紫花苜蓿UV-B光受体基因MsUVR8的克隆及功能研究北大核心CSCD

该研究采用RACE扩增技术克隆了一个紫花苜蓿UV-B光受体基因(MsUVR8),在生物信息学分析基础上,采用农杆菌介导法获得了该基因过表达愈伤组织,并对UV-B辐射处理后MsUVR8过表达愈伤组织及其野生型中的类黄酮、黄酮醇、花青素、过氧化氢(H_(2)O_(2))、超氧阴离子(O_(2)^(-·))含量以及UV-B信号通路相关基因的表达进行检测分析,以探讨MsUVR8基因的生物学功能,为揭示植物响应UV-B胁迫的分子机制奠定理论基础。结果表明:(1)成功克隆获得紫花苜蓿MsUVR8基因CDS序列834 bp,且MsUVR8与蒺藜苜蓿MtUVR8基因序列相似度高达95%以上;MsUVR8蛋白形成了不完整的β-折叠结构,系统发育分析显示其与鹰嘴豆属于同一分支。(2)对MsUVR8过表达系检测发现,紫花苜蓿MsUVR8过表达愈伤组织(UVR8-OE)中类黄酮含量较野生型愈伤组织(WT)明显升高,而且经UV-B辐射后的UVR8-OE类黄酮物质含量较WT进一步显著升高。(3)DPBA荧光标记实验发现,UV-B辐射大大促进了细胞中黄酮醇的合成,且UV-B辐射后的UVR8-OE中黄酮醇含量最高。(4)DAB和NBT染色显示,UV-B处理后WT中活性氧(H_(2)O_(2)和O_(2)^(-·))的积累增加,而在UV-B辐射处理与未处理的UVR8-OE中H_(2)O_(2)和O_(2)^(-·)的积累无明显差异,表明MsUVR8可增强植物组织细胞的抗氧化性能,并可降低UV-B胁迫引起的氧化损伤。(5)UV-B辐照后,WT中PAL、CHS和FLS表达被激活而显著提高,UVR8-OE中的4种基因表达均达到最大,且较其他3个处理组均显著增强。研究认为,紫花苜蓿MsUVR8被UV-B激活后,促进了类黄酮合成相关基因的表达,并激活了类黄酮合成关键酶的活性,从而提高了类黄酮物质的合成效率,增强了UV-B胁迫条件下植物愈伤组织的抗氧化能力。     

Early induced protein 1 (PrELIP1) and other photosynthetic, stress and epigenetic regulation genes are involved in Pinus radiata D. don UV-B radiation response

The continuous atmospheric and environmental deterioration is likely to increase, among others, the influx of ultraviolet B (UV-B) radiation. The plants have photoprotective responses, which are complex mechanisms involving different physiological responses, to avoid the damages caused by this radiation that may lead to plant death. We have studied the adaptive responses to UV-B in Pinus radiata, given the importance of this species in conifer forests and reforestation programs. We analyzed the photosynthetic activity, pigments content, and gene expression of candidate genes related to photosynthesis, stress and gene regulation in needles exposed to UV-B during a 96 h time course. The results reveal a clear increase of pigments under UV-B stress while photosynthetic activity decreased. The expression levels of the studied genes drastically changed after UV-B exposure, were stress related genes were upregulated while photosynthesis (RBCA and RBCS) and epigenetic regulation were downregulated (MSI1, CSDP2, SHM4). The novel gene PrELIP1, fully sequenced for this work, was upregulated and expressed mainly in the palisade parenchyma of needles. This gene has conserved domains related to the dissipation of the UV-B radiation that give to this protein a key role during photoprotection response of the needles in Pinus radiata.     

川草2号老芒麦（Elymus sibiricus L.）UV-B辐射敏感基因rbcL的克隆及其调控表达研究

rbcL是编码光合关键酶1,5-二磷酸核酮糖羧化酶（Rubisco）大亚基的基因。本文运用mRNA差异显示技术（DDRT-PCR）并通过5ˊRACE (Rapid Amplification of cDNA Ends) 从高原植物川草2号老芒麦（Elymus sibiricus L. cv. 'chuancao No.2'）中获得了受增强UV-B辐射抑制的rbcL基因,该基因全长cDNA为1.51kb,开放阅读框（ORF）长1.434kb,编码477个氨基酸。氨基酸序列与Elymus trachycaulus中的Rubisco大亚基具有97％的同源性、与Triticum aestivum和Hordeum comosum的Rubisco大亚基同源性均为 98％。Northern杂交分析表明,增强UV-B辐射后6h,rbcL基因表达受到强烈抑制,处理后60h,其表达几乎完全被抑制,表明即使是长期生长在高原地区、强UV-B辐射条件下的高原物种,在受到较强的UV-B辐射后,其rbcL基因的转录也会受到抑制。     

Molecular events following perception of ultraviolet-B radiation by plants

Exposure of plants to UV-B radiation (280–320 nm) results in changes in expression of a large number of genes. Before UV-B radiation or light of other wavelengths can give rise to a cellular response, it has to be perceived by some kind of receptor, and the information transduced via a signalling pathway to the target molecules, be it proteins in the cytoplasm or the genetic material in the nucleus. The perception of low levels of UV-B probably occurs via a UV-B photoreceptor followed by several different signalling pathways. These pathways include second messengers such as calcium, kinases and the catalytic formation of reactive oxygen species. High levels of UV-B, on the other hand, probably cause cellular damage and oxidative stress, thus activating a general stress signal transduction pathway which leads to a response similar to that which occurs after pathogen attack and other stresses. Some of the genes identified so far as being regulated by UV-B encode proteins involved in the biosynthesis of protective pigments, DNA repair and antioxidative enzymes, photosynthetic genes, cell cycle genes, and stress genes induced by other types of stimuli (i.e. pathogenesis-related proteins and senescence-induced genes). In the light of the information obtained on components necessary for UV-B-induced changes in gene expression, we propose in this mini-review a working model for UV-B perception and signal transduction. This model also takes into account dosage differences for the observations, which imply a separation into UV-B-specific and more general stress signal transduction.     

两个水稻GDP-甘露糖-3',5'-异构酶基因特征及表达模式的研究

GDP-甘露糖-3',5'-异构酶(GME)可以催化GDP-甘露糖转化为左旋GDP-半乳糖,该反应对于高等植物体内抗坏血酸的合成是非常重要的.但目前在分子水平上还没有对GME基因进行研究的报道.通过逆转录PCR(RT-RCR)技术从水稻成熟叶片中克隆到两个GME基因的cDNA序列,并与其他植物物种中的GMEs进行比对,结果显示,GME基因在所有植物物种中高度保守,尽管进化树分析表明单子叶植物GMEs和双子叶植物GMEs在进化上相互独立.同时,分析这两个水稻GME基因的剪切模式揭示了二者也存在高度相似性.采用半定量RT-PCR技术对两个GME基因在不同组织和不同胁迫条件下的表达模式进行研究表明,OsGME1基因在冷胁迫条件下表达水平上调,这和先前水稻冷胁迫蛋白质组学研究的结果是一致的.而OsGME2和OsGME1基因在用赤霉素处理条件下表达水平均下调,暗示赤霉素可能通过调节GME基因的表达来调控植物体内的抗坏血酸合成.     

Resistance-related physiological response of rice leaves to the compound stress of enhanced UV-B radiation and Magnaporthe oryzae

An enhanced UV-B radiation (5.0?kJ?m^?2) was supplied before, during, and after Magnaporthe oryzae infection. The effects of single and compound stress of the UV-B radiation and M. oryzae on the resistance physiology and gene expression of rice leaves were examined. Results revealed that UV-B radiation given before M. oryzae infection (UV-B?→?M.) significantly increased the pathogenesis-related proteins (PRs) activities of phenylalanine ammonialyase (PAL), lipoxygenase (LOX), chitinase (CHT), and β-1,3-glucanase, the resistance-related substances (flavonoids and total phenols) content, and resistance-related genes (OsPAL and OsCHT) expression, thereby improving the disease resistance of rice leaves. Simultaneous exposure to UV-B radiation and M. oryzae (UV-B/M.) significantly increased the OsLOX2 expression and the PRs activities. Exposure to UV-B radiation after M. oryzae infection (M.?→?UV-B) decreased the flavonoid content, did not improve the PRs activity, and increased OsLOX2 expression. Compound treatments of UV-B?→?M., UV-B/M., and M.?→?UV-B reduced the disease index by 62.3%, 40.2%, and 26.6%, respectively, indicating UV-B radiation inhibited the occurrence of M. oryzae disease, but its inhibitory effect weakened when it was provided after M. oryzae infection. Hence, rice responded to the compound stress of UV-B radiation and M. oryzae through a resistance-related physiological mechanism associated with the sequence of stress occurrence.     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Effects of supplemental UV-B radiation on primary photosynthetic carboxylating enzymes and soluble proteins in leaves of C3 and C4 crop plants

Effects of increased UV-B radiation on activities of primary photosynthetic carboxylating enzymes and on contents of soluble proteins were studied in soybean (Glycine max [L.] Merr. cv. Bragg), pea (Pisum sativum L. cv. Little Marvel), tomato (Lycopersicon esculentum L. cv. Rutgers), and sweet corn (Zea mays L. cv. Golden Cross Bantam). The purpose was to evaluate the responses of agronomic crops to increases in solar UV-B radiation. Plants were grown and exposed under greenhouse conditions for 6 h daily to supplemental UV-B radiation which was provided by Westinghouse FS-40 fluorescent sun lamps filtered with 0.127-mm film of cellulose acetate (UV-B treated) or Mylar S (Mylar control). Three UV-B levels were tested: 1.09 (treatment T₁), 1.36 (treatment T₂), and 1.83 (treatment T₃) UV-B_seu where 1 UV-B_seu equals 16.0 mW-m² weighted by EXP-[(λ-265)/21]². These UV-B levels corresponded to 6%,21%, and 36%, respectively, of decrease in stratospheric ozone content, based on the interpolations of UV-B irradiances at a solar elevation angle of 60°. Leaves of plants of soybean, pea, and tomato exposed to UV-B radiation were generally low in RuBP carboxylase activity. On a fresh weight basis, all three UV-B radiation levels significantly reduced the enzyme activity in soybean and pea, whereas tomato plants showed significant reduction in RuBP carboxylase activity only when exposed to 1.83 and 1.36 UV-B_seu. An apparent decrease in soluble proteins was observed in leaf extracts of soybean and pea plants exposed to 1.36 and 1.83 UV-B_seu whereas higher amounts of proteins were detected in leaves of tomato plants grown under UV-B radiation. Leaves of sweet corn plants grown under Mylar control were low in PEP carboxylase activity and proteins as compared with those of control plants receiving no supplemental UV and UV-B treatment. Activities of PEP carboxylase in crode extracts from leaves of sweet corn were significantly suppressed under 1.36 and 1.83 UV-B_seu as compared with the no UV control. Some stimulation of PEP carboxylase activity was observed in corn plants exposed to 1.09 UV-B_seu.     

Comparison of responses of bean, pea and rape plants to UV-B radiation in darkness and in light

Effect of UV-B radiation on leaves of bean, pea and rape plants was studied. UV-B radiation (11.2 kJ·m⁻²) induced more distinct reduction of the primary photosynthesis activity when applied in darkness than the same UV-B dose, extended in time, and applied with photosynthetic active radiation (PAR). The pea plants were more susceptible to UV-B in darkness, but in the presence of PAR their tolerance was higher. The CO₂ fixation in the bean and rape plants, exposed to UV-B was decreased, but for the pea plants it remained unchanged. The UV-B irradiation caused an increase in the content of ultraviolet-absorbing pigments. Additionally, the bean plants grown at UV-B increased the thickness of leaves, described as SLW.