【作者】 许林；

【导师】 包满珠；

【作者基本信息】 华中农业大学 ， 园林植物与观赏园艺， 2008， 博士

【摘要】 枫香（Liquidambar formosana Hance.）是我国乡土树种,分布于淮河长江流域以南地区。由于其生长迅速,抗逆性强,秋叶红色鲜艳可爱,是我国亚热带地区的典型秋色叶树种,故而在长江流域以南地区被广泛用作公路、街道、庭院和工厂绿化树种。但是在城市园林应用过程中,枫香球果存在数量多、个体大、果实硬、不易降解等问题,球果掉落时易砸伤行人及车体,给市民出行带来不便,同时也给城市环境造成了一定的污染。针对这种情况,利用基因工程培育无球果枫香成为一种行之有效的解决方法。本研究首先建立了枫香的离体微繁及植株再生体系,对影响枫香叶片再生植株的不同因素进行了系统的探讨,并在此基础上对根癌农杆菌介导枫香的遗传转化条件作了初步探索,为枫香的基因工程改良奠定了一定的基础。同时,根据拟南芥花器官发育AGAMOUS（AG）基因控制雄蕊和心皮发育的功能,我们克隆了枫香的AG同源基因LfAG,并进行了初步功能研究,最终以通过转基因抑制枫香内源AG基因的表达,获得无球果枫香为研究目的。主要结果如下:1.以优良枫香无性系的带芽茎段为外植体,建立了枫香的离体微繁体系。结果表明:将带芽茎段消毒后接种在WPM+0.1mg/LNAA+0.75 mg/L BA培养基上,诱导腋芽萌发效果最好;将萌发的幼芽切下接种在WPM+0.01mg/L NAA+0.5 mg/LBA或者WPM+0.01mg/LNAA+0.5mg/L BA+0.3 mg/LGA₃培养基上,可分别获得5.25和4.17的增殖系数以及2.57cm和2.90cm的芽丛平均高度;对比了IAA和NAA对枫香试管苗不定根的诱导效果,NAA诱导的不定根为粗短型,数量多,无二级侧根;WPM+2.0mg/L IBA诱导的不定根为细长型,根系丰富,二级侧根发达,适合作为枫香试管苗不定根诱导的培养基;将根系发育良好的再生植株移栽后,成活率可达100%,植株生长和外观形态均表现正常;2.建立了枫香叶片的植株再生体系。以5个基因型试管苗叶片为外植体,研究了NAA和TDZ对不定芽分化的作用,确定了5个基因型再生频率都较高的通用培养基为WPM+0.05mg/L NAA+0.25 mg/LTDZ,最高的再生率可达90%,平均每叶片再生3.11个不定芽;基因型P13不定芽分化的能力较差,其最高的不定芽分化率仅为32.6%,与其它4种基因型存在显著性差异（平均再生率为53.2%—90%）;光照条件对枫香叶片再生有很大的影响,最合适的光照条件为暗培养7d后转入弱光下培养;试管苗顶端第1—2片叶分化能力最强,第3—4片叶的再生能力有所下降,但仍有70%以上的再生率;3.以枫香叶片为材料,确定了其作为遗传转化受体系统的选择压力。结果表明,20mg/L的卡那霉素即可导致枫香叶片失去分化能力,15mg/L的卡那霉素即可导致枫香试管苗失去不定根分化能力。300mg/L的头孢霉素作为抑菌剂,对不定芽和不定根的分化能力均影响不大;4.以枫香叶片为受体,对根癌农杆菌介导的遗传转化条件进行了摸索。结果发现,卡那霉素对枫香侵染叶片的再生具有明显的抑制作用,共培养后直接转入选择培养外植体的再生能力急剧下降。但根据GFP基因瞬间表达的结果可知,预培养3天的叶片有利于农杆菌的附着和侵染;共培养基中AS的添加易导致叶片切口的褐化,因此在枫香遗传转化中不宜添加。对枫香叶片而言,以AB液体培养基作为侵染液,侵染10min,共培养4天的条件下进行转化,GFP表达状态最好,最有利于T-DNA的转移;5.利用同源克隆法,得到了枫香AG同源基因LfAG的全长CDS。根据GenBank中公布的美国枫香LAG基因cDNA序列设计引物,利用反转录PCR技术获得了枫香LfAG基因的全长CDS。该序列与美国枫香LAG基因核苷酸序列同源性高达98.6%,氨基酸序列同源性为96.0%,具有完整的MADS区和K区,C区具有2个AG基因的保守Motif;6.在载体pCAMBIA2300S、pART27、pHELLSGATE2的基础上,分别构建了LfAG基因的正义、反义重复和RNAi植物表达载体。利用上述3种构建转化烟草,获得的转化反义重复载体抗性植株59株和RNAi抗性植株60株均已经开花。转p2300s-LA载体的烟草抗性植株目前正在培养基中进行筛选。目前,重复反义载体转基因烟草植株已经获得了多种目的表型,包括花药花瓣化,雄蕊一端形成异域柱头和副雌蕊的形成等。此外,与野生型相比,多数转基因植株出现花药开裂推迟或者不裂的现象。各种表型的形成原因还有待于进一步分析。更多还原

【Abstract】 Formosan sweetgum distributes in the majority of temperate and subtropical regions in China.Due to its many desirable traits,such as fast growth,wide adaptability,high resistance and red leaves in autumn,formosan sweetgum is becoming popularity in forestry and is used as a street and courtyard tree in China.But the spiny fruits of the tree disintegrate very slowly and cause a nuisance on lawns and walks.So breeding for sterility or fruitless would greatly improve the usefulness of formosan sweetgum.For the aim of fruitless formosan sweetgum,an efficient in vitro propagation system and a regeneration system via organogenesis were developed firstly.Factors affecting shoot regeneration from leaf explants,including different plant growth regulators, incubation conditions,and position of explants,were investigated in detail.Subsequently, conditions suitable for Agrobacterium tumefaciens-mediated transformation of the species were preliminarily studied.Meanwhile,we isolated AG homologue gene（LfAG） from formosan sweetgum flower buds and preliminarily analyzed its functions.The exact functions of LfAG should be studied for further researches.The major results are as follows:1.A protocol for in vitro micropropagation of formosan sweetgum was established. Effects of different PGRs on sprouting of axillary buds and shoot multiplication, elongation and rooting and survival of transplanted plantlets were investigated.The results showed that the best sprouting medium WPM+0.1mg/L NAA+0.75 mg/L BA promoted sprouting in all cultured axillary buds.Young shoots were cultured on medium for shoot multiplication,and average 5.25 and 4.17 shoots,2.57cm and 2.90cm in length were obtained,respectively.The adventitious root induced by NAA was greatly different from that by IBA.The numerous roots formed on NAA were short with big callus tissue at bottom and without secondary root.Whereas the roots formed on IBA were longer with abundant secondary roots.All the rooted plantlets were transplanted to soil and grew normally outdoors.2.Plant regeneration system from leaves of formosan sweetgum was established and optimized.The morphogenic potential of leaf explants from five genotypes was tested on the 16 media containing different concentrations of TDZ and NAA.The results indicated that among five genotypes,four genotypes（P2,P6,P9,P11） showed higher regeneration rates（53.2%-90%in average）,whereas genotype J13 showed low capability of shoot regeneration on all media tested（32.6%）.In general,preferable media for inducing adventitious shoots was WPM supplemented with 0.25mg/L TDZ and 0.05mg/L NAA,on which higher regeneration rate and more adventitious shoot clumps were obtained in all five tested genotypes.The organogenesis was greatly influenced by light and the optimum condition was leaves were cultured in dark for 7d and then transferred into 50-100 lx.The upper two leaves on the stem showed higher regeneration capability（100%） than the lower two leaves,which were still over 70%in regeneration capability.Transferred to WPM basal medium containing NAA,BA and GA₃,regenerated buds elongated into shoots.3.Effects of selection markers kanamycin（Kan） on shoot differentiation of leaf explants and rooting of in vitro shoots were investigated.The results indicated that formosan sweetgum was comparatively sensitive to Kan.Selection pressures were determined as:20 mg/L Kan for shoot regeneration of leaves,and 15 mg/L Kan for rooting of shoots.300 mg/L cefotaxime（Cef） were no great influences in organogenesis of adventitious shoot and root.4.Conditions suitable for Agrobacterium-mediated transformation of formosan sweetgum were studied.The results indicated that after co-cultivation with Agrobacterium,regeneration capability of the leaves decreased dramatically when cultured in selection medium.According to the transient GFP expression,three days of pre-culture with the abaxial side of leaves touched the medium promoted adhesion and invasion of Agrobacterium evidently.For formosan sweetgum,acetosyringone（AS） in the co-cultivation medium had no promoting effect on Agrobacterium mediated transformation.Based on the transient GFP expression,10-min infection in AB liquid medium and co-culture for 4d was favorable for T-DNA transfer to plant cell of formosan sweetgum.5.We have isolated the putative AG gene from formosan sweetgum,LfAG,whose deduced protein product shares 96.0%identical amino acid residues with LAG gene product from L.styraciflua,a count-part sweetgum grown in America.LfAG included full length MADS domain and K domain,and there were two consensus AG motifs in the C termination. 6.Sense,repeated anti-sense and RNAi plant expression vectors of LfAG were construted.59 repeated anti-sense and 60 RNAi transgenic tobacco plants were obtained by the leaf discs method of Agrobacterium-mediate transforomation.Analysis of transgenic tobacco plants showed that LfAG gene was involved in the stamen and carpel of flower development.Repeated anti-sense of LfAG gene expression produced a variety of mutant phonotype with either anther transformating into petal,or stigma on anther,or secondary pistil.The reason of mutants needs further researches.更多还原