as the basic subculture medium.1mg/Lphytohormone ABA was added to the medium to improve the quality and the quantity of embryo callus.In the differentiation of callus of Puccinellia chinampoensis embryo, MS was the differentiation medium. Different ratio of hormone had different effects on differentiation. The result indicated that the best was 6-BA 0.4mg/L+IAA 0.04mg/L.Sucrose was substituted with sorbital and maltose, which could inhibit callus browning obviously and increase differentiation rate.500mg/L proline was added into induction medium, succeeded medium and differentiation medium to effectively prevent callus from browning and to improve differentiation rate.In the transformation test of Puccinellia chinampoensis callus, using kanamycin (Kana) to screen transformed callus, the optimized concentration was 50mg/L. The beat way of remove of bacterial is filtration. pBl121-GUS was transformed into the callus mediated by Agrobacter-ium tumefaciens, and the transformation rate was 47.14%.更多还原