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rol基因对741杨的遗传转化及表达研究

标题: rol基因对741杨的遗传转化及表达研究
英文标题: The Transformation and Expression of Poplar 741 with rol Gene
作者: 缪丽萍
出版时间: 2009-01-01
所在大学: 河北农业大学
关键词: rol基因,741杨,遗传转化,外源基因表达,农杆菌,残存
英文关键词: rol gene,Poplar 741,Genetic transformation,Exogenous gene expression,Agrobacterium rhizogenes,Survive
论文级别: 硕士
学位: 学位论文
导师: 杨敏生
专业: 林木遗传育种
提交时间: 2009
摘要: Ri质粒上含有诱根基因即rol基因,将含rol基因表达载体导入植物体内均能提高植物的生根能力,而rol基因转入后往往使植株表现出一些变异,对植物的生长发育产生很大的影响.为了研究rol基因对植物造成的变异,本实验通过对遗传转化条件的优化,将rolB、rolC诱根基因导入741杨,对转化植株生长变化、叶绿素含量、荧光特性,以及内源激素含量等生理指标进行检测,并对农杆菌介导法中农杆菌在转化植株上的残存状况进行研究,主要内容及结果如下: 采用含rol基因的植物表达载体感染741杨叶片外植体,优化转化体系,获得转rolB基因苗8个系号,转rolC基因苗18个系号.研究结果表明:741杨叶片在MS培养基附加6-BA 1.0和NAA 0.1时,分化率可提高至97%;筛选培养时Km临界浓度为40 mg/L;附加400 mg/L CTX可有效抑制细菌,并减少抗生素对叶片分化的影响;侵染和共培养时添加AS 150 mg/L,农杆菌侵染9-14 min,共培养3 d时,可将转化效率提升至51%;而预培养时间的延长,不利于转化效率的提高.通过Km筛选和PCR检测,初步确定rol基因已转入目的植株. 对转基因植株发根量、生长变化、叶绿素含量、荧光特性,以及内源激素含量进行检测.结果表明:转rolB基因各株系发根率,发根量提高.多数株系苗高与对照无显著差异,叶绿素a、b、a+b含量上升, Fv/Fm、PI值下降,叶绿素a、b、a+b之间呈极显著正相关性, 叶绿素a、b、a+b与PI呈负相关性,激素含量存在较大变化,IAA、ZT含量上升,ABA含量下降,IAA/ABA值上升,这与发根率和发根量的变化是一致的,IAA/ZT值上升,与苗高变化存在一定差异.转rolC基因各株系发根率、生根量提高,顶端优势受到抑制,植株矮化,叶绿素含量降低,叶绿素a、b、a+b之间呈极显著正相关性,叶绿素a、b与PI呈显著正相关性,IAA、ZT、ABA含量上升,IAA/ABA和IAA/ZT值上升,与发根量和苗高的变化一致. 对转Ri质粒741杨在继代和移栽过程中农杆菌残存状况分析.结果表明:经农杆菌介导转化后,将转化植株继代于附加300 mg/L CTX培养基可基本抑制残存农杆菌活性;随着转化植株在含抗生素培养基中继代时间的延长,残存农杆菌的数量和活性逐步下降,在继代18个月后,无法培养出目的菌落,但转化植株一旦脱离抗生素环境,残存农杆菌数量和活性又将得到恢复;农杆菌主要分布于植株体表和茎中.残存农杆菌,严重影响转化植株存活和PCR对植株的检测,并导致外源基因的漂移.
英文摘要: Ri plasmid contains rol genes which can induce formation of adventitious roots. The transformation of rol gene into plants can improve rooting ability of plants. However, some plants exhibited a series of variations, which influenced plant growth significantly. In order to study these variations, we optimize transformation systems of transfer rolB、rolC genes into poplar 741,and then get the transgenetic plants to identify the morphological character, the content of chloroplast, the character of fluorescence and the dynamic of endogenous hormones. Results were showed as follow:An efficient and stable system for poplar 741 mediated by agrobacterium rhizogenes with rolB、rolC gene was established through investigating the factors influencing the transformation efficiency. At last we obtain 8 stains of transgenetic rolB gene,and 18 stains of transgenetic rolC gene.The results showed that the optimization system is that adventitious bud differentiation of poplar 741 explants can reach to 97%with 6-BA 1.0 add NAA 0.1;the critical concentration of Km is 40 mg/L, the 400 mg/L Cef can efficacious control the Agrobacterium remain on poplar 741 explants as well as reduce the effect of CTX to adventitious bud differentiation; and the co-cultivation and liquid bacterium with AS 150 mg/L,10~15 minutes for infection, 3 days for co-culture, the conversion rate can be improved to 51%.The physiological indicators of transgenic plants showed that: Compared with poplar 741, most of transgenic plants with rolB gene increased root number and rooting rate, the high growth have no difference with poplar 741,chloroplast a,b and a+b content increased, and the chloroplast a,b and a+b have highly significant pozitive correlation. The Fv/Fm and PI value decreased,and chloroplast a,b,a+b and PI have negative correlation. There is a big change in hormone content, the IAA, ZT content increased, ABA content decreased, IAA/ABA value increased,which have consist with root number and rooting rate,while there is difference between IAA/ZT and the high growth. To transgenic plants with rolC gene, the plant increased root number、rooting rate and the highth become dwarf.Chloroplast a,b and a+b content decreased, and the chloroplast a,b and a+b have highly significant correlation. The Fv/Fm and PI value decreased,and chloroplast a,b,a+b and PI have significant pozitive correlation.The IAA,ABA,ZT content are all increased, IAA/ABA and IAA/ZT value are both increased,which have consiste with root number and high growth .Taking poplar 741 mediated by Agrobacterium rhizogenes with Ri plasmid as material, the result showed that the Agrobacterium on transgenic plant can be controlled basicly when the transgenic plants cultured in medium with 300 mg/L ceftiofur;the quantity and activity of the residual Agrobacterium on transgenic plant will be declined with the time prolong, and the Agrobacterium can not be cultured in YEB when the transgenic plant cultured in 18 month later. The residual Agrobacterium rhizogenes mainly distributed on transgenic plant surface and stem inside ,which seceriously effect the survival of transgenic plants and the PCR result .While the Agrobacterium on transgenic plant will be resume when the transgenic plant cultured in medium without ceftiofur;and the foreign Gene would flow once there are Agrobacterium rhizogenes residual on transgenic plants.