【作者】 孙迎坤；

【导师】 李纪元；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2013， 博士

【摘要】 山茶花（Camellia japonica）是山茶属（Camellia）的模式植物，该物种的许多品种花器官变异极其丰富，是我国传统的十大名花之一。同时该物种还存在非常普遍的萼片瓣化和雄蕊瓣化的表型变异，这对于研究山茶花重瓣花形成的分子机理和起源方式均具有重要意义。分子植物育种可以缩短观赏植物育种的周期，人为定向进行新品种培育。根据花器官发育的“ABC”模型，A类和C类功能基因可在植物山茶重瓣花形成中起到关键调控作用。研究A类和C类基因在山茶重瓣花形成中的分子调控机理，将为山茶和其他观赏植物的花型育种提供依据。采用同源克隆方法和RACE（Rapid Amplification of cDNA Ends）技术，从山茶托桂型重瓣类栽培品种‘金盘荔枝’的发育早期花芽中分离到了山茶花花器官发育同源基因cDNA全长，序列分析表明分别为A类的AP1同源基因CjAPL1（GenBank登录号JX657332）和CjAPL2（GenBank登录号JX843815）、 AGL6同源基因CjAGL6（GenBank登录号JX657333），C类的AG类同源基因CjAG1（GenBank登录号JX843816）。生物信息学和保守域分析表明，克隆的4个基因皆为典型的Type II类型的MADS-box家族花器官发育调控基因。以六种典型花型的山茶原种和品种为材料，利用实时荧光定量方法对山茶中获得的4个基因的时空表达模式进行了研究。在时间表达模式中，随着花芽发育成熟度的增加和花瓣数量的增多，CjAPL1和CjAPL2基因的高表达趋势表现为由花芽发育早期转至中期，但在半重瓣品种的花芽发育中后期也进行了高表达。CjAGL6基因在单瓣花型中的最高表达量是在花芽发育早期，随花瓣数量增多高表达转至发育中后期，但半重瓣品种的后期表达量最高。CjAG1基因除在半重瓣品种的花芽发育中期进行了高表达外，在其他5种花型中普遍为花芽发育的后期表达量最高。在不同品种的山茶花器官中，CjAPL1基因在萼片或瓣化萼片、外轮花瓣中的表达量高于雄蕊和内轮花瓣，同时也在花柱、子房和瓣化雄蕊中存在高表达，表明CjAPL1基因不仅对萼片和外轮花瓣花器官调控功能比较明显，而且对于花柱和子房也具有明显的调控功能。CjAPL2基因与CjAPL1基因的表达模式相似，在山茶萼片、瓣化萼片、外轮花瓣和心皮中的表达量普遍高于内轮花瓣和雄蕊，且在玫瑰重瓣型和完全重瓣型中该基因调控效果更加显著。CjAGL6基因在单瓣型、半重瓣型和托桂型中的高表达集中于心皮、萼片或瓣化萼片部位，其次为内轮的花瓣、瓣化雄蕊和雄蕊；在其他3个品种中的高表达量部位为雄蕊和内轮花瓣。CjAG1基因的表达量较一致，主要集中于心皮、雄蕊、瓣化雄蕊，且在内轮花瓣和瓣化萼片中的总体表达量高于外轮花瓣；这说明CjAG1主要对内轮花器官起调控作用。但在‘红十八学士’中该基因对于对于外轮花瓣和瓣化萼片的调控作用却较内轮花瓣明显，显示出山茶完全重瓣品种基因表达的特异性。利用酶切方法，分别构建了4个正义植物表达载体和4个干扰植物表达载体,利用农杆菌介导的花序浸染法分别转化拟南芥野生型、ap1-3和ag-1突变体植株。经表型观察统计，获得了含CjAPL1、CjAPL2、CjAGL6和CjAG1正义基因的拟南芥野生型阳性植株分别为65株、42株、73株和102株；含CjAPL1、CjAGL6和CjAG1干扰基因的拟南芥阳性植株数量各45株、21株和25株；含CjAPL1正义基因的拟南芥ap1-3突变体和含CjAG1正义基因的拟南芥ag-1突变体阳性植株分别为36株和28株。从每个转基因表型为阳性类型的植株中随机挑选3株，经基因组PCR扩增和Southern杂交鉴定确认为阳性转基因植株，荧光定量结果表明，与对照相比较，所有目的基因在转正义和干扰基因的阳性植株中分别出现相应表达量显著升高和下降的结果。经表型观察统计，转CjAPL1和CjAPL2基因拟南芥野生型植株典型的表型变异为：萼片边缘演变为花瓣质地的白色，花内都出现花柱增加1枚，但雄蕊数量前者增加4枚，后者2-3枚；此外，转CjAPL2的植株单朵花花径变大，花瓣增加1-3枚，萼片数量不变或减少1枚。CjAPL2还能够促进花瓣形成，且这两个基因还可以调控内轮雌、雄蕊的器官数量增加。转CjAGL6正义基因的植株雄蕊和雌蕊数量保持不变，但萼片增加1枚，花瓣数量和形态没有发生变化。转CjAG1正义基因的植株单朵花花径缩小明显，雄蕊增多2-4枚，萼片先端出现柱头状乳突，花瓣数量减少甚至无花瓣发育，雌蕊数量没有变化。观察到转CjAPL1和CjAG1干扰基因的阳性植株都出现雄蕊减少1-2枚的表型；转CjAGL6干扰基因的花径变大，雄蕊退化，花柱为1枚且上部分弯曲，萼片和花瓣各发育为3枚、内弯，花瓣外表面的上半部分颜色变红，表明所干扰的基因有调控萼片、花瓣和花柱生长及伸长的功能；雄蕊退化也表明CjAGL6对于花器官的形态和数量发育起到重要的调控作用。在正义表达载体转拟南芥突变体实验中，转山茶CjAPL1正义基因的拟南芥ap1-3突变体，花序恢复为总状花序，单花花径变大，花瓣数量也恢复为4枚；同时，株型也得到恢复。通过转拟南芥ap1-3突变体的花瓣数量增加，再次表明CjAPL1基因有增加花瓣数量的调控功能。转山茶CjAG1正义基因的拟南芥ag-1突变体，雄蕊数量也恢复为6枚，这也证实了CjAG1基因具有调控雄蕊数量增加的功能。综合荧光定量、模式植物功能验证和表型观察统计分析结果，认为山茶重瓣花的起源方式可能包括萼片起源、雄蕊起源和重复起源等多种，且这些起源方式对于不同花型重瓣花形成的作用是不同的。半重瓣型品种的重瓣性为单一的萼片起源；牡丹重瓣型品种重瓣性为雄蕊起源为主，雄蕊起源和萼片起源并存；托桂型品种中重复起源与雄蕊起源并存，辅以萼片起源。玫瑰重瓣型和完全重瓣型山茶品种重瓣性以重复起源作为主要的重瓣花起源方式，辅以雄蕊起源和萼片起源。本研究表明，山茶CjAPL1、CjAPL2基因不仅能促进萼片向花瓣属性的转化，而且可以调控花瓣和雄蕊、雌蕊的数量增加；CjAGL6基因能够调控雄蕊数量，同时促进增萼片和花瓣数量增多；CjAG1基因对于雄蕊数量增多调控效果显著，并能引起花瓣突变为雄蕊、萼片向心皮演变。对所克隆的山茶AP1、AGL6和AG同源基因的时空表达模式和模式植物功能基因验证结果表明这4个基因在山茶重瓣花形成过程中具有花器官属性决定及花器官数量增加的调控功能。更多还原

【Abstract】 Camellia japonica, with plentiful floral organ varitions, is the modle plant of Camelliaand one of the ten famous flowers in China. Meanwhile as there are very common phenotypicvariation of sepals and stamens petalody, it is significant to studies on the double flowerformation. Molecular breeding could shorten breeding cycle and orientedly cultivate newspecies. Based on the "ABC" model of floral organs development, class A and C genes couldhave the key function during the double flower formation of C. japonica. The molecularregulation mechanisim discussion of A and C genes for doule flower formation, could providea basis for the flower type breeding of Camellia and other ornamental plant in the future.Firstly, four homologous gene were isolated from early flower bud of C. japonica ‘JinpanLizhi’, a amenone form double flower cultivar by homologous sequence cloning and RACE.The result of sequences analysis showed that they are CjAPL1(GenBank accession#JX657332)and CjAPL2(GenBank accession#JX843815) belonging to AP1homologous genes, CjAGL6(GenBank accession#JX657333) belonging to AGl6homologous gene and CjAG1(GenBankaccession#JX843816) belonging to AG homologous gene. They were floral organsdevelopment genes of typical Type II kind in MADS-box family by bioinformatics andconservative domain analysis.Then in six typical flower types of protospecies and cultivars of C.japonica, spatial andtemporal expression patterns of CjAPL1、CjAPL2、CjAGL6和CjAG1genes were studied byReal-time quantitative PCR. Along with the flower bud maturity and petal quantity increase,the high expression trend of CjAPL1and CjAPL2genes changed from early flower bud tomidlle one, but at midlle and later flower bud in semidouble flower. In single flower thehighest expression level of CjAGL6gene presented at early flower bud, and changed to midlleand later flower bud along with the quantity increase, but at later flower bud in semidouble flower. The high expression level of CjAG1gene were most at later flower bud in C. japonicaexcept of at midlle flower bud in semidouble flower.In floral organs spatial expression pattern of thses six samples, the expression level ofCjAPL1gene were higher at sepal, petalody sepal and outer petal than at stamen and innerpetal. Meanwhile it were also higher at style and ovary. This indicated that CjAPL1gene hadrelative obvious regulatory function at sepal and overy and style. CjAPL2gene had the sameexpression pattern as CjAPL1, the higher expression level at sepal, petalody sepal, outer petaland carpel than at inner petal and stamen, and obvious regulatory effect presented in rosedouble form and fully double form.The highest expression level of CjAGL6gene were at carpel, sepal and petalody sepal,next at inner petal and stamen in protospecies, semidouble double flower and amenone form.While it were at stamen and inner petal in other cultivars.The expression level of CjAG1gene were mainly at outer petal. This indicated thatCjAG1had a main regulatory function to inner floral organs. But in fully double form thisgene had stronger regulatory function to outer petal and petalody sepal than inner petal,verifying the expression specificity in fully double flower cultivar of C. japonica.Four sense plant expression vectors and four RNAi plant expression vectors wereconstructed by restriction enzyme digestion, then respectively transformed into Arabidopsisthaliana of wild type (WT), ap1-3and ag-1mutant plants by inflorescence soak mediatedwith Agrobacterium tumefaciens. The positive plants quantity of transgenic WT Arabidopsisthaliana were65of transgenic sense CjAPL1gene,42of transgenic sense CjAPL2,73oftransgenic sense CjAGL6and102of transgenic sense CjAG1,45of transgenic RNAi CjAPL1gene,21of transgenic RNAi CjAGL6and25of transgenic RNAi CjAG1,36of transgenicap1-3with sense CjAPL1gene and28of transgenic ag-1with sense CjAG1gene. The positiveplants with hygromycin resistance were identified by PCR amplication and Southern blotting.The result of Real time-PCR detection showed that all aim genes expression level increased intransgenic sense gene plants and reduced in in transgenic RNAi gene plants. The results of phenotypic observation and statistics showed that in transgenic senseCjAPL1and CjAPL2plants there were same type varitions of edge developed petaloidmorphology,1increased styles and1-4or2-3increased stamens each. Moreover, there werelarger flower diameter,1-3increased petals and1reduced sepal in transgenic CjAPL2plants.These indicated that both genes could indirectly regulate stamen and pistil development, inaddition and CjAPL2gene could promote petal formation.In transgenic sense CjAGL6plants though the quantity stamen and pistil did not changed,sepals increated1. Meanwhile the quantity and shape of petals did not changed. In transgenicsense CjAG1plants the diametre of one flower shrinked distinctly, stamens increased2-4,carpelloid-sepal beared stigmatic papillae, but petals reduced or disappear.There was Phenotype change of1-2stamens reduced in plants of RNAi genes. In positiveplants of RNAi CjAGL6gene the single flower diametre increased, no stamen presented,1style with inflexed upper part, and the rest floral organs included3inflexed sepals and3inflexed petals with red color in upper part of surface. These showed this RNAi genes couldregulate growth and stretch of sepal, petal and style. The vestigial stamen also indicated thedisturb effect was obvious.The inflorescence of ap1-3Arabidopsis thaliana with transgenic CjAPL1gene reverted toraceme from dense corymb, the diametre of one flower increased, the petals quantity revertedto4. Meanwhile the plant type reverted to WT. The result of petals quantity increase showedthat CjAPL1gene could regulate the petal formation. The stamen of ag-1Arabidopsis thalianawith transgenic CjAG1gene reverted to6. This indicated CjAG1gene could regulate stamenformation and laid a solid foundation for stamens petalody.The results of Real-time quantitative PCR, function verification in modle plant,phenotypic observation and statistics showed that the origin way of double flower in Camelliamay include some ways incluing sepals origin, stamens origin and repeat origin, and so on.These ways were different in various flower types. Semidouble type flower was the singlesepals origin. Peony double form flower were sepals origin and stamens origin, and stamens origin as main origin way. Repeat origin and stamen origin were both important in amenoneform flower, and assisted with sepals origin. Rose double form and double form flowers maderepeat origin as main origin way, assisted with stamens origin and sepals origin.All study showed that CjAPL1and CjAPL2genes in C. japonica could not only promotethe character conversion from sepal to petal, but also regulate the quantity increase of the petal,stamen and pistil. CjAGL6gene could regulate stamen quantity increase, also promote thequantity increase of sepal and petal. CjAG1had obvious regulative effect to stamen quantityand caused the petal reverted to stamen, sepal to carpel. The result of spatial and temporalexpression patterns and transgenic studies showed that AP1, AGL6and AG homologous genesisolated from C. japonica could regulate the floral organs character and increase floral organsquantity during doule flower formation of C. japonica.更多还原