缺铁胁迫对梨叶片中GA信号转导相关基因的影响

以不同程度缺铁的‘砀山酥梨’组培苗为实验材料,应用ELISA法测定叶片中内源GA含量,并依据NCBI上GA氧化酶GA2ox同源基因的保守序列,采用RACE技术克隆其基因全长,从梨基因组数据库中比对获得GA受体GID1的4个等位基因和DELLA蛋白的4个等位基因,通过实时RT-PCR分析GA2ox基因和GID1的4个等位基因和DELLA蛋白的4个等位基因的相对表达量,以探讨缺铁对梨叶片GA含量及其信号转导相关基因表达的影响。结果表明:(1)梨叶片中GA含量随着其缺铁程度的加重而增加。(2)克隆出梨叶片中GA2ox基因,其cDNA全长为1 014bp(GenBank登录号为KJ008976)。(3)GA2ox基因的表达量并未随梨缺铁程度增加而上升;GA受体GID1的4个等位基因相对表达量均随梨缺铁程度的加重而增加,其相对表达量与GA含量呈正相关关系;在DELLA蛋白的4个等位基因中,仅DELLA1相对表达量随着梨缺铁程度的加重而逐渐增加,说明DELLA1对缺铁胁迫最敏感。推测梨缺铁诱导了GA合成,但并没有促进活性GA向无活性GA转化。     

果树赤霉素代谢与信号途径研究进展

赤霉素作为5大植物激素之一,在果树的花芽分化、花序发育、开花坐果、果实的生长发育及植株的形态建成等方面扮演着重要的角色,但对果树赤霉素的分子生物学研究与其他大田作物相比差距较大。为了在果树生产中能更加合理有效地利用赤霉素调控果树花果发育,研究果树赤霉素的合成及其信号转导途径的分子调控机制十分必要。研究发现GA合成的关键酶KO、GA2ox及GA20ox的表达均与果树矮化呈负相关,而KS的含量则与植株高矮呈正相关,板栗雄性不育现象也与KO、KAO的表达量密切相关。GAMYB基因及LFY基因则在果树的成花诱导和雄蕊发育等生殖生长过程中发挥重要作用。DELLA蛋白在果树的GA信号途径中作为负调控因子可致使矮化植株形成,在果树的细胞周期循环过程、转录调控、花的形成、细胞的信号转导及许多生理过程中,DELLA蛋白泛素化降解均扮演着至关重要的角色。主要从果树赤霉素的合成及赤霉素的信号途径两大方面,着重对果树赤霉素合成过程中的关键酶基因及其定位、果树赤霉素信号途径的重要元件如赤霉素受体GID1、DELLA蛋白等进行了综述,以期为高效利用赤霉素调控果树生长发育提供重要的理论参考。     

DELLA 蛋白在植物生长发育中的作用

DELLA蛋白作为GA信号转导通路中起抑制作用的转录因子,是一类定位在核内的生长抑制蛋白,可以直接与植物体内关键转录因子的蛋白互作,进而在许多植物信号活动中发挥核心作用。该文对近年来国内外有关模式植物及果树、蔬菜、花卉、粮食作物等植物DELLA蛋白基因家族的鉴定、时空表达模式、蛋白结构、参与的GA信号转导机理、与光敏色素互作因子PIF及F box蛋白的互作及DELLA蛋白在植物种子萌发、形态建成、豆科植物根瘤菌共生、气孔关闭、植物抗逆反应等过程中的重要作用等方面的研究进展进行综述,并比较了DELLA蛋白基因家族在不同物种中的差异,对其今后的研究热点和方向进行了展望,为进一步探讨DELLA蛋白的功能提供信息。     

鸭梨PPO与GFP融合基因的烟草转化及亚细胞定位观察

将鸭梨PPO基因与绿色萤光蛋白GFP基因相融合共同进行遗传转化的方式,对鸭梨多酚氧化酶开展细胞定位研究。通过克隆该酶基因除终止密码子TAA外长度为1 779bp的CDS序列,与绿色荧光蛋白基因重组构建了荧光表达载体pBI121-PPO-GFP,借助农杆菌转化烟草,转基因烟草叶片细胞经激光扫描共聚焦显微镜观察,绿色荧光蛋白荧光与叶绿体自发荧光相重合。结果表明鸭梨多酚氧化酶为叶绿体蛋白质。     

巴西橡胶树赤霉素信号转导途径HbRGA1结构和功能分析

DELLA蛋白是赤霉素激素信号负调控因子,具有抑制植物生长发育的作用。解析其家族成员结构与功能将有助于揭示橡胶树DELLA蛋白家族成员调控橡胶树生长发育的机制。本研究从橡胶树热研73397叶片中克隆HbRGA1的cDNA全长序列。该基因长为2136 bp,含1839 bp的ORF,编码613个氨基酸。HbRGA1蛋白序列包含DELLA和GRAS保守结构域,与杨树、木薯和橡胶树DELLA基因相似性高达82.5%。qRT-PCR分析发现HbRGA1在橡胶树叶片中表达量高,在树皮和胶乳中表达量极低。叶片中HbRGA1表达量受喷施赤霉素和脱落酸等诱导显著上调。本研究表明HbRGA1与橡胶树赤霉素等激素信号密切相关,为深入研究其在橡胶树生长发育中的结构和功能打下良好基础。     

橡胶树DELLA蛋白编码基因HbGAI的克隆与表达分析

该研究采用RT-PCR与RACE技术,从橡胶树‘热研7-33-97’胶乳中克隆了1个DELLA蛋白编码基因HbGAI(GenBank登录号为KT696439)。HbGAI全长cDNA序列2 050bp,包含1个长1 842bp的完整开放阅读框。序列分析显示,HbGAI基因编码613个氨基酸,其推导的蛋白含有DELLA和GRAS结构域,分子量为66.476kD,理论等电点为5.19,无跨膜结构域,属于亲水性蛋白。进化树分析表明,HbGAI蛋白与其他植物中DELLA蛋白具有较高的相似性,与麻疯树JcGAI和蓖麻RcGAI亲缘关系较近。荧光定量PCR结果显示,割胶和茉莉酸甲酯处理下调胶乳中HbGAI基因的表达,乙烯利处理4h内显著上调胶乳中HbGAI基因的表达,表明HbGAI基因可能在橡胶树割胶、茉莉酸、乙烯响应中发挥作用。     

蒺藜苜蓿株高控制基因COSTIN的克隆及功能研究

株高是影响植物株型建成的重要农艺性状之一,直接决定作物的倒伏性和生物产量,但目前关于苜蓿等豆科牧草株高性状形成的分子调控机制尚不清楚。通过定向筛选豆科模式植物蒺藜苜蓿Tnt1逆转座子插入突变体库,分离鉴定了一个蒺藜苜蓿矮化突变体compact stalk internodes(costin),该突变体的矮化表型是由于茎节伸长受到抑制所致。通过基因表型连锁分析成功克隆了COSTIN基因,该基因编码一个钙离子交换蛋白,与拟南芥的CALCIUM EXCHANGER 7(CAX7) 基因高度同源。qRT-PCR检测发现COSTIN基因在茎、叶和果荚等组织中有较高的表达。进一步研究发现在costin突变体中赤霉素合成途径关键基因MtCPS、MtKAO1、MtGA20ox4、MtGA20ox7和MtGA3ox1表达下调;外施赤霉素GA3可以恢复costin突变体的矮化表型。上述研究表明COSTIN基因通过影响植物激素赤霉素的生物合成来调控蒺藜苜蓿的茎节伸长。     

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。     

葡萄VvRGL基因应答GA调控无核果实发育的研究

DELLA蛋白是GA信号传导途径的核心作用元件,属于GRAS核转录调节因子家族。该研究以‘魏可’葡萄品种为试材,通过基因克隆、启动子分析、染色体定位、基因蛋白结构及系统进化分析,鉴定VvRGL1和VvRGL2精确序列,预测其潜在功能,并采用qRT-PCR技术检测VvRGL基因应答GA在果皮、果肉及种子(区)的时空表达特征。结果表明:(1)VvRGL1及VvRGL2的染色体定位为Chr14和Chr7,开放阅读框(ORF)为2 007bp和1 815bp,编码氨基酸数量为668和604个;二者均含有GRAS保守结构域,但却不具备DELLA结构域,属于GRAS转录因子大家族成员,而不属于DELLA亚家族;基因结构分析表明,VvRGL1的DNA序列有1个内含子,2个外显子,而VvRGL2的DNA序列无内含子,有1个外显子,基因结构高度保守;进化分析表明,VvRGL1与拟南芥和柑桔的亲缘关系较近,而VvRGL2与草莓和杨树的亲缘关系较近。(2)2个基因的启动子均含有响应赤霉素和胚乳发育相关的作用元件,表明它们可能参与响应GA信号传导和种子胚乳发育过程。(3)qRT-PCR结果显示,外源GA处理均不同程度降低了2个基因在葡萄果皮和种子区的表达,但却上调了其在幼果期果肉中的表达,表明在果皮与种子(区)中,GA可能通过抑制葡萄VvRGL1/RGL2基因的表达参与调控葡萄无核果实的发育。本研究结果为进一步阐明VvRGL在赤霉素信号传导及葡萄无核果实发育机理中的作用机制提供重要依据。     

珍珠黄杨ITS-PCR体系的建立与优化

本研究利用改良CTAB法从珍珠黄杨叶片中提取基因组DNA作为模板,在TaqDNA聚合酶量不变的基础上,利用正交设计L_9(3~4)对4个因素(模板DNA、Mg~(2+)、dNTP和引物)在3个水平上对珍珠黄杨ITS-PCR反应体系进行优化.实验结果表明,在总体积50μL的反应体系中,建立了最佳ITS-PCR扩增条件:Mg~(2+)浓度2.0 mmol/L、引物浓度0.3 μmol/L、dNTP浓度0.3 mmol/L、DNA模板浓度240 ng/50μL、ToqDNA聚合酶的用量1.75 U/50μL和退火温度56℃,该优化体系保证了珍珠黄杨ITS-PCR产物的纯度和质量要求.珍珠黄杨ITS片段克隆测序后获得的序列长度为642 bp,其系统学信息将为珍珠黄杨的起源进化提供有力的分子水平证据.     

皱叶黄杨的一个新异名

根据对小叶黄杨(Buxus sinica (Rehder & Wilson)Cheng ex M.Cheng var.parvifolia M.Cheng)模式标本的研究, 并比较皱叶黄杨(Buxus rugulosa Hatusima)的标本, 而将小叶黄杨作为皱叶黄杨的新异名。     

Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1

This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein-protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.     

A novel dwarfing mutation in a green revolution gene from Brassica rapa

Mutations in the biosynthesis or signaling pathways of gibberellin (GA) can cause dwarfing phenotypes in plants, and the use of such mutations in plant breeding was a major factor in the success of the Green Revolution. DELLA proteins are GA signaling repressors whose functions are conserved in different plant species. Recent studies show that GA promotes stem growth by causing degradation of DELLA proteins via the ubiquitin-proteasome pathway. The most widely utilized dwarfing alleles in wheat (Triticum aestivum; e.g. Rht-B1b and Rht-D1b) encode GA-resistant forms of a DELLA protein that function as dominant and constitutively active repressors of stem growth. All of the previously identified dominant DELLA repressors from several plant species contain N-terminal mutations. Here we report on a novel dwarf mutant from Brassica rapa (Brrga1-d) that is caused by substitution of a conserved amino acid in the C-terminal domain of a DELLA protein. Brrga1-d, like N-terminal DELLA mutants, retains its repressor function and accumulates to high levels, even in the presence of GA. However, unlike wild-type and N-terminal DELLA mutants, Brrga1-d does not interact with a protein component required for degradation, suggesting that the mutated amino acid causes dwarfism by preventing an interaction needed for its degradation. This novel mutation confers nondeleterious dwarf phenotypes when transferred to Arabidopsis (Arabidopsis thaliana) and oilseed rape (Brassica napus), indicating its potential usefulness in other crop species.     

Overexpression of a GRAS protein lacking the DELLA domain confers altered gibberellin responses in rice

The rice SLR1 (SLENDER RICE 1) gene encodes a DELLA protein that belongs to a subfamily of the GRAS protein superfamily and that functions as a repressor of gibberellin (GA) signaling. Based on the constitutive GA response phenotype of slr1 mutants, SLR1 has been thought to be the sole DELLA-type protein suppressing GA signals in rice. However, in rice genome databases we identified two sequences homologous to SLR1: SLR1-like1 and -2 (SLRL1 and -2). SLRL1 and SLRL2 contain regions with high similarity to the C-terminal conserved domains in SLR1, but lack the N-terminal conserved region of the DELLA proteins. The expression of SLRL1 was positively regulated by GA at the mRNA level and occurred preferentially in reproductive organs, whereas SLRL2 was moderately expressed in mature leaf organs and was not affected by GA. Transformation of SLRL1 into the slr1 mutant rescued the slender phenotype of this mutant. Moreover, overexpression of SLRL1 in normal rice plants induced a dwarf phenotype with an increased level of OsGA20ox2 gene expression and diminished the GA-induced shoot elongation, suggesting that SLRL1 acts as a repressor of GA signaling. Consistent with the fact that SLRL1 does not have a DELLA domain, which is essential for degradation of DELLA proteins, a level of SLRL1 protein was not degraded by application of gibberellic acid. However, the repressive activity of SLRL1 against GA signaling was much weaker than a truncated SLR1 lacking the DELLA domain. Based on these characteristics of SLRL1, the functional roles of SLRL1 in GA signaling in rice are discussed.