【作者】 陶均；

【导师】 李玲；

【作者基本信息】 华南师范大学 ， 植物学， 2004， 硕士

【摘要】 本实验研究了（1）发根农杆菌的生理生化、分子生物学鉴定及生物检测和活力比较；（2）发根农杆菌介导的蓝猪耳遗传转化和蓝猪耳毛状根的植株再生，为蓝猪耳的遗传转化和其它分子生物学研究奠定基础。 采用3-酮乳糖产物法、差异酸生成实验和游动性实验鉴定6种发根农杆菌供试菌株A4、R1205、R1000、R1601、R1022和15834。结果表明R1205、R1601、R1000和A4为Ⅱ型农杆菌，其活力大小依次是R1000，R1205，A4，R1601。R1022和15834不是Ⅱ型菌。通过不同培养时间下菌液的浓度测定表明，所有测试菌株均在16—32h时呈现对数生长，培养32h时的菌液浓度为R1000＞R1205＞R1601＞R1022＞A4＞15834，但培养40h时A4菌液浓度达到最大。利用PCR方法鉴定结果表明，所有供试菌株中，A4、R1205、R1000和R1601为含Ri质粒的发根农杆菌，而R1022和15834未出现阳性结果。利用黄瓜作为菌株遗传转化率鉴定的模式系统来研究A4，R1205，R1000，R1601活力，结果表明R1000的遗传转化率最大，达到79.16％，其余依次是R1205、R1601和A4。 R1000、R1205、R1601和A4转化蓝猪耳形成的根PCR和Southern blot检测表明，除R1205转化形成的根的一个克隆外，所有的毛状根克隆都呈现PCR阳性。在PCR检测阳性的克隆中，随机挑选2个做Southern blot，结果均呈阳性。 不同菌株对蓝猪耳的遗传转化能力不同，R1000和A4明显高于R1205和R1601。蓝猪耳遗传转化的最适菌液OD₆₀₀值为1.0（6×10⁷个细菌／ml）。2～3天共培养有利于蓝猪耳的遗传转化，10～30．M外源乙酰丁香酮能有效地提高毛状根的形成率，但是在高于40．M时，转化率不能显著提高。4m幼以下的硝酸银便能有效地提高遗传转化率，但高剂量（10m幼）时作用却相反。预培养和共培养培养基pH从4.5提高到6.5时，毛状根诱导率显著提高，但是当提高到7.5时，诱根率却显著下降。在最适转化条件下，发根农杆菌介导的蓝猪耳遗传转化率可达90%， 经Klll筛选的蓝猪耳毛状根扩大培养7天后切下转入诱芽和诱导愈伤组织培养基中，3周后没有发现愈伤组织形成。培养3周后在1/2Ms+4一Pul.0培养基上大约2.5%毛状根形成芽。芽经过250m幼Km抗性筛选28天后86.7%的再生芽能在抗性培养基上存活，但生长不明显。筛选后的芽在1/2 MS+NAAO.1培养基上生长40天后无根形成，并且6株芽中2株黄化死亡，其余4株高度不到2 cm。存活的4株芽转至1/2MS+GAI.0培养基上，28天后芽仍无明显生长。更多还原

【Abstract】 In this study, the screening and identification of the different Agrobacterium rhizogenes strains were carried out using physiological, chemical , molecular and bioassay methods, and the vigor of different strains was also compared based on their characters; the transformation of torenia mediated by Agrobacterium rhizogenes, the hairy root formation and its regeneration were importantly studied. The purpose of this study is to provide theoretical and technical support for torenia transgenic hairy root and other molecular researches used by torenia.The comparation and identification of Agrobacterium rhizogenes strains were investigated using 3-ketolactose test, the differential acid production assay and the motility assay. The results showed that these physiological tests were quickly, sufficiently to identify the Agrobacterium rhizogenes strains; and the vigor of strains decreased in the turn of R1000, R1205, A4 and R1601. The logarithmic growth of the strains was from 16 h to 32 h. The OD600 value at 32 h from the highest to the lowest was R1000 R1205, R1601, R1022, A4 and15834. But the concentration of A4 at 40 h was higher than that of other strains. Four strains (A4, R1205, R1000, R1601) in this study were Agrobacterium rhizogenes according to PCR assay. Using cucumber seedlings as the transformatic model mediated by the strains (A4, R1205, R1000, R1601), the transformatic frequency of R1000 for cucumber seedlings was the highest (79.6%), which was followed by R1205, R1601and A4.The transformation of Torenia fournieri L mediated by Agrobacterium rhizogenes is reported. Almost all roots induced by four different bacterial strains R1000, R1601, A4 and R1205 were found to harbor the rolB gene according to the PCR assay and Southern blot analysis, the results showed that all roots that could grew on selection medium containing 250 mg/1 kanamycin were putative hairy roots.The effects of varying conditions on torenia transformation were investigated. Torenia transformation of R1000 and A4 were more efficient than that of R1025 or R1601. The optimum concentration of the strain was about 1.0(OD600 value). 2 ~3 days for co-cultivation were found to be sufficient for this transformation. The best concentration of acetosyringone was about 30 M. Silver nitrate below 4 mg/L caused little change in efficiency and higher concentration (above 10 mg/L) strongly inhibited hairy root induction. pH value increase from 4.5 to 6.5 strongly stimulated hairy root formation and higher pH value (above 7.5) inhibited it. Under the optimized condition, the transformation frequency of torenia approached 90%.No Calli were formed from any hairy root after 3 weeks culture. 7 buds were induced from 2.5% transformatic roots on 1/2 MS containing 1.0 mg/1 4-PU after 3 weeks culture. 86.7% buds surrived on 1/2 MS containing 250 mg/1 Km after 28d. Two of six buds were dead 40d after that were put on 1/2MS medium containing 0.1 mg/1 NAA, and the tall of other four buds was less 2 cm. The four buds still grew very slowly on the 1/2 MS containing 1 mg/1 GA.更多还原