【作者】 蒋学美；

【导师】 蔡礼鸿； 向旭；

【作者基本信息】 华中农业大学 ， 果树学， 2008， 硕士

【摘要】 多胺(Polyamines,PAs)作为具有调控作用的生理活性物质,在果树开花坐果中的作用的研究日益引起人们的重视。本研究紧密围绕荔枝正常坐果与内源多胺的关系,分析坐果过程中内源多胺的变化、外源多胺处理对荔枝果实发育启动和正常坐果过程的影响,并克隆荔枝组织中多胺合成途径的关键酶S-腺苷甲硫氨酸脱羧酶(SAMDC)和乙烯合成途径的关键酶ACC合成酶(ACS)的基因序列,拟开展荔枝果实发育过程中此类酶基因的功能分析和表达分析,在理论上,可能阐明荔枝果实发育中SAM代谢流向与多胺和乙烯生物合成及其在荔枝正常坐果和生理落果过程中作用的分子机理,丰富荔枝果实发育的理论;在实践上,可能为栽培技术创新提供理论依据,从外源多胺应用技术和内源多胺代谢调控的角度,为荔枝高效低耗和优质果品生产提供新的途径。本研究获得如下主要结果:1.套袋不授粉加人工处理外源多胺的诱导荔枝单性结实的试验表明,桂味和糯米糍不可诱导单性结实,妃子笑可以用外源多胺人工诱导单性结实,三种多胺的诱导效果次序为:精胺(Spermine,Spm)＞亚精胺(Spermidine,Spd)＞腐胺(Putrecine,Put)。谢花后外源多胺处理表明,三种外源多胺均可明显提高妃子笑荔枝的坐果率,尤其以Put处理效果最显著。2.建立起适合荔枝材料中Put、Spd、Spm三种内源多胺含量分析的高效液相色谱测定方法,一次样品的测定可在15 min内完成;Put、Spd和Spm在0.03125～2 nmol线性范围内,相关系数分别为0.9996、0.9996、0.9995;精密度和稳定性实验相对标准偏差均小于5%,说明测定方法稳定、分析精密度高。3.分析测定三个荔枝主栽品种在花穗发育与坐果初期其叶片与花器官和幼果中内源多胺含量的变化:在叶片中,正常成花的妃子笑三种多胺含量先升后降,在盛花期出现高峰;桂味未成花的叶片三种多胺的持续下降则与其此期新梢大量发生密切相关。在花器官和幼果中,三个荔枝品种多胺总量要明显高于叶片中;妃子笑三种多胺含量以Spd含量最高,且Spd含量在坐果初期均保持较高水平。4.在果实膨大期,分析外源多胺处理对内源多胺含量的影响:外施高浓度的Spd使内源Spd的含量明显下降;外施Spm则可以大大提高内源Spd的含量;外施Put只能提高内源Put的含量,对Spd和Spm含量影响不大;三种多胺的混合使用对提高内源Put的效果显著,可以缓和Spd和Spm的变化幅度。在这三种多胺中,Spm与Spd相互影响,作用显著。5.采用同源序列扩增法,首次从荔枝中克隆分离得到一段728bp的DNA序列,命名为LcACS1。序列分析表明,该基因片段编码区493bp,推测其编码165个氨基酸残基,Blast结果显示,该基因片段推导的氨基酸序列与其他9种植物ACS基因编码的氨基酸序列同源性在82%-89%之间。并且有4个保守结构域,在保守区1与保守区3分别插入了一段109bp与125bp的内含子。6.采用RT-PCR的方法,首次从荔枝中克隆分离到584bp SAMDC基因片段,命名为LcSAMDC1,序列分析表明,该基因片段编码194个氨基酸残基。Blast结果显示,该基因片段与已报道其他植物的SAMDC基因的核苷酸序列同源性在78%以上,其推导的氨基酸序列与其他9种植物SAMDC基因编码的氨基酸序列同源性在75%-85%之间,并且有4个较长的保守结构域。更多还原

【Abstract】 Polyamines,as physiological active materials with broad functions of regulation,play a critical role in flowering and fruit setting of fruit trees.In the present paper,the relationship between fruit setting and endogenous polyamine was studied.The changes of endogenous polyamine contents in fruit setting and effects of exogenous polyamines on the promotion of fruit growth as well as fruit setting were investigated.We also report the cloning of S-adenosylmethionine decarboxylase(SAMDC) cDNA fragment from litchi (Litchi chinensis ’Guiwei’) leaves,an enzyme involved in the polyamine biosynthesis pathway,and 1-aminocyclopropane-1-carboxylicacidsynthase(ACS) cDNA fragment,a key enzyme responsible for the ethylene biosynthesis.In theory,SAM metabolism and biosynthesis of polyamine and ethylene during litchi fruit growth and their molecular mechanism in fruit setting and physiological fruit dropping can be established.In practice, this research can also offer theoretical foundation for developping a new way to produce high quality litchi fruit with low investment.The main results were listed as follows:1.Use of exogenous polyamines on bagged flower without pollination showed that parthenocarpy can be induced in Feizixiao but not in Guiwei and Nuomici.Spm was more effective for the induction,followed by Spd and Put.The three polyamines all can obviously improve percentage of fruit setting especially Put.2.HPLC analysis of Put,Spd and Spm contents in litchi was established,which was completed in 15 minutes.All the relative coefficients were above 0.999 when the linear arrange of the three polyamines was from 0.03125 to 2 nmol.The relative standard deviation of precision and stability experiments was lower than 5%,indicating that the method was a simple and rapid method with more accuracy and stability.3.The contents of Put,Spm and Spd in the leaf,floral organ and fruitlet of three litchi varieties were analyzed during their development.Put,Spm and Spd in the leaf of normal flowering Litchi plant increased and then declined after a abrupt peak at anthesis. However,the contents of polyamines in the leaf of non-flowering ’Feizixiao’ changes in an opposite trend.The contents of polyamine in the leaves of non-flowering ’Guiwei’ continuously declined which may be related to new flushing in this phase.The contents of total PAs in floral organ and fruitlet were much higher than in leaves.In floral organ and fruitlet of Litchi chinensis ’Feizixiao’,Spd was the most abundant and kept high during the initial stage of fruitset. 4.The influence of exogenous polyamines on endogenous polyamine contents was analyzed.Exogenous Spd decreased the endogenous Spd content,and exogenous Spm enhanced endogenous Spm greatly.Exogenous Put can only improve endogenous Put and did not alter endogenous Spd and Spm.A combination of these three polyamines notably increased the content of endogenous Put and mildly changed Spd and Spm.Among the three polyamines,Spd and Spm influenced each other.5.A 728bp ACS gene fragment named LcACS1 was isolated from litchi for the first time using a homology-based method.Sequence analysis revealed that the coding sequence fragment ofACS gene was 493bp which deduced 165 amino acids.The result of BLAST showed the ACS amino acid showed 82%to 89%homology with ACS genes from other 9 plants reported.Four conserved catalytic domains in the ACS gene were found and there is respective 109bp intron and 125bp intron in the domain l and domain3.6.A 584bp SAMDC gene fragment named LcSAMDC1 was isolated from litchi via RT-PCR for the first time.Sequence analysis revealed that the fragment of SAMDC gene encode product of 194 amino acids.BLAST seach demonstrated that LcSAMDC1 showed above 78%sequence identity with other plants SAMDC genes.The deduced amino acids of LcSAMDC1 showed 75%to 85%identity with SAMDC from 9 other plants.Four conserved catalytic domains in the SAMDC gene were found.更多还原