甘蓝型油菜BnCYP79B1基因的克隆与表达

硫苷是十字花科植物的一种次生代谢产物,其合成途径受细胞色素P450的CYP79家族蛋白的调控,该实验采用同源克隆技术在甘蓝型油菜中克隆到了CYP79B1基因,命名为BnCYP79B1(GenBank登录号为JX535391.1)。BnCYP79B1基因cDNA全长1 625bp,编码一个含有541个氨基酸、理论等电点为8.88。序列对比结果显示,BnCYP79B1与花椰菜CYP79B1在DNA序列上的相似性为98.83%,推测蛋白氨基酸序列的相似性为99.26%。通过不同时期不同部位BnCYP79B1基因表达量的分析,发现BnCYP79B1基因在高秆高硫苷品系的根中表达量较高,而对矮秆高硫苷品系则是叶中表达量较高。在BnCYP79B1表达总量上,高秆品系较矮秆品系高,高硫苷品系较低硫苷品系高。     

油菜矮秆突变WRKY转录因子cDNA克隆及表达分析

以甘蓝型油菜为材料,利用已建立的抑制性消减文库(SSH),采用RACE技术克隆到1个植物WRKY转录因子相关基因,命名为BnD11,其cDNA全长1034 bp,含有810 bp的完整开放阅读框,编码269个氨基酸。该基因编码的氨基酸序列与拟南芥WRKY40氨基酸序列相似性为79%,与拟南芥中编码WRKY-DNA结合蛋白40基因的氨基酸序列相似性达78%,与其它多种植物的WRKY转录因子的氨基酸序列也有较高的相似性。半定量RT-PCR对BnD11进行组织特异性表达分析显示:在正常生长条件下,BnD11在野生型和矮秆油菜的各个组织中均有表达,但在矮秆突变的根、茎、茎尖的相对表达量明显高于野生型。研究表明,BnD11功能区段具有很高的保守性,可能参与了油菜的茎秆发育。     

水母雪莲通气组织形成的相关基因SmLSD1克隆及表达

该研究以水母雪莲为实验材料,通过RT-PCR结合RACE技术克隆了通气组织形成相关基因SmLSD1(GenBank登录号为OL690334),并对该基因在不同胁迫下的表达量及编码蛋白结构进行测定分析。结果表明：(1)水母雪莲SmLSD1基因全长965 bp,包含537 bp的开放阅读框,编码178个氨基酸。(2)同源序列比对发现,水母雪莲SmLSD1蛋白与菊科植物牛蒡LSD1的氨基酸序列相似性最高,达到98.31%。(3)亚细胞定位显示SmLSD1基因主要在细胞核和细胞膜上表达;原核表达显示,SmLSD1基因编码氨基酸的分子量约为18 kD。(4)荧光定量分析显示,SmLSD1基因在根、茎、叶中均有表达,且在叶片中表达量最高;在低温、低氧及紫外胁迫下,SmLSD1基因的表达量下调。研究推测,SmLSD1基因在水母雪莲通气组织的形成以及对逆境胁迫的响应中发挥着重要作用。     

无芒隐子草CsPEAMT基因克隆及表达特性分析

以无芒隐子草(Cleistogenes songorica)干旱胁迫下的cDNA文库中磷酸乙醇胺N-甲基转移酶(phosphoethanolamine N-methyltransferase,PEAMT)基因的EST序列为基础,采用RACE方法克隆该基因编码区序列,该序列全长为2 104bp,开放读码框1 506bp,编码501个氨基酸。无芒隐子草PEAMT蛋白编码的氨基酸序列与多种植物的PEAMT氨基酸序列有较高相似性,其中与高粱SbPEAMT、玉米ZmPEAMT的蛋白序列相似性最高(93%),说明PEAMT基因在植物进化中非常保守。采用实时定量RT-PCR分析无芒隐子草幼苗在干旱过程中CsPEAMT基因的表达结果显示,干旱胁迫诱导CsPEAMT基因在根和叶中大量表达,且在干旱第8天时CsPEAMT基因在叶和根中表达量分别是未干旱对照的43.35倍和13.25倍,复水后CsPEAMT基因的表达量开始下调。研究表明CsPEAMT基因可能是无芒隐子草抗旱性相关的基因。     

紫花苜蓿赤霉素受体基因的克隆及表达分析

赤霉素受体(GID)是赤霉素信号转导途径的重要成员,直接影响着赤霉素对植物体效应的发挥。该研究利用同源克隆的方法,首次从紫花苜蓿中克隆得到1个赤霉素受体基因,命名为MsGID1b。序列分析发现,MsGID1b基因开放阅读框长度为1 053bp,编码350个氨基酸,推测其蛋白质分子量为39.839kD,是一个无信号肽和跨膜结构的亲水性蛋白。序列比对结果表明,MsGID1b基因与蒺藜苜蓿MtGID1b基因的核苷酸序列相似性为98%,氨基酸序列相似性为99%,且具有HSL家族典型的HGG和GXSXG保守结构域及GA、DELLA蛋白结合位点。荧光定量PCR分析表明,MsGID1b基因在紫花苜蓿各组织中的表达丰度依次为:根盛花初花茎叶荚果;经GA3、ABA、NaCl、PEG和黑暗诱导后该基因表达上调,尤其是在GA3诱导下,MsGID1b基因的表达量一直维持在较高水平,表明MsGID1b基因可能参与紫花苜蓿的抗逆调控。     

雷公藤鲨烯环氧酶基因克隆与表达分析

以雷公藤(Tripterygium wilfordii Hook.f.)发状根为试验材料,采用RT-PCR方法,克隆得到2个雷公藤鲨烯环氧酶编码基因,命名为TwSE1(GenBank登录号MG717395)和TwSE2(GenBank登录号MG717396)。序列分析表明,TwSE1和TwSE2的开放阅读框分别为1 578和1 584bp,分别编码525和527个氨基酸,2条序列相似性为76.18%,但N端序列不保守。实时荧光定量PCR检测雷公藤鲨烯环氧酶在不同组织部位的表达模式,以及雷公藤发状根受茉莉酸甲酯(MeJA)诱导后基因表达的结果表明,TwSE1、TwSE2基因在雷公藤根、茎、嫩叶、老叶、花中均有表达,TwSE1在花中表达丰度最高,在根中表达丰度最低;但TwSE2在花和嫩叶中表达量最高,在老叶中表达量最低,且TwSE2在各组织部位的表达量都低于TwSE1。雷公藤发状根经MeJA诱导后,TwSE1、TwSE2基因表达量均上升,都表现为表达量先上升后下降再上升的趋势,并于诱导后3h达到一个高水平表达,之后下降,在诱导12h后表达量又迅速上升;在相同诱导条件下,TwSE2基因表达水平的提高小于TwSE1基因。     

马铃薯糖转运蛋白基因的克隆及表达分析

植物SWEET基因家族是一类糖转运蛋白,在植物的生理活动和生长发育过程中发挥着重要功能。为了解马铃薯SWEET基因的相关信息,探究其在马铃薯不同组织以及在生物胁迫与非生物胁迫下的表达特性。该研究采用同源克隆技术从马铃薯‘青薯9号’中克隆了StSWEET5基因(GenBank登录号为MN295671),其CDS序列长度为717 bp,编码238个氨基酸。系统进化树分析结果表明,StSWEET5与番茄的氨基酸序列相似性最高(97.06%)。qRT-PCR分析表明:StSWEET5基因在马铃薯各组织(根、茎、叶、花、块茎、匍匐茎)中均有表达,且在花中的表达显著高于其他组织;糖胁迫下,StSWEET5基因在根、茎、叶中均有表达,尤其在根中的表达差异最为显著(P0.05)。在晚疫病菌(Phytophthora infestans)诱导后36 h时,表达量达到最高,随后急剧下调。推测StSWEET5基因参与了马铃薯糖胁迫以及响应了晚疫病诱导的过程。     

烟草FIE和MSI1基因的克隆及序列分析

采用电子克隆与实验克隆结合的方法获得了烟草胚乳发育相关基因NTFIE和NTMSI1的cDNA序列,序列号分别为EU375458和EU375459.序列分析结果表明,这两个cDNA序列均含有完整的开放读码框,分别编码370和424个氨基酸,含有保守的WD基序.氨基酸序列比对和系统发育分析结果显示,不同物种之间FIE和MSI1基因编码氨基酸序列同源性都较高.组织表达分析结果表明,这两个基因均具有一定程度的组织表达特异性,NTFIE cDNA基因在花中的表达量最多,但在根和茎中未检测到表达,而NTMSI1 cDNA基因只在离体培养的细胞和根中特异性表达.     

梭梭肌动蛋白基因HaACT1全长的克隆与表达分析

根据本实验已克隆得到的梭梭肌动蛋白基因Ha ACT1部分序列,通过5'RACE技术获得HaA CT1的cDNA全长序列。序列分析表明,该基因c DNA全长序列1 534 bp,其中5'UTR序列为75 bp,3'UTR序列为325 bp,开放阅读框序列为1 134 bp,编码376个氨基酸。该序列与Gen Bank中收录的其它植物Actin基因核苷酸序列的相似性均在84%以上,并且它们的氨基酸序列的相似性达95%以上,Gen Bank登录号为KM886609。根据得到的c DNA序列设计全长引物,进一步克隆了Ha ACT1的基因组DNA序列,由4个外显子和3个内含子组成。利用半定量和绝对荧光定量PCR对Ha ACT1的表达进行分析,发现该基因在梭梭的不同组织及各种非生物胁迫下均能稳定表达,适合作为梭梭中其它功能基因表达研究中的内参基因。     

滨梅2个肌动蛋白cDNA片段的克隆与表达

目的:克隆耐盐碱果树滨梅的肌动蛋白基因actin,为该物种优异性状基因的功能鉴定提供内参.方法:利用一对actin简并引物克隆滨梅actin cDNA片段,对其进行序列和表达分析.结果:得到2条743 bp的cDNA片段,两条核苷酸序列相似性为82％,命名为PmAct1和PmAct2并在GenBank登录(分别为JX855160和JX855161);序列比对发现2条actin片段氨基酸序列与其他植物同源性均在96％以上;根据不同果树Actin相似性构建进化树,表明2个滨梅actin明显分为两种类型,但均与蔷薇科果树亲缘关系较密切;半定量RT-PCR表达谱发现PmAct1可能为组成型表达类型,而PmAct2特异地在根和叶组织中表达量较高.结论:首次获得了2个滨梅actin基因,为该物种其他功能基因的挖掘和表达分析奠定了基础.     

The suppressive function of the rice DELLA protein SLR1 is dependent on its transcriptional activation activity

When the gibberellin (GA) receptor GIBBERELLIN INSENSITIVE DWARF 1 (GID1) binds to GA, GID1 interacts with DELLA proteins, repressors of GA signaling. This interaction inhibits the suppressive function of DELLA protein and thereby activates the GA response. However, how DELLA proteins exert their suppressive function and how GID1s inhibit suppressive function of DELLA proteins is unclear. By yeast one-hybrid experiments and transient expression of the N-terminal region of rice DELLA protein (SLR1) in rice callus, we established that the N-terminal DELLA/TVHYNP motif of SLR1 possesses transactivation activity. When SLR1 proteins with various deletions were over-expressed in rice, the severity of dwarfism correlated with the transactivation activity observed in yeast, indicating that SLR1 suppresses plant growth through transactivation activity. This activity was suppressed by the GA-dependent GID1-SLR1 interaction, which may explain why GA responses are induced in the presence of GA. The C-terminal GRAS domain of SLR1 also exhibits a suppressive function on plant growth, possibly by directly or indirectly interacting with the promoter region of target genes. Our results indicate that the N-terminal region of SLR1 has two roles in GA signaling: interaction with GID1 and transactivation activity.     

Computational insight into novel molecular recognition mechanism of different bioactive GAs and the Arabidopsis receptor GID1A

Gibberellin (GA) is an essential plant hormone and plays a significant role during the growth and development of the higher plants. The molecular recognition mode between GA and receptor Arabidopsis thaliana GIBBERELLIN INSENSITIVE DWARF1 A (AtGID1A) was investigated by molecular docking and dynamics simulations to clarify the selective perceived mechanism of different bioactive GA molecules to AtGID1A. The 6-COOH group of GA, especially its β configuration, was found to be an indispensable pharmacophore group for GA recognition and binding to AtGID1A. Not only does a strong salt bridge interaction between the 6β-COOH group of GA and Arg244 of AtGID1A play a very important role in the GA recognition of the receptor, but also an indirect water bridge interaction between the pharmacophore group 6β-COOH of GA and the residue Tyr322 of AtGID1A is essential for the GA binding to the receptor. The site-directed residues mutant modeling study on the receptor-binding pocket confirmed that the mutations of Arg244 and Tyr322 decreased the GA binding activity due to the disappearances of the salt bridge and the hydrogen bond interaction. The 3β-OH group of GA was well known to be necessary for the GA bioactivity due to its forming a unique hydrogen bond with Tyr127 of AtGID1A. In addition, the hydrophobic interaction between GA and AtGID1A was considered a necessary factor to lock the GA active conformation and stabilize the GA-GID1A complex structure. The novel molecular recognition mode will be beneficial in elucidating the GA regulation function on the growth and development of the higher plants.

高等植物赤霉素代谢及其信号转导通路

赤霉素是一类重要的植物激素,对植物的生长发育,如种子的萌发、茎的延展、叶片的生长、休眠芽的萌发以及植物的花和种子的发育等生理具有重要的调控作用。从1926年被发现至今,阐明了赤霉素代谢机理及调控机制,明确了赤霉素在植物体内的信号转导途径。本文综述了赤霉素的生物合成途径及其平衡的调节;赤霉素受体GID1、DELLA蛋白在赤霉素信号转导途径中的作用及相关研究;泛素介导的DELLA蛋白降解在赤霉素信号转导中的研究进展。     

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.     

能源植物小桐子赤霉素合成代谢及信号转导相关基因的鉴定及序列分析

赤霉素(gibberellin,GA)是一类非常重要的植物激素,在植物种子萌发、茎干伸长、叶片生长、腺毛发育、花粉成熟、开花诱导和果实成熟等生长发育过程中都发挥着重要的作用。GA在一年生草本植物中可以促进开花,而在大多数多年生木本植物中则抑制成花诱导。为了更好地研究赤霉素在木本油料能源植物小桐子(Jatropha curcas)开花调控方面的作用机理,我们对小桐子整个基因组中参与GA合成代谢和信号转导的全部基因进行了鉴定和序列分析。这些基因包括6个多基因家族编码的蛋白,即GA2氧化酶(GA2-oxidase,GA2ox)、GA3氧化酶(GA3-oxidase,GA3ox)、GA20氧化酶(GA20-oxidase,GA20ox)、GID1(GIBBERELLIN INSENSITIVE DWARF1)、DELLAs和F-box蛋白,以及2个单基因编码的蛋白,EL1(EARLY FLOWERING1)和SPY(SPINDLY)。采用拟南芥和水稻中已经鉴定的上述基因编码的蛋白序列在小桐子基因组序列数据库和本实验的小桐子转录组数据库中进行BLASTP分析,找到17个同源蛋白的全长序列,并将其与28个拟南芥的、16个水稻的、24个葡萄的和22个蓖麻的同源蛋白构建系统发育树进行比对分析。结果表明,小桐子中参与赤霉素合成代谢及信号转导的大多数基因与蓖麻和葡萄同源基因的相似度更高。     

Tall or short? Slender or thick? A plant strategy for regulating elongation growth of roots by low concentrations of gibberellin

Background

Since the plant hormone gibberellin (GA) was discovered as a fungal toxin that caused abnormal elongation of rice shoots, the physiological function of GA has mainly been investigated in relation to the regulation of plant height. However, an indispensable role for GA in root growth has been elucidated by using severely GA-depleted plants, either with a gene mutation in GA biosynthesis or which have been treated by an inhibitor of GA biosynthesis. The molecular sequence of GA signalling has also been studied to understand GA functions in root growth.

Scope

This review addresses research progress on the physiological functions of GA in root growth. Concentration-dependent stimulation of elongation growth by GA is important for the regulation of plant height and root length. Thus the endogenous level of GA and/or the GA sensitivity of shoots and roots plays a role in determining the shoot-to-root ratio of the plant body. Since the shoot-to-root ratio is an important parameter for agricultural production, control of GA production and GA sensitivity may provide a strategy for improving agricultural productivity. The sequence of GA signal transduction has recently been unveiled, and some component molecules are suggested as candidate in planta regulatory sites and as points for the artificial manipulation of GA-mediated growth control.

Conclusions

This paper reviews: (1) the breakthrough dose–response experiments that show that root growth is regulated by GA in a lower concentration range than is required for shoot growth; (2) research on the regulation of GA biosynthesis pathways that are known predominantly to control shoot growth; and (3) recent research on GA signalling pathways, including GA receptors, which have been suggested to participate in GA-mediated growth regulation. This provides useful information to suggest a possible strategy for the selective control of shoot and root growth, and to explain how GA plays a role in rosette and liana plants with tall or short, and slender or thick axial organs.     

Chemical screening and development of novel gibberellin mimics

Gibberellin (GA) plays versatile roles in the regulation of plant growth and development and therefore is widely used as a regulator in agriculture. We performed a chemical library screening and identified a chemical, named 67D, as a stimulator of seed germination that was suppressed by paclobutrazol (PAC), a GA biosynthesis inhibitor. In vitro binding assays indicated that 67D binds to the GID1 receptor. Further studies on the structure–activity relationship identified a chemical, named chemical 6, that strongly promoted seed germination suppressed by PAC. Chemical 6 was further confirmed to promote the degradation of RGA (for repressor of ga1-3), a DELLA protein, and suppress the expression levels of GA3ox1 in the same manner as GA does. 67D and its analogs are supposed to be agonists of GID1 and are expected to be utilized in agriculture and basic research as an alternative to GA.     

Understanding gibberellic acid signaling--are we there yet?

The phytohormone gibberellic acid (GA) controls important aspects of plant growth such as seed germination, elongation growth, and flowering. The key components of the GA signaling pathway have been identified over the past 10 years. The current view is that GA binds to a soluble GID1 receptor, which interacts with the DELLA repressor proteins in a GA-dependent manner and thereby induces DELLA protein degradation via the E3 ubiquitin ligase SCF(GID2/SLY1). GA-dependent growth responses can generally be correlated with and be explained by changes in DELLA repressor abundance, where the DELLA repressor exerts a growth restraint that is relieved upon its degradation. However, it is obvious that other mechanisms must exist that control the activity of this pathway. This review discusses recent advances in the understanding of GA signaling, of its homeostasis, and of its cross-talk with other signaling pathways.     

Molecular determinants that convert hormone sensitive lipase into gibberellin receptor

Gibberellins (GAs) are tetracyclic, diterpenoid plant hormones, essential for many developmental processes in higher plants. Plants perceive GA through a nuclear-localized GA receptor, GA INSENSITIVE DWARF1 (GID1). From sequence similarity, it is suggested that GID1 evolved from a hormone-sensitive lipase (HSL), and recent x-ray crystallography of the GA-GID1 complex has given insights into how GID1 recognizes GA. Analyses of the GA signaling pathway in several plant species further suggest that the GID1-mediated GA signaling pathway emerged in the vascular plant lineage and since then regulation of GA recognition specificity seems to have been fine tuned to strictly regulate the on-off GA signal.