【作者】 韩雪；

【导师】 柳参奎；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2013， 博士

【摘要】 杨树是木本植物的模式植物,其遗传转化机理、转化方法和转化范围已有深入研究,并取得了较大的发展,同时也获得了不同品种的转基因植株,但在杨树遗传转化过程中,实际上是在进行大量的重复实验后,而得到了的少数几个转化子。FOX-Hunting System技术,即Full-length cDNA Over-eXpressor gene Hunting System(全长cDNA高效表达功能基因探索系统),该方法通过把全长cDNA高效率地导入植物中,使其基因过量表达并导致表型发生变化,达到明确基因功能的目的。本文为将FOX hunting system技术应用在木本植物杨树中,建立高效率的杨树遗传转化体系具有现实意义。成功的植物基因转化首先需要依赖一个良好的再生体系的建立,而理想受体系统的建立则与植物的组织培养分不开。本文以山新杨(Populus davidiana Dode X Populus bollena Lauche)叶片为实验材料,建立了高效的山新杨再生体系,在此基础上,建立了一个高效的遗传转化体系。使用建立的山新杨高效的遗传转化体系,将已构建的蒙古柳cDNA农杆菌表达文库,转入山新杨中,获得了大量的蒙古柳FOX山新杨抗性植株,并对获得的大量的抗性植株进行耐盐碱性的初步筛选,获得了较为耐盐碱的转基因品种,为在木本植物中利用FOX hunting system技术筛选功能基因打下基础。具体实验结果如下：建立了一个高效的山新杨再生体系,为山新杨的遗传转化奠定基础。(1)以2%的NaCIO作为消毒剂,消毒不同的时间,筛选较好的消毒时间,结果显示使用2%的NaCIO作为消毒剂,消毒15min可以达到较好的消毒效果。(2)使用6-BA(0.1,0.3,0.5mg/L)和NAA (0.01,0.03,0.05,0.08,0.1mg/L)两种激素进行不同浓度的组合,筛选出具有较高分化率的山新杨叶片分化培养基,结果显示,山新杨叶片在1/2MS+6-BA0.3mg/L+NAA0.08mg/L分化培养基上,具有最高的分化率,分化率达到90%以上。(3)将分化出丛生芽的叶片外植体接种于含有6-BA (0.3,0.5,0.8mg/L)和NAA(0.05,0.1,0.15mg/L)两种激素进行不同浓度组合的壮苗培养基上进行壮苗,以提高生根率,筛选得到较优的壮苗培养基。实验结果表明最佳壮苗培养基为1/2MS+6-BA0.05mg/L+NAA0.1mg/L。(4)我们将分化出的丛生芽分成单芽,接种于含有不同浓度的NAA (0.1,0.2,0.25,0.3,0.4mg/L)培养基中,筛选较优的生根培养基,结果显示,山新杨芽在1/2MS+NAA0.25mg/L培养基中生根,生根率为100%,且根系粗细适中,分根多且根系长势快。(5)将在培养基中生根长大的山新杨组培苗进行炼苗和移栽,获得盆栽山新杨植株,移栽成活率达到90%以上。以高效的山新杨再生体系为基础,建立了一个高效的山新杨遗传转化体系,为获得大量的cDNA文库转基因植株提供成熟的技术。(1)卡那霉素(Kan)作为遗传转化过程中的筛选标记,对Kan的不同浓度进行筛选,选择较适合的浓度应用于遗传转化中。实验结果表明,30mg/L作为筛选培养山新杨叶片分化的筛选压较为合适；40mg/L作为山新杨生根筛选的适宜浓度。头孢噻亏钠(Cef)作为遗传转化中的抑菌剂,可以抑制根癌农杆菌的生长,但也影响植物的正常生长发育,实验结果表明选用200mg/L的Cef作为筛选山新杨抗性苗的抑菌剂浓度较为合适。(2)对不同菌液浓度(OD600=0.6,0.8,1.0,1.2)和侵染时间(10min,20min30min,40min)对山新杨进行遗传转化的影响进行了实验,并对实验样品表面的农杆菌的附着情况进行了扫描电镜的观察,实验结果表明在遗传转化过程中菌液浓度选定在0.8-1.0之间,侵染时间在20-30min为适宜。(3)对乙酰丁香酮(AS)在共培养培养基中所起的效果做了研究,结果显示在遗传转化过程中,共培养培养基中添加AS对遗传转化效率存在一定的影响,但效果不明显。(4)在上述(1)一(3)中筛选的最优条件处理下,研究了不同叶龄(30d,60d,90d)的山新杨叶片对遗传转化效率的影响,并对实验样品表面农杆菌的附着情况和内部农杆菌的侵入情况进行了扫描电镜和透射电镜的观察,筛选出了适合于杨树遗传转化的叶片外植体年龄。实验结果表明30d大小的叶片作为遗传转化的外植体可明显提高山新杨的遗传转化效率。扫描电镜实验结果观察到30d大小的叶片表面在遗传转化过程中所附着的农杆菌的量较60d,90d的叶片外植体多。透射电镜的进一步实验结果表明30d大小的叶片作为外植体时,叶片内部侵入的农杆菌的数量均多于60d和90d的叶片外植体。实验结果表明使用30d大小的山新杨叶片作为遗传转化的外植体,可提高遗传转化效率。综上优化因素,遗传转化效率高达30%以上。本部分实验使用pBI-121-MD-GFP基因作为植物遗传转化的指示报告基因,进行遗传转化,分化出的芽可在荧光显微镜下进行观察,激发出荧光。并选择部分转基因植物进行Southern Blot和Northern Blot实验,进一步验证该基因转入了山新杨中并得到了表达。实验使用建立的高效的遗传转化方法,将已构建得到的蒙古柳(Salix linearistioularis Hao) cDNA农杆菌文库转入到山新杨中,获得了218株蒙古柳FOX山新杨抗性植株。同时将抗性植株进行耐盐碱性的初步筛选,初步获得了2个抗NaCl,2个抗NaHCO3的山新杨抗性品种。并将具有抗性的植株进行移栽,以备后续实验的进行。更多还原

【Abstract】 Poplar is the most useful model plants. Transgenic poplar genetic transformation mechanism, methods and the scope of transformation have been studied, and made a great progress, and also received many different varieties poplar transgenic plants. In fact, in the process of the experiment of poplar genetic transformation, only a few of transgenic poplar was obtained according to repeated experiments. FOX-Hunding System, namely the Full-length cDNA_Over-eXpressor gene Hunding System (full-length cDNA efficient expression of functional genes discovery System). The full length cDNA was transfer into plants efficiently. The phenotype was changed according to the gene expression and achieve the gene function. In this part the FOX hunting system technology was applied in the woody plants of poplar, and a efficient poplar genetic transformation method was established. Success of plant genetic transformation was based on a efficient plant regeneration system. The establishment of the transformation system and the regeneration system is inseparable. The Populus davidiana Dode×Populus bollena Lauche leaf as the experiment material, efficient regeneration system was established. Based on the efficient regeneration system, the efficient genetic transformation system was established. Poplar as a woody-plant model can be used in mutant library construction and FOX hunting system, improve the transformation frequency is significant. The salix cDNA Agrobacterium library has been built, and the library was transfer into populus using the efficient genetic transformation system. A great many of salix FOX populus was obtained. The resistance plants were treated by NaCl and NaHC03, and the salt-stress populus was generated. It was the foundation of screening functional genes using the FOX hunting system technology. The main research was as follows:A efficient regeneration system was established, and lay a foundation for the genetic transformation.(1) At2%NaCIO for sterilization, different time was used for screening sterilization time, the results showed that using2%NaCIO,15min for sterilization of populus can achieve a good effect.(2) Different concentration of6-BA (0.1,0.3,0.5mg/L) and NAA (0.01,0.03,0.05,0.08,0.1mg/L) were studied in the process of shoot differentiation. The results showed that the highest frequency of shoot regeneration (90%) from leaf was obtained on1/2MS medium supplemented with0.3mg/L6-BA and0.08mg/L NAA.(3) For elongation and proliferation of the differentiation shoot, the shoot explants were put on the medium containing6-BA (0.3,0.5,0.8mg/L) and NAA (0.05,0.1,0.15mg/L), the result showed that1/2MS+6-BA0.05mg/L+NAA O.lmg/L was the most suitable for elongation and proliferation.(4) Effect of NAA on rooting capacity was investigated. The medium containing NAA (0.1,0.2,0.25,0.3,0.4mg/L) was used for rooting. The result showed that NAA at0.25mg/L was most effective for root regeneration(100%), and the growth status of roots were length and polyrhizal.(5) The plants of the populus in the medium was transplanted after hardening, and the potted seedlings was obtained. The survival rate was upto90%.On the basis of the efficient regeneration system, a efficient genetic transformation system was established for obtaining a great many of cDNA transgenic plants.(1) Kan as a selection marker in genetic transformation. Screening a suitable concentration was important in the genetic transformation. Experimental result showed that30mg/L Kan was suitable for differentiation selection,40mg/L Kan was appropriate for rooting. Cef as bacteriostatic in the genetic transformation. Cef can restrain the growth of the Agrobacterium, but also affected the normal growth and development of plants. Experimental result showed that200mg/L Cef for bacteriostatic was suitable.(2) The different bacteria concentration (OD600=0.6,0.8,1.0,1.2) and infection time (10min,20min,30min,40min) were carried on the experiment. The experimental samples were observated by scanning electron microscopy (SEM). The experimental result showed that in the process of genetic transformation bacteria concentration between OD600=0.8-1.0, and the infection time20-30min were suitable for transformation.(3) The effect of AS on the transformation was studied, the result showed that in the process of genetic transformation, adding AS in the cocultivation medium was no effect on the genetic transformation efficiency.(4) Based on above (1)-(3) of the optimal conditions, the effect of different leaf age (30days,60days,90days) on transformation efficiency was studied. The experimental sample was observated by the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for selecting the suitable leaf explant for poplar genetic transformation. The experimental result showed that30-day-old leaf as explant for genetic transformation improved the genetic transformation efficiency obviously. The status of Agrobacterium attachment to leaf surface was observed by scanning electron microscopy. The result showed that Agrobacterium attachment gradually reduced with increasing leaf age of explants. The30-day-old leaf surface showed substantial Agrobacterium attachment. There were fewer Agrobacterium cells attached to leaf surfaces of60-day-old and90-day-old. According to transmission electron microscopy observations, there were more Agrobacterium invasions in the30-day-old leaf explants than60-day-old and90-day-old explants. Our result suggested that younger leaves had higher transformation efficiency (-30%) than older leaves (10%).Using the green fluorescent protein (GFP) marker, the expression of pBI121-MD-GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode x P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants.In this section, Salix linearistioularis Hao as the research object. The. Agrobacterium cDNAs library of Salix was transformated into the populus and obtained the218Salix FOX Populus resistance plants. The resistance plants was treated by NaCl and NaHCO3.2resistance plants stress to NaCl and NaHCO3respectivly. It is a foundation for using FOX hunting system technology screening functional genes in woody plants更多还原