水稻萌发期激素信号转导和谷胱甘肽代谢转录分析

【目的】利用转录组测序技术,探究水稻萌发过程中激素信号转导和细胞内部氧化还原平衡的调控机理,以期增加对萌发过程中复杂调控机制的理解,促进萌发期基因组转录调控网络的构建,并挖掘调控种子萌发的相关基因,为水稻直播稻新品种选育提供理论参考。【方法】利用萌发0、24和48 h的种子进行动态转录组测序分析,以差异倍数≥2、错误发现率≤0.05为阈值筛选差异基因,并利用Gene Ontology(GO)和KEGG Pathway数据库对萌发不同阶段的差异基因进行分析注释;同时利用实时荧光定量PCR对测序结果进行验证;最后运用String蛋白互作数据库以combined_score≥0.9为阈值分析差异基因的蛋白互作网络。【结果】在种子萌发前期鉴定到8719个差异基因,而在萌发后期仅鉴定到3480个。GO和KEGG富集结果均显示与激素信号转导相关的基因主要在萌发前期被诱导,特别是生长素信号转导途径中的GH3家族基因在萌发前期均受到显著诱导;而谷胱甘肽代谢途径中的基因在萌发后期转录更为活跃,其中谷胱甘肽-S-转移酶基因富集最多。此外,两个异柠檬酸脱氢酶基因在萌发过程中被显著诱导,经蛋白互作预测发现两个异柠檬酸脱氢酶基因与GH3家族基因可能存在相互作用。【结论】在种子萌发前期,生长素信号转导途径中的GH3家族基因可能在减弱生长素信号以及降低生长素活性方面发挥着重要作用,其高表达能降低生长素对种子的休眠作用,促进萌发启动;在种子萌发后期,谷胱甘肽代谢途径中的谷胱甘肽-S-转移酶基因可能在细胞抵抗氧化胁迫中发挥主要作用;此外,在整个萌发过程中,GH3和异柠檬酸脱氢酶家族基因的相互作用可能在实现激素转导途径和谷胱甘肽代谢途径的交互串联作用、共同调控种子萌发方面具有重要意义。     

Levels of Crotonaldehyde and 4-hydroxy-(E)-2-nonenal and Expression of Genes Encoding Carbonyl-Scavenging Enzyme at Critical Node During Rice Seed Aging

The critical node(CN) is an important stage during seed aging, which is related to effective genebank conservation. Previous studies have demonstrated that proteins undergo carbonylated modification at the CN in rice, indicating oxidative damage. However, the levels of reactive carbonyl species(RCS) and the associated scavenging system at the CN are largely unknown. In this study, we optimized methods for the extraction and analysis of RCS from dry rice embryos. In order to acquire seeds at the CN, rice seeds were subjected to natural conditions for 7, 9, 11 and 13 months, and the seed germination rates were reduced to 90%, 82%, 71% and 57%, respectively. We chose the stage with seed germination rate of 82% as the CN according to the rice seed vigor loss curve. The levels of crotonaldehyde and 4-hydroxy-(E)-2-nonenal(HNE) were significantly increased at the CN. In addition, genes encoding carbonyl-scavenging enzyme, including Os ALDHs and Os AKRs, were significantly down-regulated at the CN, and reductions in the expression of Os ALDH2-2, Os ALDH2-5, Os ALDH3-4, Os ALDH7, Os AKR1 and Os AKR2 in particular could be responsible for RCS accumulation. Thus, the accumulations of crotonaldehyde and HNE and down-regulation of genes encoding carbonyl-scavenging enzyme might be related to an accelerating loss of seed viability at the CN.     

引发提高水稻劣变种子发芽率的蛋白质组学分析

引发处理是目前简便有效的提升种子发芽率和整齐度的方法,但引发提高水稻劣变种子发芽能力的机理并不清楚。利用蛋白质组学方法,分析清水引发和藤茶提取物二氢杨梅素引发2种处理方法与对照的差异蛋白,初步揭示引发提升种子发芽能力的机制。分析双向电泳图谱差异获得2倍以上显著差异蛋白点79个,通过MALDI TOF/TOF MS质谱分析鉴定出74个蛋白,其中2种引发处理与未引发对照相比同步上调或下调的共有蛋白,即引发相关蛋白有57个。分析引发相关蛋白可知,绝大多数的逆境防御蛋白类、能量相关蛋白类以及蛋白质合成和目标类蛋白丰度在引发处理中显著增加,推测引发提高种子发芽率的原因与逆境记忆、能量代谢活动和蛋白质合成能力增强等密切相关。     

高温处理对不同状态水稻种子发芽率的影响

以2个早籼稻品种的超干种子、干种子、鲜种子、湿种子、露白种子为材料,研究了高温处理对不同状态水稻种子发芽率的影响。结果表明,水稻不同种子状态耐受高温能力存在极显著差异,从强到弱依次为超干种子干种子鲜种子湿种子露白种子;水温对种子发芽率的影响比气温更显著;处理温度超过一定范围,发芽率随着温度升高和时间延长极显著下降;不同品种高温处理后的种子发芽率表现相同规律趋势,但也存在极显著个体差异;种子水分制约种子耐受高温能力,种子水分含量越高,耐受高温能力越弱。     

转Pi9抗稻瘟病基因水稻株系的比较转录组分析

【目的】在转录水平上解析Pi9基因介导的稻瘟病抗性调控机理,为培育抗病水稻品种提供理论依据。【方法】向水稻品种日本晴(NPB)及其转Pi9抗稻瘟病基因株系(NPB/Pi9)接种稻瘟菌。分别于接种后0 h、12 h、24 h、36 h提取叶组织样品,选取12503个水稻基因定制基因芯片,进行水稻基因转录组分析,并通过qRT-PCR对部分差异表达基因进行验证。【结果】NPB/Pi9在接种后12 h、24 h和36 h的基因表达量分别与其接种0 h表达量比较,共检测到7754个差异表达基因;相应地,感病水稻NPB在以上时间点共检测到7385个差异表达基因;在接种后36 h,NPB/Pi9的差异表达基因数目显著多于NPB。比较NPB/Pi9和NPB相同时间点的基因表达量,共获得4065个差异表达基因,其中接种后36 h的差异表达基因显著多于接种后0 h、12 h或24 h。因此,NPB/Pi9的稻瘟病防御反应更强烈。对NPB/Pi9与NPB相同时间点的差异表达基因进行GO和KEGG分析,细胞外区域、植物对刺激应答、转录调控、氧化还原、离子结合、次生代谢和植物激素相关的GO分类在接种后呈显著富集,苯丙氨酸代谢、类黄酮生物合成和植物激素信号途径的KEGG通路在接种后显著富集。与效应分子触发的免疫反应(ETI)相关的水杨酸信号途径、几丁质酶,以及与病原相关分子模式触发的免疫反应(PTI)相关的胞外区域、对刺激的应答、木质素合成等,均在抗感水稻之间差异表达。而且PTI/ETI共有的WRKY转录因子、MAPK激酶、茉莉酸和乙烯信号途径等发生差异表达。综上所述,NPB/Pi9和NPB的差异表达模式与ETI和PTI相关,两者相互联系并在Pi9介导的稻瘟病抗性中发挥作用。【结论】与日本晴比较,抗病基因型NPB/Pi9对稻瘟病防御反应更强烈。转录因子、激酶、NBS-LRR基因、几丁质酶、水杨酸、茉莉酸和乙烯信号途径,以及植物次生代谢在Pi9介导的稻瘟病抗病反应中发挥重要作用。     

NADPH Oxidases Act as Key Enzyme on Germination and Seedling Growth in Barley (Hordeum vulgare L.)

Reactive oxygen species (ROS) play an important role in seed germination. Although hydrogen peroxide (H₂O₂), a type of ROS, enhances the germination rate of various plant seeds, little is known about the mechanism. NADPH oxidases catalyze the production of superoxide anion (O₂^-) that is one of the ROS and the enzymes regulate plant development. We, therefore, investigated the role of NADPH oxidases in seed germination and seedling growth in barley (Hordeum vulgare L.). The production of O₂^- was observed both in embryo and aleurone layers in barley seeds treated with distilled water (DW). However, it was suppressed in seeds treated with diphenylene iodonium (DPI) chloride, NADPH oxidase inhibitor. Moreover, DPI markedly delayed germination and remarkably suppressed α-amylase activity in barley seeds, indicating the importance of NADPH oxidases in germination of barley seeds. The gene expression and the enzyme activity of NADPH oxidases gradually increased after imbibition, and the enzyme activities were closely correlated with seedling growth after imbibition. Besides, DPI markedly suppressed the seedling growth. These results indicated that NADPH oxidases perform a crucial function in germination and seedling growth in barley. These facts clearly reveal that O₂^- produced by NADPH oxidases after imbibition regulates seed germination and seedling growth in barley.     

Genetic Control of Germination Ability under Cold Stress in Rice

An F₉ recombinant inbred lines (RIL) population, derived from a cross between IR28 (Oryza sativa L. spp. indica) and Daguandao (O. sativa L. spp. japonica), was used to construct a molecular linkage map and to identify germination ability including the traits of imbibition rate, germination rate, germination index, root length, shoot length and seed vigor at 14°C for 23 d. A composite interval mapping approach was applied to conduct genetic analysis for germination ability. The frequency distributions of the germination ability traits under the cold stress in the RIL population showed continuous segregation, suggesting they were quantitative traits controlled by several genes. A total of seven QTLs were identified on chromosomes 4, 6 and 9, including two for imbibition rate (qIR-6, qIR-9), one for germination rate (qGR-4), two for germination index (qGI-4-1, qGI-4-2) and two for root length (qRL-4-1, qRL-4-2). There were no detected QTLs controlling shoot length and seed vigor. The phenotypic variance explained by a single QTL ranged from 9.1% to 37.0%, and two major QTLs, qIR-6 and qGI-4-2, accounted for over 30% of the phenotypic variance. The expressions of QTLs were developmentally regulated and growth stage-specific. Most of the QTLs observed here were located in the regions similar to the QTLs for rice cold tolerance reported previously, indicating that these QTLs were reliable. However, qRL-4-2 is not reported before.     

脱落酸对水稻种子萌发的影响

以3种水稻种子培两优288、R402、LP04为材料,用不同浓度外源脱落酸(ABA)作浸种处理,研究其对水稻种子萌发的影响.结果表明:低浓度ABA(10mg/L)对水稻种子LP04的萌发具有促进作用,20～90mg/L的高浓度ABA对水稻种子的萌发具有明显的抑制作用.结合水稻种子萌发过程中可溶性糖含量、α-淀粉酶活性和内源植物激素等生理指标的检测结果表明,20～50mg/L的ABA能有效抑制水稻种子的萌发,可作为外源ABA在水稻种子储藏中使用的参考浓度.     

不同水势环境对大豆种子活力的影响

试验用7种浓度的PEG溶液来创造大豆种子吸水的不同水势环境。结果表明:种子在低渗透势溶液中缓慢吸水比在清水中快速吸水,其发芽势、发芽率、发芽指数和活力指数都明显提高,田间出苗率达极显著差异,同时提高幼苗干重、鲜重。大豆种子在-1.1860MPa的PEG溶液中吸水48小时,种子活力提高。     

水稻幼穗响应稻曲病菌毒素胁迫早期的转录组分析

【目的】由稻曲病菌引起的稻曲病不仅造成水稻减产,而且还会产生对动物和植物有毒的真菌毒素。探明水稻幼穗对稻曲病菌毒素胁迫响应的分子机制,可为发掘水稻抗稻曲病基因以及抗病分子育种开辟新的思路。【方法】用稻曲病菌毒素处理水稻幼穗,采用转录组测序技术对水稻幼穗进行转录组测序,以水稻9311基因组作为参考基因组进行对比,利用TPM法计算基因表达量,设定参数（差异倍数的绝对值不小于2,且q值不大于0.05）筛选差异表达基因。结合基因差异表达分析、富集功能分析,鉴定水稻响应胁迫的关键基因,并利用实时荧光定量PCR技术对差异表达基因进行验证。【结果】在稻曲病菌毒素胁迫12 h后,水稻幼穗出现2526个差异表达基因（DEG）;通过GO富集、KEGG代谢途经和KOG功能分析,将差异基因划分为GO功能下的64个条目、32个代谢途径和KOG功能下23个类别,包括淀粉和蔗糖代谢、苯丙类生物合成、碳代谢、糖酵解/糖异生、氨基糖和核苷酸糖代谢等生物学过程。DEG中有66个植物转录因子,分属7种植物转录因子家族,包括WRKY和Myb两大转录因子。分析二萜类生物合成与淀粉和蔗糖代谢途径相关基因发现,OsCPS2、OsKSL4和细胞色素P450等基因表达量上调,而淀粉酶、β-呋喃果糖苷酶和UDP-焦磷酸化酶等基因表达量下调,推测这些基因在水稻响应稻曲病菌毒素胁迫时发挥重要的作用。【结论】稻曲病菌毒素作为非生物胁迫因素对水稻幼穗具有毒性;通过干扰淀粉和蔗糖代谢等途径而影响种子营养物质的合成,降低水稻抵抗病原菌侵染水稻的能力。     

"920"处理对水稻eA系种子耐贮藏性研究

用不同尝试的“920”对含euil基因的水稻长穗颈不育系（eA系）与不含该基因的不育系（A系）进行处理,收获种子储藏6个月、9个月和12个月后分别测试发芽率的发芽势。结果表明,无论eA系和A系,其种子储藏时间不超过9个月时,发芽率、发芽势不发生显著变化;储藏时间达1a(12个月）时,个别浓度处理间或材料间开始出现显著差异。但喷施“920”不会eA系种子发芽率和发芽势,个别浓度处理还有提高发芽率和发芽势的趋势。     

Water Uptake by Seeds in Yellow-seeded Soybean (Glycine max (L.) Merrill) Cultivars with Contrasting Imbibition Behaviors

Abstract

The rate of water uptake by seeds is assumed to be an important factor affecting the susceptibility of seeds to flooding injury, but the traits which could contribute to restricting rapid imbibition by seeds remain undetermined in yellow-seeded soybeans. This study was conducted to determine the possible factors controlling the rate of imbibition in soybeans with yellow seed coats. The imbibition behavior of two yellow-seeded cultivars, Tamahomare and Tachinagaha, with different flooding susceptibilities was examined in relation to initial seed moisture content, the hydration location of the seed surface during imbibition, and the state of the seed coat. Low seed moisture increased the water uptake by Tamahomare intact seeds and even allowed substantially faster imbibition than occurred in its embryos, while in Tachinagaha seed moisture content had little influence on seed imbibition. This rapid imbibition by low-moisture Tamahomare seeds was not due to alterations in the permeability of the seed coat nor in water uptake by the embryo, but rather to increased movement of water along the abaxial sides of the cotyledons. The seed coat of Tamahomare loosened readily upon submergence whilst that of Tachinagaha rather tightly adhered to the embryo, suggesting that the seed coat of Tamahomare adheres very loosely to the embryo and may aid in conducting water laterally between the seed coat and embryo. The degree of adherence of the coat to the embryo and/or the ease with which the coat can be loosened upon hydration appear to play a crucial role in determining the rate of water uptake in yellow-seeded soybeans.