玉米温敏自交不亲和系的鉴定与生态学机制分析

在玉米繁殖过程中发现了一种生态自交不亲和材料HE 97,为了证明其不亲和类型及其生态学机制,通过2 a田间分期播种和花丝切片观察,对该材料的自交不亲和性进行了研究.结果表明,HE 97的自交不亲和性主要受温度影响,表现为高温自交亲和,低温自交不亲和,亲和性转换的敏感时期是雄穗的小花分化期/雌穗生长锥伸长期,临界温度为20～24℃.花粉在其相同基因型花丝上不能萌发可能是导致HE 97自交不亲和的主要原因.     

玉米温敏自交不亲和材料花丝SSH-cDNA文库的构建

为了解玉米温敏自交不亲和材料HE97的表达机制,构建了该材料自交亲和与不亲和阶段花丝的正向与反向抑制差减杂交文库,并对其中的219个正向克隆和52个反向克隆进行了测序分析,分别获得了49个和20个Unique ESTs,涉及到代谢、光合作用、信号转导、物质运输、蛋白质合成加工、防御反应、细胞的发育和衰老、基因表达等重要生命过程中的12种不同功能类型.通过电子克隆获得了包含1090 bp的烯脂酰辅酶A水合酶基因,并对其蛋白质功能进行了分析.     

A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants

Self-incompatibility, the rejection of self pollen, is the most widespread mechanism by which flowering plants prevent inbreeding. In Brassica, the S receptor kinase (SRK) has been implicated in the self-incompatibility response, but the molecular mechanisms involving SRK are unknown. One putative downstream effector for SRK is ARC1, a protein that binds to the SRK kinase domain. Here it is shown that suppression of ARC1 messenger RNA levels in the self-incompatible Brassica napus W1 line is correlated with a partial breakdown of self-incompatibility, resulting in seed production. This provides strong evidence that ARC1 is a positive effector of the Brassica self-incompatibility response.     

等电聚焦电泳法测定羽衣甘蓝自交不亲和性的研究

利用聚丙烯酰胺凝胶等电聚焦电泳法,研究了羽衣甘蓝强自交不亲和系、弱自交不亲和系与自交亲和系的柱头和花粉蛋白质谱带,并进行自交,以考察亲和指数。结果表明:强自交不亲和系的成熟柱头(S)和弱自交不亲和系的成熟柱头的蛋白谱带有明显差异;强自交不亲和系有特异谱带的出现,而弱自交不亲和系和自交亲和系柱头上没有特异谱带。对比自交不亲和系开花前2~3 d的柱头(S′)与成熟柱头(S)的蛋白质谱带发现了S特异蛋白质的准确位置,其等电点为8.5~9.1。此法可用于鉴定羽衣甘蓝的自交不亲和性。用同种方法处理花粉,没有发现自交不亲和系的特征谱带。     

Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A. lyrata

Transitions from cross-fertilizing to self-fertilizing mating systems have occurred frequently in natural and domesticated plant populations, but the underlying genetic causes are unknown. We show that gene transfer of the stigma receptor kinase SRK and its pollen-borne ligand SCR from one S-locus haplotype of the self-incompatible and cross-fertilizing Arabidopsis lyrata is sufficient to impart self-incompatibility phenotype in self-fertile Arabidopsis thaliana, which lacks functional orthologs of these genes. This successful complementation demonstrates that the signaling cascade leading to inhibition of self-related pollen was maintained in A. thaliana. Analysis of self-incompatibility will be facilitated by the tools available in this species.     

香水柠檬RHF2A的克隆与互作蛋白的筛选

【目的】以香水柠檬（Citrus limon (L.) Burm. F.）为研究对象,分析两个RHF2A的时空表达规律,利用酵母双杂交技术和双分子荧光互补试验（BiFC）筛选、验证其互作蛋白,为深入研究RHF2A在香水柠檬自交不亲和过程中的分子作用机制奠定基础。【方法】从前期转录组和泛素修饰组（未公开）筛选获得2个E3泛素连接酶RHF2A（RING-H2 Zinc Finger2A）基因RHF2A-1、RHF2A-2,并克隆其全长序列,通过生物信息学分析2个RHF2A的序列和蛋白质结构,预测其启动子顺式作用元件,构建35S-RHF2A-GFP融合蛋白表达载体用于亚细胞定位分析,采用实时荧光定量PCR分析两个RHF2A的时空表达模式,构建酵母双杂诱饵载体从香水柠檬酵母文库筛选互作蛋白。构建BiFC载体,在洋葱活体细胞内对目标蛋白进行互作验证。【结果】从香水柠檬克隆获得RHF2A-1、RHF2A-2,ORF全长分别为1 161和1 134 bp;NCBI结构域预测发现其具有Ring/U-box结构域。启动子分析发现其具有花粉特异性表达相关元件POLLEN1LELAT52、GTGANTG10。组织表达分析发现RHF2A-1在花粉特异性表达,RHF2A-2在叶中特异性表达;时空表达分析结果显示,RHF2A-1在自交柱头表达量从第1天开始升高,第3天达到峰值,是杂交柱头表达量的5倍以上。亚细胞定位显示RHF2A-1和RHF2A-2定位在细胞核。经过Uniprot网站预测其互作蛋白显示RHF2A能与KRP6、AT3G57370、UBA1、FBL17、SK11蛋白相互作用,推测其参与自交不亲和泛素化反应途径、配子体发育调控、花粉的生长发育等生物学过程。通过酵母双杂交技术筛选出72个克隆,对其测序并进行Blast比对后排除重复克隆,最终得到ABCF3等20个候选互作蛋白。经过一对一互作验证、双分子荧光互补实验确定RHF2A-1与ABCF3-2存在互作关系。【结论】RHF2A-1的时空表达规律与自交不亲和过程中花粉在雌蕊上的萌发规律相吻合;筛选得到柠檬授粉过程中直接影响花粉生长发育的互作候选蛋白,初步证明RHF2A-1在自交不亲和过程中具有重要作用。     

Recognition and rejection of self in plant reproduction

Plant self-incompatibility (SI) systems are unique among self/nonself recognition systems in being based on the recognition of self rather than nonself. SI in crucifer species is controlled by highly polymorphic and co-evolving genes linked in a complex. Self recognition is based on allele-specific interactions between stigma receptors and pollen ligands that result in the arrest of pollen tube development. Commonalities and differences between SI and other self/nonself discrimination systems are discussed.     

甘蓝型油菜自交不亲和系1010及其在杂种优势育种中的利用研究

本文报告了甘蓝型油莱(Brassica napus L.)自交不亲和系1010的选育经过和方法,分析和评价了该系的配合力、自交不亲和性的保持和繁殖体系等问题。     

大果黄花梨自交亲和性变异机制研究

梨品种大果黄花由黄花芽变而来.田间授粉试验表明,黄花梨自花授粉结实率仅为1.5%,属于梨自交不亲和性品种;而大果黄花自花授粉的结实率高达60.0%,属于自交亲和性品种;相互授粉时,黄花×大果黄花组合的坐果率达70.0%,为杂交亲和,但反交时坐果率为1.0%,表现为杂交不亲和.荧光显微镜观察这些组合授粉后花柱内花粉管生长情况,也得到相同的结果.这些结果表明,两品种雌蕊的自交不亲和性性状正常.进一步鉴定出了黄花和大果黄花是基于S-RNase基因的S-基因型,发现两者均含有S1-RNase和S2-RNase基因;而且两品种的这1对雌蕊S-RNase基因均特异性地在花柱中表达,表达量没有明显差异,表明大果黄花和黄花的雌蕊S-RNase基因并无差异.由此推断:大果黄花的自交亲和性突变,是由于花粉自交不亲和性功能丧失,从而表现出自花授粉能够结实.     

芸芥自交不亲和系S等位基因分析

[目的]在利用芸芥自交不亲和系途径开展育种研究的过程中,为了避免在人工试配组合时遇到双亲蕾期杂交结籽率较好,但在自然隔离条件下配制杂交种时出现双亲花期交配不亲和现象的发生。[方法]对11份不同芸芥材料进行了系内株间和系间的完全双列杂交研究。[结果]7-5-1-1、7-5-1-2、芸芥SI-1、和田芸芥I、87-86I、定西芸芥I、渭芸-7I、87-85I、9421I这9份材料系内株间异交和自交的亲和指数均小于1,表现为不亲和,说明它们为S等位基因纯合、自交不亲和性稳定的自交不亲和系,这9份材料中存在6种S等位基因型;06武芸3和马厂芸芥I这2份材料的自交不亲和性尚未稳定。[结论]在利用自交不亲和系途径培育一代杂种时,在农艺学性状和自交不亲和性基本稳定时,有必要进一步测定自交不亲和系S等位基因的纯合性和基因型,这一工作可以有效地指导育种实践。     

2D-DIGE技术研究自交不亲和杏品种‘新世纪’花柱表达蛋白

 【目的】从蛋白质组水平探索杏树自交不亲和的分子遗传机制。【方法】采用双向差异凝胶电泳和质谱检测技术,对自交不亲和杏品种‘新世纪’在小蕾期、大蕾期、自花授粉后24 h及异花授粉(‘新世纪’ב巴旦水杏’)后24 h的花柱进行差异性蛋白质组学研究。【结果】在‘新世纪’杏的小蕾期、大蕾期、自花授粉后24 h、异花授粉24 h后的花柱中共检测到约1 500个蛋白点,其中有66个差异表达的蛋白点。自交不亲和杏柱头对自体与异体花粉感应在蛋白质组水平有差异,其中异花授粉后花柱中特异表达的蛋白点1个,异花授粉后表达丰度明显升高的蛋白点4个,表达丰度明显降低的蛋白点29个;质谱分析只有13个蛋白质点得到归属鉴定。【结论】明确‘新世纪’杏花花柱对自体与异体花粉感应在蛋白质组水平上有差异。     

外源多胺对不同苹果品种自花授粉花粉管生长的影响

[目的]研究外源多胺处理苹果花柱后自花授粉花粉管生长情况,获得能提高不同苹果品种自交坐果率的外源多胺适宜浓度,为提高苹果自交亲和率提供科学依据.[方法]以红奥、早红香、嘎拉、斗南、富士、金冠和夏光7个苹果品种的花柱和花粉为试验材料,用花柱半离体方法研究外源多胺对苹果自花授粉花粉管生长的影响.[结果]适宜浓度的外源Spm、Spd和Put处理自交不亲和苹果花柱后,均能提高花柱伸出花粉管比率和每根花柱管伸出花粉管数,其中红奥苹果花柱最适宜的Spm浓度为0.100 mmol/L,其他自交不亲和品种均为0.005 mmol/L;除金冠和夏光苹果适宜的Put浓度为0.25 mmol/L外,其他自交不亲和品种为0.01 mmol/L;适宜红奥和富士苹果的Spd浓度为0.01 mmol/L,适宜斗南、金冠和嘎拉苹果的Spd浓度为0.50 mmol/L,适宜夏光苹果的Spd浓度为0.25 mmol/L.[结论]一定浓度的外源多胺能够提高自交不亲和苹果品种的自交坐果率.     

铁皮石斛自交和杂交亲和性的细胞学研究

为了探讨铁皮石斛Dendrobium officinale自交和杂交亲和性的生物学机制,利用荧光显微镜观察铁皮石斛自交、种内杂交后柱头性状和花粉管的生长情况。结果发现:自花授粉1 h后,花粉粒萌发,花粉管到达花柱基部接近子房处,4 h后花粉管进入子房,8 h后停止生长,72 h后子房萎缩,花粉管消失,不同的亲本间略有差别。种内杂交后的花粉管能够顺利进入子房,发育良好。由于铁皮石斛自交花粉管进入子房后停止生长,导致子房萎缩,可以认为铁皮石斛属于配子体自交不亲和。研究结果为铁皮石斛的自交不结实机理研究提供了细胞学基础,也为铁皮石斛种质资源保育、杂交育种等研究提供理论依据。     

樱桃自交不亲和S9-单元型特异的F-box基因克隆及其表达分析

【目的】克隆李属甜樱桃自交不亲和性花粉S-决定子基因，为今后果树配子体自交不亲和性机理研究奠定理论基础。【方法】根据GenBank登录的16个S-locus F-box同源基因保守区设计兼并引物，利用RT-PCR、RACE等手段，从甜樱桃品种红灯花粉cDNA中克隆到两个编码376-氨基酸多肽的全长基因。【结果】GenBank Blast分析显示,克隆的两个基因中一个基因编码的蛋白产物与数据库甜樱桃自交不亲和性S3-单元型特异的PaSFB3（AB096857）编码的氨基酸序列完全一致。另一个基因编码一新的PaSFB同源序列，其推测的氨基酸序列N-端同SFB3一样具有明显的F-box基序，与PaSFB1～6的一致性为76%～82%。研究显示该基因在花粉组织中特异性表达，并表现出S9-单元型特异的连锁信号。【结论】新基因为甜樱桃自交不亲和性花粉S-决定子候选基因PaSFB家族中一新成员，命名为PaSFB9 （GenBank登录号：DQ422809），红灯自交不亲和基因型确定为S3S9。     

A pollen factor linking inter- and intraspecific pollen rejection in tomato

Self-incompatibility (SI)--intraspecific pollen recognition systems that allow plants to avoid inbreeding--in the Solanaceae (the nightshade family) is controlled by a polymorphic S locus where "self" pollen is rejected on pistils with matching S alleles. In contrast, unilateral interspecific incompatibility (UI) prevents hybridization between related species, most commonly when the pollen donor is self-compatible (SC) and the recipient is SI. We observed that in Solanum, a pollen-expressed Cullin1 gene with high similarity to Petunia SI factors interacts genetically with a gene at or near the S locus to control UI. Cultivated tomato and related red- or orange-fruited species (all SC) exhibit the same loss-of-function mutation in this gene, whereas the green-fruited species (mostly SI) contain a functional allele; hence, similar biochemical mechanisms underlie the rejection of both "self" and interspecific pollen.     

The male determinant of self-incompatibility in Brassica

In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor.     

甘蓝型油菜自交不亲和系柱头和花粉粒不亲和互作的扫描电镜研究

用扫描电镜观察研究了甘蓝型油菜(Brassica napus L.)自交不亲和系柱头乳突细胞和花粉粒及其相互识别的形态以及用NaCl溶液处理后的效应。讨论了乳突细胞的结构,用NaCl溶液克服柱头自交不亲和性的理化机理以及花粉粒形态与授粉作用的关系等问题。     

低能Ar+注入对玉米花粉萌发及Ca2+浓度分布的影响

To investigate the effect of low energy ion implantation on maize pollen germination and cytosolic Ca2+ distribution during pollen germination process, the argon ion (Ar+) with energy of 30 keV, dose of 0.78 ×1015-13×1015 ion/cm2 was implanted into maize pollen by irradiation, and the germination of pollen and cytosolic Ca2+ distribution during pollen germination process of the Ar+ implanted pollen were studied. The results showed that when been irradiated with Ar+ with dose of 5.2×1015 ion/cm2, the germination rate of maize pollen increased remarkably, while implantation of ions with dose exceeding 5.2×1015 ion/cm2 sharply decreased the germination rate of maize pollen. Meanwhile, tracing of esterified Ca2+ fluorescence probe fluo-3 AM for intact pollen showed that variation of cytosolic Ca2+ concentration was consistent with the change of pollen fertility. The dynamics of cytosolic Ca2+ concentration caused by low energy ion implantation may be concluded as the initial effects of pollen germination.     

芸薹属自交不亲和功能因子及分子机制研究进展

芸薹属的自交不亲和性受S位点基因控制,其上的SRK基因和SCR/SP11基因是自交不亲和反应中的两个关键因子,另外所发现的其他因子也对自交不亲和发挥了重要作用。文章介绍了影响自交不亲和的功能因子及其分子结构特征和功能,对自交不亲和反应的分子机制作了进一步阐述,同时对这些功能基因在芸薹属植物甘蓝染色体组上的定位研究作出展望。     

植物自交不亲和机制研究进展

植物自交不亲和反应(self-incompatibility,SI)是开花植物防止自交退化,保持遗传多样性的一种常见的生殖隔离现象。自交不亲和的机制具有多样性及独立性,各个科之间的自交不亲和机制起源不同而且不亲和基因之间也不存在同源性。归纳总结了植物自交不亲和的几种分类情况,并按其分类对不同种类的自交不亲和机制分别进行了阐述。在此基础上,对植物自交不亲和领域研究的难点和方向进行了分析和展望。