【作者】 齐秀娟；

【导师】 张绍铃；

【作者基本信息】 南京农业大学 ， 果树学， 2013， 博士

【摘要】 本研究主要以全红型软枣猕猴桃品种‘天源红’为试材,通过田间调查、石蜡切片等技术来探讨影响‘天源红’落果率相对较高的原因。同时,采用焦锑酸盐沉淀法研究了猕猴桃有性生殖过程中的花粉管生长机理。应用荧光显微镜、石蜡切片技术、差异凝胶电泳(DIGE)、质谱(MALDI-TOF/TOF)和生物信息学技术,对其授粉、受精前后花柱和子房蛋白质组进行比较研究,以期寻找授粉、受精时花柱及子房中特异表达的蛋白质,进一步研究了猕猴桃有性生殖过程中授粉受精的分子机理。主要结果如下：1.对全红型软枣猕猴桃‘天源红’花器结构、雌花开放动态及寿命、花期温度、柱头可授性、有效授粉期、授粉受精、胚胎发育、果实生长速率、落果率等进行了系统研究。(1)柱头属于干性柱头,具有一道浅裂沟,乳突呈长圆柱形；开花过程可分为以下五个阶段：花萼开裂期、花萼大裂期、花瓣变色期、大蕾期、开花期；整个开花过程温度平稳,开花整齐,雌花单花期为1-2d；柱头可授性在花后1-2d最强,花开后3-5d可授性逐渐降低,花后第6d丧失可授性；有效授粉期为开花第1d和第2d。(2)受精作用属于有丝分裂前配子融合类型。一个精子与卵细胞融合形成合子；另一个精子与次生极核融合形成初生胚乳核。(3)胚发育属茄型。卵细胞受精后,合子经过3d左右的休眠期进行第一次分裂。合子分裂通常发生在初生胚乳核分裂之后,经棒形胚、球形胚、鱼雷形胚和心形胚,至果实成熟时发育为子叶形胚。小球胚时期胚柄最为发达,球形胚末期胚柄开始退化,心形胚时期胚柄解体。(4)胚乳发育类型为细胞型。初生胚乳核先于合子分裂,胚乳细胞分裂较快,胚周围的胚乳细胞有降解现象。至胚成熟形成种子时,胚乳细胞始终分布在种皮内侧周围,因此‘天源红’属于有胚乳种子。(5)花期、有效授粉期以及柱头可授性时间短,同一子房受精不同步,胚囊、胚胎发育异常,这是‘天源红’落果率相对较高的主要原因。2.应用扫描电镜和荧光显微镜对‘天源红’同种花粉在柱头上原位萌发及花粉管生长情况进行了观察,并用焦锑酸盐沉淀法对其授粉前后柱头及花柱中Ca2+进行超微细胞化学定位,研究结果表明：(1)授粉后1h,花粉管生长穿过柱头表面,授粉后10h,生长到达花柱底部。(2)授粉前后,柱头接受面靠近柱头外围细胞的角质层一侧细胞器内含有丰富的钙,而柱头非接受表面钙颗粒分布很少。(3)授粉前和授粉后1h,花柱顶端钙颗粒较少,基部钙颗粒较多；授粉后10h,花柱顶端和基部钙分布密度无明显差别。(4)授粉前后花柱顶端钙主要均匀分布在细胞质膜位置；在花柱基部授粉前主要在引导组织胞间隙中,授粉后1h主要在细胞质内,授粉后10h主要存在于细胞质、内质网上。猕猴桃授粉前后,柱头和花柱组织中均含有钙,授粉前和授粉后1h花柱中的钙呈现出梯度分布,授粉后10h钙的梯度分布现象减弱甚至消失。3.应用荧光显微镜、石蜡切片技术、差异凝胶电泳(DIGE)、质谱(MALDI-TOF/TOF)和生物信息学技术,对其授粉、受精前后花柱和子房蛋白质组进行比较研究。(1)采用DIGE进行分离后,成功获得了蛋白质组分辨率高、蛋白点数多、分布均匀且清晰的花柱、子房双向凝胶电泳图谱。(2) DeCyder V6.5软件分析获得授粉前后花柱差异2.5倍以上的蛋白质点共有24个,质谱鉴定成功了18个点,其中有8个差异蛋白点属于actinidin家族的蛋白,另有两个差异点属于同一种蛋白质,这些蛋白质主要与水解、细胞壁代谢、逆境胁迫响应、糖类和能量代谢等有关；10个具有统计学意义的表达差异蛋白点,其中5个在花柱中表达上调,5个表达下调。根据猕猴桃授粉前后花柱差异蛋白表达谱比较,筛选出与授粉相关的花柱蛋白。(3)用DIGE进行分离并用DeCyder V6.5软件分析,在授粉前、授粉后120h的子房中共检测到约1500个蛋白质点,其中有55个差异表达的蛋白点；质谱鉴定了差异2.0倍以上的蛋白质点13个,得到了8个鉴定结果,分别属于6种蛋白质；6种差异蛋白质点,其中5种在授粉后子房中表达上调,只有1种表达下调。根据猕猴桃受精前后子房差异蛋白表达谱比较,筛选出与双受精相关的子房蛋白。更多还原

【Abstract】 The all red Actinidia arguta’Tianyuanhong’were studied by field investigation or using fluorescence paraffin technique, to explore the factors that caused its high fruit drop rate. At the same time, Stigmatic and stylar proteins play a pivotal function in plant reproduction during pollination and fertilization. We compared kiwifruit(Actinidia. arguta) Stigmatic and stylar proteins before and10h after pollination. Several specific proteins were identified using fluorescence differential in-gel electrophoresis (DIGE), Matrix-Assisted laser Desorption/Ionization Time of Flight Mass Spectromety (MALDI-TOF/MS) and bioinformatic technology. We also observed the growth of kiwifruit pollen tubes in the style by fluorescence microscope. The main results were as follows:1. The study included flower structure, floral dynamic and life, bloom temperature, stigma receptivity, effective pollination period and embryogenesis of the all red Actinidia arguta’Tianyuanhong’.(1) Female flower branches include the following five types:Stigma of Tianyuanhong’ was dry, it was long cylindrical shape and had a crack ditch. Flowering process is divided into the following five stages:calyx cracking time, calyx large cracking time, petal color change, large flower buds and flowering time.’Tianyuanhong’flowers were neat in steady temperature, its life span of one single flower was approximately1-2days.’Tianyuanhong’receptivity is strong during1st-2nd day and begins to decline during3rd-5th day, and stigma com pletely lose its receptivity on6th day.’Tianyuanhong’ effective pollination period was approximately1-2days. (2) Double fertilization is the type of the Premitotic gametogamy. A sperm and egg cell fusion formation of homozygous; another sperm and secondary Primary endosperm formation of a nuclear fusion.(3) The development of embryo belongs to solanad type. The fertilized egg usually underwent a rest Period about3days before embarking on its first division. And the first division of the zygote took Place after the division of Primary endosperm nucleus. After the club-shaped embryo, the globular embryo, the fish-torpedo and the heart-shaped embryo, the cotyledon-shaped embryo developed when the fruit became ripe. At the Early globular embryo stage, the suspensor was well developed. From the late globular to early heart-shaped embryo stage, the suspensor grew to its minimum length.(4) The endosperm development of’Tianyuanhong’was cellular type. The Primary endosperm nucleus division was often Prior to zygote, and it divided quickly. The endosperm cells surrounding the embryo were disintegrated during the development of embryo.(5) flowering stage, effective pollination period and stigma receptivity with short time are the main factors that affect the female success of’Tianyuanhong’.2. Fluorescence microscope and scanning electron microscopy were used to observe pollen germination and pollen tube growth, Potassiam antimonate was used to localize calcium in stigma and style tissue of’Tianyuanhong’ before and after pollination.(1)1h after pollination, pollen tube grew through stigma surface, and got to basal style10h after pollination.(2) Receptive surface of stigma, Cuticle organelles were rich in calcium granules before and after pollination, but no-receptive surface of stigma, organelles had little calcium granules.(3) Top style had little calcium, but basal style had more calcium before and1h after pollination. The calcium contents were briefly identical in top and basal style10h after pollination.(4) The calcium distributed in plasma membrane of top style before and after pollination, in intercellular gap of basal style before pollination, in plasma membrane of basal style1h after pollination, and in plasma membrane and endoplasmic reticulum of basal style10h after pollination. Stigma and style tissue of’Tianyuanhong’were rich in calcium before and after pollination. Calcium of style showed the gradient distribution before and1h after pollination, it weakened or disappeared10h after pollination. 3. Compared with kiwifruit (Actinidia. arguta) stylus and ovary proteome before and after pollination. Several specific proteins were identified using fluorescence microscope, paraffin technique, differential in-gel electrophoresis (DIGE), Matrix-Assisted laser Desorption/Ionization Time of Flight Mass Spectromety (MALDI-TOF/MS) and bioinformatic technology.(1) According to the DIGE profiles, We gained the two-dimensional gel electrophoresis, which had high resolution ratio and more protein spots proteome.(2) We picked24differential protein spots (ratio>2.5) by DeCyder6.5(Amersham Bioscience), and identified18differential proteins through MALDI-TOF/MS. Of these proteins,8belong to the actinidin family, and2were identified as being the same protein. Among the10significantly different protein spots,5were up-regulated while5were down-regulated, compared with stigmatic and stylar proteins before pollination. Most of the identified proteins play an important role during kiwifruit polliantion. Their putative functions in these processes are discussed.(3) About1500protein points were detected by DeCyder6.5(Amersham Bioscience), and55different proteins were expressed at ovary proteins before and120h after pollination. Thirteen protein spots (ratio>2.0) were identified through MALDI-TOF/MS. Of these proteins,8belong to6kinds of proteins. Among the6significantly different protein spots,5were up-regulated while1were down-regulated. Compared with differences ovary proteins expression before and after Fertilization, we can filter out the double fertilization of ovary proteins.更多还原