【作者】 张焕玲；

【导师】 李周岐；

【作者基本信息】 西北农林科技大学 ， 森林培育， 2010， 博士

【摘要】 杨树是世界范围内广泛栽培的林业和能源农业首选树种,是我国重要的短周期工业用材林树种,新品种杨树的选育一直是林木育种学者研究的热点。但杨树有较长的幼年期,许多性状在成年期才才能表现出来,这在很大程度上限制了该树种遗传育种及相关研究工作进展。因此,促进杨树提早开花,缩短其幼年期,对加速杨树遗传改良周期具有积极作用。近年来,许多与植物花发育相关基因的克隆与在部分植物中的转化成功,为人们通过转基因技术来改造植物花期奠定了基础。FT(Flowering Locus T)是在拟南芥中克隆得到的促花基因,FT基因的表达产物就是人们长期寻找的“成花素”,转FT基因的植物大都能表现明显的早花性。本论文在克隆毛果杨(Populus trichocarpa)中FT的同源基因FT1和FT2,以及黄豆热激启动子(HSP)控制下的FT基因植物表达载体PHSP::FT基础上,构建了热激启动子控制下的FT1和FT2基因植物表达载体PHSP::FT1和PHSP::FT2;建立了白杨杂种无性系717-1B4(雌株,P. tremula x P. alba ,以下简称717)和353-53(雄株,P. tremula x P. tremuloides,以下简称353)的高效遗传转化受体系统;采用农杆菌克隆AGL1介导的叶盘转化方法,优化了FT基因转化2种杨树无性系的遗传转化条件,获得了191个转FT基因的PCR阳性植株;并利用热激诱导处理成功诱导了转基因植株早期开花,筛选了可用于2杨树无性系的FT基因,探讨了影响FT基因热激诱导表达促进杨树开花的各种因素,阐述了热激诱导条件下产生的花的发育过程,并对FT促花信号嫁接转移进行了初探。论文主要取得了如下结果。1、构建了杨树FT1和FT2基因热激启动子控制下的植物表达载体PHSP::FT1和PHSP::FT2。2、通过系统试验,研究了生长素NAA、细胞分裂素6-BA和TDZ、叶片刻伤方式、接种方式、培养条件等对717、353白杨杂种无性系离体叶片再生的影响。同时研究了两无性系离体叶片再生和不定芽生根的卡那霉素临界浓度。结果表明,同时适合717、353白杨杂种无性系离体叶片再生的培养基组成为MS+0.10 mg/L NAA+ 0.5 mg/L 6-BA+ 0.05 mg/L TDZ。在该培养基中,适合两无性系叶片再生的处理是:叶片用打孔器打成4mm的叶盘、叶正面接触培养基、5d暗培养后每天16h光照培养。此离体再生系统下717和353无性系的离体叶片不定芽再生频率分别为85.6%和77.5%,叶片分化的平均不定芽数分别为12.4各8.6个。适合717、353杨树无性系离体叶片不定芽再生的卡那霉素敏感临界浓度为100 mg/L,不定芽生根卡纳霉素敏感临界浓度为25 mg/L。3、研究了影响PHSP::FT基因转化717无性系的各种因素,优化了转化条件,并用该优化条件,实现了PHSP::FT1和PHSP::FT2基因对717无性系,以及三种基因载体对353无性系的转化,获得了转PHSP::FT、PHSP::FT1、PHSP::FT2三种基因的717、353无性系PCR阳性植株191个。结果表明,短时间的预培养有利于提高PHSP::FT基因对717无性系的转化率;PHSP::FT基因载体转化717无性系,需要较高的AGL1菌液浓度、较长时间的AGL1浸染、以及较短的共培养时间;乙酰丁香酮(AS)对PHSP::FT基因转化717无性性转化率无明显影响;优化的AGL1介导的FT基因转化717无性系的条件是:预培养3d、菌液活化至OD600=0.5-0.6、浸染60min、共培养2d、不添加AS,在该条件下转化率为31.7%,优化的转化条件适用于其他FT基因对353无性系的转化。4、筛选了能在杨树无性系717和353中顺利表达促进其开花的FT基因,探讨了影响FT基因热激诱导表达的各种因素,得到了能顺利诱导FT基因表达的热激条件,并对转FT基因的杨树产生的花发育过程进行了详细观察。结果表明,热激启动子控制的FT基因在转基因植株中不存在基因的泄漏表达情况;不同来源的FT基因诱导杨树早期开花效果差异较大,来自拟南芥的FT基因促花效果优于来自毛果杨的FT1和FT2基因,任何基因对353无性系的促进开花效果优于717无性系;影响FT基因热激诱导表达的主要因素有:受体基因型、同一基因型的不同转基因植株、热激植株的年龄与高度、热激处理的条件等;FT基因与受体基因型之间互作明显,开花最好的3个基因与受体基因型组合是353/PHSP::FT、717/PHSP::FT和353/PHSP:: FT1;FT基因的热激诱导效果在转基因克隆间和克隆内均存在差异,且这种差异会随着热激材料的年龄与大小而发生变化。热激材料的高度与开花率成正比关系,高的植株比低的更倾向于开花;且不同的基因-受体组合,对热激材料高度的要求不一样,353/PHSP:: FT (或FT1)组合要求热激植株的高度大于20cm,717 /PHSP::FT组合要求热激植株高度大于30cm;热激材料的年龄主要影响转基因植株初始花发生时间,对基因-受体组合的终开花率影响不显著,但影响同一基因不同克隆的开花率。每天的热激时间对转基因克隆开花率没有显著影响,热激持续的时间对花序的正常发育影响较大,较长时间的持续热激,可以抑制花序回复营养生长,增加正常花序数目;较高的热激温度(40℃)可以提前转基因植株的初始花发生时间,促进FT基因表达,增加花序数目,抑制花序回复营养生长。热激时较低的环境温度有利于FT基因的热激诱导表达;热激后,FT基因诱导的花芽均产生于热激后枝条顶端延伸的新生枝条叶腋处,FT基因不能促使热激前已有的叶芽转变成花芽;花芽分化的花器官存在广泛变异,花器官的类型取决于其花芽在枝条上的位置或花芽产生的时间。热激诱导产生的花芽只有20-40%最终分化成花序,5%的花序、花序上30%的花朵有花药散粉。花序中的两性花较单朵花中的两性花发育正常。5、劈接的转基因植株未能引起非转基因接穗开花,但转基因砧木同对照相比开花率下降,说明嫁接导致转基因砧木中FT信号部分转移。更多还原

【Abstract】 Poplar(Genus Populus)is a preferred tree species in forestry and engery agriculture and has planted worldwide, is a major timber wood species for short peroiod industry in China, also is a hot interesting sopts for tree breeders to select and cultivate new species . Poplar has a long juvenile period , whereas many traits only express when adult , which resulting in limitation of progress in genetics and breeding and associated researches around this species. Therefore, stimulate precocious flowering and shorten breeding period will play a positive role in poplar genetic improvement. In recent years, successful of cloning and transfermation of many genes associated with flower development, laying foundation for plant flowering stage modification through transgenic technology. FT(Flowering Locus T)is a floral stimulating gene isolated from Arabidopsis thaliana, the expression product of FT gene is the“florigen”that researchers seeked over a long period. Researchs about FT transgenic plants mostly showed early flowering ability. In this paper , based on sucessfully cloning of Arabodipsis FT gene homologs FT1 andFT2 from Populus trichocarpa , and construct of PHSP::FT driven by a heat shock promoter(HSP)from Soybean, we created constructs of PHSP::FT1 and PHSP::FT2 of FT1 and FT2 genes driven by the same heat shock promoter, developed effencient transfation system on hybird aspens either from Section Leuce including 717-1B4( female ,Populus. tremula x P. alba, following Abbreviated as 717)or 353-53(male ,P. tremula x P. tremuloides,following Abbreviated as 353). 191 PCR positive clones of FT transgenes were obtained via leaf disc incubated in agrobacterium strain AGL1, and optimization of transform conditions for the 3 FT genes on 2 poplar clones. Early flowering were successfully induced on FT transgenes after heat induction system , best flowering FT genes were selected for 2 poplar clones, all factors affect FT gene expression and transgenes flowering capacity were discussed , flower development process under heat induction treatment were set forth, and finally graft-transmissible of FT induced flowering signal was preliminary explored. Results obtained from our experiments as following:1.Created constructs of PHSP::FT1 and PHSP::FT2 driven by a heat shock promoter of FT1 and FT2 genes in poplar.2.Factors e.g. auxin NAA, cytokinin 6-BA and TDZ, cutting styles of leaf explants, inoculation style, and culture situation etc, affect on shoots regeneration on hybrid aspen clones 717 and 353 were studied through systematic tests. Also threshold sensitive concentration of canamycin was tested in shoot regeneration and rooting in 2 poplar clones. Results showed appropriate culture medium for shoots regeneration on leaf explants is MS containing 0.10 mg/L NAA, 0.5 mg/L 6-BA and 0.05 mg/L TDZ for both poplar clones 717 and 353. The likely treatments for shoot regeneration were 4mm leaf discs punched form leaf explants, leaf adaxial touch medium, and 16 hr lights/d after initial 2 days in darkness in the above medium composition. Under the above regeneration system, shoots regeneration frequency on leaf explants were 85.6% and 77.5%, with average number of shoots per explant were 12.4 and 8.6 on poplar clones 717 and 353, respectively. The threshold sensitive concentration of canamycin was 100 mg/L in shoot regeneration on leaf explants, and 25 mg/L in shoots rooting for both poplar clones 717 and 353.3.Factors affect PHSP::FT transformation on clone 717 was tested and optimized, successfully transformation of PHSP::FT1 and PHSP::FT2 on 717, and three FT constructs transformation on clone 353 via optimized system were achived, 191 PCR positive clones obtained from PHSP::FT , PHSP::FT1 and PHSP::FT2 transgenes on clones 717 and 353. Results indicated short time pre-culture enhanced transfornmation ratio on clone 717, high concentration of AGL1, long time of transformation in AGL1 solution, relative short time incubatation were essential for PHSP::FT transformation on clone 717. AS (Acetosyringone) has less effect on transformation frequency of PHSP::FT construct on clone 717. Optimized transformation condition for FT gene on clone 717 via AGL1 is, pre-culture 3 days, agrobacterium activation of OD600=0.5-0.6, transformation 60 minutes, incubation 2 days in darkness, no AS attendance, would reach a transformation frequency of 31.7%. In additional, the optimized system also appropriate for other FT constructs on clone717 and the 3 FT constructs on clone 353.4.FT gene that could express smoothly and induced transgenes flowering after heat induction on both poplar clones 717 and 353 was selected, Factors that affect FT gene expression following heat induction were discussed, appropriate heat induction situations in smooth induction express of FT gene were found, and flower development process on FT transgenic poplar were observed in detail.Heat induction results showed, no leaky expression of the FT genes under HSP promoter was observed, great diversity among early flowering induction in various FT gene origins, FT gene has better flowering ability than genes FT1 and FT2 from P. Trichocarpa, and each FT gene prefer flowering in clone 353 to clone 717. Factors affect FT gene expression following heat induction including, receptor genotype, clones within the same genotype, ages and height of transgenes when heat induction started, heat induction treatments etc. Interaction of FT gene and receptor genotype was obvious, the 3 best flowering combinations of FT genes and receptor genotype are 353/PHSP:: FT, 717 /PHSP::FT and 353/ PHSP:: FT1;FT gene expression following heat induction was varied within clones and ramets inside clone, and such variation changes associated with ages and height of transgenes when heat induction began. .Flowering frequency is positive related to plant height, larger plants appeared to flower more readily than did smaller plants, variable height were required based on different gene/clone combination, plants must higher than 20cm in 353/PHSP:: FT(or FT1), or than 30 cm in 717 /PHSP::FT were considerably more likely to flower. Ages of transgenes mainly affect flowering initiation following heat induction, whereas less affect final ramets flowering frequency, but influence clones flowering frequency within same transgene.Heat induction hours per day has less effect on transgenes flowering frequency, duration of heat induction treatment greatly affected normal development of catkin , increased duration time of heat induction could prevent catkins reverted to vegetative growth, whereas increased normal catkin products. Increased heat induction temperature (40℃)would advance FT gene expression and initial flowering on transgenes, more production of catkins, and less catkin reverted to vegetative growth.Lower room temperature in GH is favorable for FT gene expression following heat induction. Floral buds initiated from leaf axils in newly sprouted shoots which elongated in original shoots apex after heat induction started, floral organ differentiated from floral buds varied widely, kinds of floral organs differentiation depends on location of floral shoots initiated on shoots or initiation time. Only 20-40% floral buds induced following heat induction formed into catkins, 5% catkins and 30% individual flowers on catkins dispersed pollen grain. Bisexual flowers on male catkin were more normal than those on single flowers.5.Cleft grafted transgenes failed to induce non-transgenic scions to flower after heat induction, but transgenic rootstocks had less flowering frequency than did controls, indicating grafting might resulted in transmission of part of FT product in grafted transgenic rootstocks.更多还原