外源亚精胺对鸭梨自花授粉花粉管伸长及花柱蛋白表达的影响

为探讨外源多胺调控鸭梨自交不亲和的生理生化机制,分别以自花授粉和外源亚精胺处理后1~3 d的花柱为试材,测定了不同处理不同时期花粉管生长发育动态,利用双向电泳技术和质谱分析技术分析了外源亚精胺处理前后不同时期鸭梨花柱蛋白的差异表达。结果显示:与对照相比,不同浓度的Spd处理均能极显著促进花粉管生长,0.25 mmol/L Spd处理72 h后可使大量花粉管到达花柱基部;在自交花柱电泳图谱中发现了3种特异蛋白质,A蛋白质Mr A=28.0 k Da,p I=5.7;B蛋白质Mr B=28.5 k Da,p I=5.7;C蛋白质Mr C=22.0 k Da,p I=5.3,这3种特异蛋白质可能与自交不亲和性相关;质谱分析获得了2个较好的蛋白点,分别为推定和假定蛋白。     

小报春类钙调素蛋白基因PfCML41的克隆及序列分析

CMLs是一类钙离子传感器,能与Ca~(2+)结合,对促进花粉萌发和花粉管生长发挥重要作用。为了探究CMLs对小报春(Primula forbesii)异型自交不亲和系统的影响,在小报春以长花柱做母本时的亲和授粉与不亲和授粉的转录组数据中发现了一个差异表达基因PfCML41,并对其进行了CDS序列全长克隆和基因序列分析。结果表明,PfCML41基因的CDS序列全长为555 bp,编码184个氨基酸。编码的蛋白无信号肽和跨膜区域,属于亲水性蛋白。PfCML41含有EF-hand结构,能与Ca~(2+)结合。通过系统进化树分析,PfCML41与茶(Camellia sinensis)的CsCML41亲缘性更近。实时荧光定量PCR结果表明PfCML41在不亲和授粉组合中的相对表达量高于亲和授粉组合,且差异显著。本研究为小报春PfCML41的表达分析与功能验证提供了理论依据。     

油茶CoSAUR32和CoSAUR50基因的克隆及表达分析

本研究以油茶‘华硕’为试验材料,通过PCR克隆获得两个生长素响应SAUR (Small auxin upregulated RNA)基因家族的基因,命名为CoSAUR32和CoSAUR50。CoSAUR32基因开放阅读框(ORF)为372 bp,编码123个氨基酸;CoSAUR50基因开放阅读框(ORF)为318 bp,编码105个氨基酸。序列分析发现CoSAUR32和CoSAUR50蛋白均无信号肽与跨膜结构,在叶绿体和细胞核中均可表达。对其进行同源性比对及进化树分析发现,油茶两个SAUR与茶树SAUR亲缘关系最近。实时荧光定量PCR结果表明,CoSAUR32和CoSAUR50基因在自交授粉子房中36 h时的表达量显著高于异交子房,在自交授粉花柱中24 h时的表达量显著高于异交花柱。推测CoSAUR32和CoSAUR50可能参与油茶自交不亲和过程。本研究为挖掘SAUR基因的功能及解析油茶自交不亲和分子机制提供参考依据。     

槲皮素对SI甘蓝授粉引起的PK活性及相关指标的影响

通过槲皮素处理,研究了自交不亲和甘蓝自花授粉和异花授粉与蛋白激酶的关系。结果表明,槲皮素能明显抑制自交不亲和甘蓝自花授粉引起的蛋白激酶活性,导致蛋白质磷酸化明显受阻,进而促进自花诱导的花粉萌发和花粉管生长,同时使SOD、POD、CAT活性升高,Ca2+含量下降,Ca2+在胞内的分布明显改变,而槲皮素对异花授粉抑制作     

油茶SnRK2基因家族的克隆及表达分析

以油茶(Camellia oleifera)主栽品种‘华硕’为试验材料,通过RT-RCR的方法克隆出油茶蔗糖非酵解相关蛋白激酶(sucrose non-fermenting-1-related protein kinase 2, SnRK2)基因家族,命名为CoSnRK2.5,CoSnRK2.8。利用不同的在线网站对CoSnRK2基因家族进行生物信息学分析,通过实时荧光定量PCR对CoSnRK2基因家族的表达模式进行研究分析。结果表明,CoSnRK2基因家族的CDS长度为1 035～1 092 bp,编码344～363个氨基酸,具有SnRK2基因家族2个特有的结构域:相对保守的N端结构域和特异性明显的C端结构域。实时荧光定量PCR结果表明,CoSnRK2.5基因在异交(‘华硕’ב华鑫’) 72 h花柱中的表达量明显高于自交,自交12 h子房中的表达量远高于异交;CoSnRK2.8基因在自交24 h花柱中的表达量远高于异交,异交72 h花柱中的表达量远高于自交;异交84 h子房中的表达量远高于自交。本研究对进一步挖掘该基因家族在油茶后期自交不亲和过程中发挥的作用提供研究依据,对油茶自交不亲和分子机制解析具有重要的意义。     

自交亲和与自交不亲和日本梨中S4基因的研究进展

综述了近20年日本梨中与自交亲和和自交不亲和有关的S4基因的研究进展。这些结果包括：“二十世纪”(基因型为S2S4)为自交不亲和品种,其突变品种“奥嗄二十世纪”(基因型 为S2S4SM,SM=stylar part mutant;花柱部分突变)为自交亲和型。“奥嗄二十世纪”自交亲和的原因有三点：(1)S4SM基因被删除或者是低水平表达。(2)S4SM基因仅在柱头表达。(3)S4SM基因表达较迟。这主要是由于“二十世纪”S基因座上的S4等位基因发生了高度突变成为S4SM基因或者S4等位基因被删除,导致其基因产物花柱中的S4-RNase受到高度抑制或缺乏的缘故。S4基因由997bp组成,其中85~768bp为684bp的阅读框,925~931bp和943~950bp为多聚腺苷酸信号。S4基因的表达产物为S4蛋白,S4蛋白由228个氨基酸残基组成,其中N端的27个氨基酸残基为信号肽,成熟的S4蛋白由201个氨基酸残基组成。S4蛋白的生理功能是抑制花粉的萌发和花粉管的伸长。     

玉米BEL1-like基因家族的鉴定、表达和调控分析

BEL1-like(BELL)家族蛋白是植物中普遍存在的一类具有同源异型结构域的转录因子。拟南芥中BELL家族蛋白能与KNOTTED1-like蛋白互作形成异源二聚体,并结合到特异顺式作用元件来调控基因的表达,从而影响植物生长发育进程。本文采用隐马可夫(HMM)模型,在玉米基因组中鉴定到15个BELL家族基因(Zm BELL),分布于7条玉米染色体。通过与拟南芥BELL基因的序列比较将这些基因分为两大类。在玉米8种组织中Zm BELL有不同的表达模式,具有明显的组织表达特异性。基于基因共表达分析及BELL-like蛋白特异结合的顺式元件分析,预测到86个可能受Zm BELL调控的下游靶标基因。这86个基因和12个Zm BELL表达模式相同,并且在基因启动子区存在与BEL1-like蛋白结合的顺式元件。这些结果为进一步解析玉米BELL家族基因的功能和作用机制积累了有价值的资料。     

玉米SSⅡb蛋白多克隆抗体的制备及其应用

抗体是蛋白质功能研究的重要试剂。玉米蛋白的功能研究因缺乏相应的抗体而难以开展。玉米淀粉合酶Ⅱb与淀粉合酶Ⅱa均属于淀粉合酶,其主要负责支链淀粉的延伸。但对淀粉合酶Ⅱb的报道较少,本研究以原核表达的淀粉合酶Ⅱb的C端(455～704 aa) GST融合蛋白为抗原,免疫新西兰大白兔,分离血清制备SSⅡb多克隆抗体。Western blot试验发现, SSⅡb多克隆抗体不但能识别SSⅡb-C (455～704 aa)抗原,而且能识别玉米不同发育阶段胚乳中SSⅡb蛋白。利用该多克隆抗体的Western blot试验表明,玉米SSⅡb蛋白的表达在授粉后10 d到30 d的胚乳中表达模式是先增后降,授粉后15 d表达量最高,而半定量RT-PCR的结果表明授粉后20 d SSⅡb的转录水平最高。这些结果表明成功制备了特异的玉米SSⅡb的多克隆抗体,并能在Western blot中特异识别玉米内源SSⅡb蛋白,为进一步深入研究SSⅡb蛋白功能奠定基础。     

陕农138小孢子发育过程中蛋白表达差异的比较分析

为了寻找小麦单核中晚期愈伤组织诱导率较高的原因,探讨小麦花药发育的蛋白质代谢机制,本试验利用双向电泳技术对陕农138小麦小孢子单核中晚期和双核期的全蛋白分析表明,在等电点4~7之间可识别约450个以上清晰的蛋白质点,检测到差异点26个,对其中14个质量较好、重复性较高的差异表达的蛋白质点采用基质辅助激光解吸分离飞行时间质谱(MALDI-TOF-MS)进行肽指纹图谱分析,并利用Mascot软件在NCBInr数据库搜索,鉴定出11个蛋白质点,另3个蛋白质点未得到有意义的鉴定,11个蛋白质点分别被鉴定为UDP-葡萄糖焦磷酸化酶(1个蛋白点)、叶绿素抗体结合蛋白(1个蛋白点)、pentatricopeptide重复蛋白PPR566-6 (1个蛋白点)、半胱氨酸蛋白酶抑制剂(1个蛋白点)、泛素(1个蛋白点)、S-腺苷-L-半胱氨酸水解酶(2个蛋白点)、放氧增强蛋白(2个蛋白点)和假定蛋白(2个蛋白点)。这些蛋白质点的功能涉及到糖代谢、蛋白质降解、细胞防卫等代谢过程。     

玉米C型细胞质雄性不育系C48-2及其保持系线粒体差异蛋白分析

采用固相pH梯度-SDS PAGE双向电泳, 对玉米C型细胞质雄性不育系(C48-2)及其保持系(N48-2)单核期花药线粒体蛋白质进行了分离, 经考马斯亮蓝染色, 得到重复性较好的双向电泳图谱。PDQUEST软件可识别约260个蛋白质点, 对其中10个重复性比较好且差异表达在2倍以上的蛋白质点, 采用基质辅助激光解析分离飞行时间质谱进行肽指纹图谱分析, 然后用Mascot软件对NCBI数据库搜索, 其中2个蛋白质点被鉴定为谷氨酸脱氢酶(GDH)和依赖电压阴离子通道蛋白-1(VDAC1)。GDH的高表达会影响正常的氮代谢, 导致细胞的能量供应发生障碍, 有可能导致雄性不育; VDAC1是线粒体外膜上控制细胞通透性的蛋白, 与植物的程序性死亡密切相关。     

Genotype × environment interactions in populations possessing <Emphasis Type="Italic">Ga1</Emphasis>-<Emphasis Type="Italic">s</Emphasis> and <Emphasis Type="Italic">ga1</Emphasis> alleles for cross incompatibility in maize

Use of cross incompatibility in corn (Zea mays L.) by the Ga1-s allele may reduce cross-fertilization in specialty and conventional organic corn with pollen from genetically-modified (GM) corn. For effective use, information about environment, and genotype × environment effects on cross-fertilization by ga1 as well as heritability of cross incompatibility in maize is necessary. Our objective was to obtain this information. Four population pairs differing in their genotype at ga1 were evaluated for cross incompatibility with ga1 pollen in different environments. Populations were derived by crossing the recurrent parents B116, PHG35, ARZM16035:S19, and (CHZM05015:Mo17)Mo17 to Ga1-s donor parent Mo508W/Mo506W. Two replicates of each treatment were grown in the center of 952 m² fields planted with purple corn as an adventitious source of ga1/ga1 pollen. Open pollination was allowed and the amount of cross-fertilization estimated by averaging the percentage of purple seeds. Environment and genotype × environment effects were not significant. Contrasts to evaluate differences in cross-fertilization between Ga1-s and ga1 populations revealed that mean percentages of cross-fertilization in Ga1-s populations of B116, ARZM16035:S19, and (CHZM05015:Mo17)Mo17 were significantly lower than in ga1 populations. The estimated broad-sense heritability on an entry-mean basis for cross incompatibility was 0.81. Results suggest differences in genotype at ga1 played a major role in cross-fertilization of populations differing in their genotype at the ga1 locus. Incompatibility may be selected effectively over different environments and the Ga1-s system may be of value to reduce cross-fertilization with GM corn pollen.     

Crossability of American tree peony ‘High Noon’ as seed parent with Japanese cultivars to breed superior cultivars

The tree peony (Paeonia suffruticosa Andr.) is a valuable ornamental plant. American and French cultivars have desirable flower colors, while, Japanese cultivars have larger flowers with better presentation. We hybridized an American tree peony cultivar ‘High Noon (HN)’ (the seed parent) with 57 different Japanese cultivars (pollen parents) to investigate cross compatibility, with the ultimate goal of obtaining improved hybrids. Of the 1,927 crosses performed, 135 (38 cross combinations) yielded a total of 181 seeds (86 mature) and 22 seedlings. Five of the hybrids have already flowered and exhibited their parent character with large yellow flowers. To investigate the causes of cross incompatibility, we examined pollen tube growth in a cross between ‘HN’ and a Japanese cultivar. The result indicated that there are three most important causes of incompatibility, namely, abnormal pollen tube growth, the failure of fertilized ovules, and poor seed germination. Although cross compatibility in each combination was low, crosses between ‘HN’ and Japanese cultivars might be successful with the right paternal plants. In this study, 13 Japanese cultivars had higher cross compatibility with ‘HN’ than the others. Our results will enhance tree peony breeding and guide the selection of parents for hybridization.     

Inheritance of Maysin Content in Silks of Maize Inbreds Resistant to the Corn Earworm

Genetic information is needed on maysin, a compound in maize, Zea mays L., silks that is antibiotic to corn earworm larvae, Helicoverpa (formerly Heliothis) zea (Boddie), to assist in the process of breeding for resistance to this pest. This study was initiated to elucidate the inheritance of maysin content in the silks of a cross between two maize inbreds (GT114 and GT119) having resistance to the corn earworm. Reversed phase high performance liquid chromatography (HPLC) procedures were used to quantify the maysin concentration of silks from individual plants in the parent, F₁, F₂, and first backcross generations of the cross GT114 × GT119. A model assuming dominance at a single locus for low maysin content which is expressed only when a dominant modifier is present was proposed to explain the segregation ratios obtained in the F₂ and first back-crosses of GT114 × GT119. This model also provided a fit for maysin data previously generated from a cross between inbreds F6 and F44. Observations of selfed families in the backcross of GT114 × GT119 to the low maysin parent confirmed the two-locus model (one factor with dominance for low maysin expressed only when a modifier gene is also present) as a plausible explanation for inheritance of silk maysin in the cross. The process of selection for high maysin will be complicated somewhat by the presence of a gene which modifies the expression of dominance unless a method of easily identifying the gene, independent of maysin content, can be developed.     

Pollen–pistil interaction in the inter-specific crosses of Sesamum sp

The wild species of Sesamum have long been recognized as an important source of many valuable pest and disease resistance genes and other novel traits such as male sterility and plant type for the cultivated taxon. Utilization of these species in crop improvement is marginal because of their inherent low crossability with the cultigen. An understanding of the biological nature of incompatibility systems that impede hybridization offers tools for successful introgressions into cultivated sesame. The objective of this investigation was to observe pollen germination and pollen tube growth in cross pollinated S. indicum pistils as an indicator of pre-zygotic barriers operating in wide crosses involving S. indicum. Three wild species: S. alatum, S. radiatum and S. laciniatum were used as pollen donors in hybridization with S. indicum. In the cross S. indicum × S. alatum 85.23% of pollen grains germinated and pollen tubes reached ovaries within 8 h after pollination. In other two crosses, S. indicum × S. laciniatum and S. indicum × S. radiatum the level of incompatibility was high as evidenced by a reduced pollen germination and slow rate of pollen tube growth. Measures to circumvent incompatibility in these crosses to produce interspecific hybrids are discussed.     

Pollen pistil interactions and interspecific incompatibility in Pennisetum

Summary The causes of low crossability between Pennisetum species were studied. In the compatible cross P. typhoides x P. violaceum, the pollen tube growth was normal and there was good seed set. In P. typhoides x P. cenchroides, pollen tubes reached the ovary but no seed set was obtained indicating ovarian incompatibility. The stylar incompatibility was found in the crosses of P. typhoides with P. schweinfurthii, P. hohenackeri, P. orientale and P. squamulatum. In crosses P. typhoides x P. polystachyon and P. typhoides x P. pedicellatum, mostly pollen tubes did not grow beyond stigmatic surface and only few reached the style. Stigmatic as well as stylar incompatibilities seem to be the reasons for no seed set in these crosses.     

弱光胁迫对不同基因型玉米雌雄花发育和授粉结实能力的影响

以不耐阴型玉米豫玉22和耐阴型玉米郑单958为试验材料,设置自然光照和弱光胁迫两个处理,研究弱光胁迫对不同基因型玉米雌雄花发育的影响。研究结果表明,弱光胁迫延缓了雌雄穗的生长发育,表现为抽雄、吐丝和盛花期延迟,花期和雌雄间隔期延长。弱光胁迫下,在吐丝期不耐阴型玉米花粉量比对照显著增加,耐阴型玉米花粉量与对照差异较小;不耐阴型玉米花粉表面网纹在弱光胁迫下变粗且间隙增大,花粉萌发孔及其附近严重畸形,有的明显内陷,花粉内淀粉粒数目显著减少,营养供应能力减弱,耐阴型玉米在弱光胁迫下花粉表面网纹略有加粗或没有变化,萌发孔略微凹陷且程度远低于不耐阴型玉米,花粉中淀粉粒密度略有降低。遮光处理后,不耐阴型玉米的花粉活力、花粉萌发率和萌发速率表现为下降,而耐阴型玉米表现为上升。花丝的生长速率及伸出苞叶数量在弱光胁迫下显著下降,果穗上部花丝受到的影响最大,向下逐渐减弱。弱光胁迫下两个基因型玉米穗长和穗粒数减少,不耐阴型玉米受弱光胁迫影响的程度高于耐阴型玉米。雌雄间隔期延长、营养供应能力减弱导致的花粉畸形、花丝生长速率和可授粉花丝数目的减少以及籽粒IAA含量的下降和ABA含量的增加是弱光条件下玉米穗粒数显著减少的主要原因。     

玉米与摩擦禾、薏苡的杂交不亲和性

采用荧光显微技术, 对摩擦禾、薏苡花粉在玉米柱头上的萌发和生长过程进行了观察。摩擦禾花粉粒在玉米柱头上均能萌发, 花粉管在柱头中伸长并到达花柱基部, 且可将雄配子送入胚囊内, 玉米果穗顶端有受精结实痕迹, 说明摩擦禾与玉米的杂交障碍不是杂交不亲和, 而是胚囊不亲和或杂种衰亡。薏苡花粉粒在玉米柱头也能萌发, 花粉管能伸入花柱, 但玉米与薏苡杂交生殖隔离较摩擦禾严格, 杂交极其困难, 杂交障碍为胚囊不亲和或花柱不亲和。玉米与薏苡杂交时, 薏米花粉管能到达玉米花柱基部, 而川谷花粉管却在花柱中停止生长, 杂交障碍与薏苡种类有关。玉米与薏苡杂交的花粉管异常率高于玉米与摩擦禾杂交花粉管异常率, 反映了玉米与摩擦禾的亲缘关系较与薏苡近。     

Investigation on pre-zygotic barriers in the interspecific crosses involving Gossypium barbadense and four diploid wild species

Interspecific hybridisation has played significant role in the improvement of economically useful traits such as productivity, earliness, fibre quality and resistance to pests and diseases in cotton. However, wide crosses are often limited by the operation of either pre or/and post-fertilization barriers. The present study on pollen tube behaviour of four diploid wild species viz., G. triphyllum, G. davidsonii, G. thurberi and G. armourianum in the pistils of G. barbadense was taken up to determine the extent of pre-zygotic barriers operating in these crosses. High rate of pollen germination (∼80%) and normal growth of pollen tubes were observed upon selfing of both G. barbadense and four wild species. Pollen tubes reached the pistils and fertilization was accomplished within 8 HAP in all the five parental species. In the interspecific crosses, pollen germination was normal in the cross involving G. armourianum and drastically inhibited in the crosses involving G. triphyllum, G. davidsonii and G. thurberi. Even though delayed pollen tube growth was a general feature in all the four crosses, successful fertilization was observed only in the cross involving G. armourianum. In crosses involving G. triphyllum, G. davidsonii and G. thurberi, pollen tubes failed to reach the ovary even at 24 HAP due to the presence of strong stylar and ovarian incompatibility. Abnormalities in pollen tubes such as swelling, twisting, intense callose plug formation, breakage, branching and reverse orientation were frequently observed among the incompatible crosses.     

Prezygotic interspecific hybridization barriers between kenaf (Hibiscus cannabinus L.) and four wild relatives

Use of interspecific hybridization in genetic improvement of kenaf (Hibiscus cannabinus), a multipurpose crop, is limited by low cross‐compatibility. Previous hybridization studies suggest that part of incompatibility barrier is prezygotic. Pollen germination, stigma receptivity and growth of alien pollen on kenaf pistil were studied using wild relatives of kenaf. Four wild and semi‐domesticated species, H. surattensis, H. acetosella, H. radiatus and H. vitifolius, were used as pollen donors in the present experiment. H. radiatus exhibited highest pollen germination (65.89%) on H. cannabinus stigma. For closely related species (H. surattensis, H. acetosella, H. radiatus), stylar incompatibility was noticed, while for distantly related species H. vitifolius, the incompatibility was stigmatic. Number of pollen tubes entering ovule of H. cannabinus pistil was higher for H. radiatus (1.30). Alien pollen growth and penetration of pollen tube through ovule were higher in species sharing same genome of H. cannabinus. High callose deposition was observed in incompatible crosses, suggesting a general mechanism for prezygotic incompatibility in Hibiscus section Furcaria. Callose deposition in incompatible crosses increased with time.     

Effect of humidity on intergeneric pollinations of iap (Inhibition of Alien Pollen) sorghum [Sorghum bicolor (L.) Moench]

The production of interspecific or intergeneric progeny involving Sorghum spp. is greatly enhanced by the presence of the iap (Inhibition of Alien Pollen) allele in sorghum [Sorghum bicolor (L.) Moench]. Variation in the expressivity of the iap phenotype has been observed in previous studies, leading to the hypothesis that the humidity during pollination is affecting the degree of alien pollen adhesion and pollen tube growth. The objective of this study was to determine the optimum humidity for maximum maize pollen tube growth on sorghum florets homozygous for iap. Growth chambers were used to create environments of 45, 65, and 85 % humidity with reciprocal crosses made between the 65 and 85 % humidity chambers. Maize pollen was dusted onto the stigmas of Tx3361 (iap iap) and ATx623 (Iap Iap). Pollen adhesion and germination were higher for pollinations with Tx3361 at all humidity levels but the greatest adhesion and germination was observed at 45 % humidity for Tx3361. Under this humidity, pollen tube growth to the style and ovary was observed. The results indicate that lower humidity maximizes maize pollen adhesion and germination on the stigmas of Tx3361. Analysis of the differences in adhesion between Tx3361 and ATx623 indicate that Iap affects pollen adhesion at the stigma surface and may be controlling pollen–pistil incompatibility in interspecific crosses in sorghum.