GmSTF accumulation mediated by DELLA protein GmRGAs contributes to coordinating light and gibberellin signaling to reduce plant height in soybean

Plant height influences plant architecture,lodging resistance,and yield performance.It is modulated by gibberellic acid(GA)metabolism and signaling.DELLA proteins,acting as central repressors of GA signaling,integrate various environmental and hormonal signals to regulate plant growth and development in Arabidopsis.We examined the role of two DELLA proteins,GmRGAa and GmRGAb,in soybean plant height control.Knockout of these proteins led to longer internodes and increased plant height,primarily by increasing cell elongation.GmRGAs functioned under different light conditions,including red,blue,and far-red light,to repress plant height.Interaction studies revealed that GmRGAs interacted with the blue light receptor GmCRY1b.Consistent with this,GmCRY1b partially regulated plant height via GmRGAs.Additionally,DELLA proteins were found to stabilize the protein GmSTF1/2,a key positive regulator of photomorphogenesis.This stabilization led to increased transcription of GmGA2ox-7b and subsequent reduction in plant height.This study enhances our understanding of DELLA-mediated plant height control,offering Gmrgaab mutants for soybean structure and yield optimization.

Origin, evolution, and molecular function of DELLA proteins in plants

Gibberellic acid(GA), a ubiquitous phytohormone, has various effects on regulators of plant growth and development. GAs promote growth by overcoming growth restraint mediated by DELLA proteins(DELLAs). DELLAs, in the GRAS family of plant-specific nuclear proteins, are nuclear transcriptional regulators harboring a unique N-terminal GA perception region for binding the GA receptor GIBBERELLIN INSENSITIVE DWARF1(GID1) and a C-terminal GRAS domain necessary for GA repression activity via interaction with multiple regulatory proteins. The N-terminal conserved region of DELLAs evolved to form a mode of GID1/DELLA-mediated GA signaling originating in bryophytes and ferns. Binding of GA to GID1 increases the affinity between DELLAs and a SCF E3 ubiquitin–ligase complex, thus promoting the eventual destruction of DELLAs by the 26 S proteasome. DELLAs negatively regulate GA response by releasing transcription factors to directly activate downstream genes and indirectly regulate GA biosynthesis genes increasing GA responsiveness and feedback control by promoting GID1 transcription. GA communicates extensively with other plant hormones and uses crosstalk to regulate plant growth and development. In this review, we summarize current understanding of evolutionary DELLA-mediated gibberellin signaling and functional diversification of DELLA, focusing primarily on interactions of DELLAs with diverse phytohormones.

Identification of PtGai(a DELLA protein)in trifoliate orange and expression patterns in response to drought stress

Gibberellins(GAs)are an important hormone in regulating plant growth and development,and DELLA protein is an essential negative regulator of GA signal transduction.The aim of the study was to clone a GAinhibiting protein DELLA from trifoliate orange(Poncirus trifoliata L.Raf.)and to analyze the bioinformations and expression patterns of the protein gene in tissues and in response to drought stress.A DELLA protein was isolated from trifoliate orange and named as PtGai(Genebank number:MZ170959).The PtGai protein had 1731 bp open reading frames,along with 576 amino acid codes,and also grouped with sweet orange(XM_006430552.4).The PtGai protein sequence was 65%homology with the sequences of DELLA proteins in other plant families.PtGai protein existed in the nucleus based on the prediction of subcellular localization.PtGai protein could be expressed in roots,stems,and leaves,along with the highest expression in stems.PtGai was upregulated by drought stress in leaves and roots,along with the decrease of root total GA concentration and the inhibition of shoot and root biomass production.It indicated the characteristics of PtGai protein and the roles of PtGai in GA synthesis and plant growth.

Identification of a novel gain-of-function mutant allele,slr1-d5,of rice DELLA protein

Controling the height of crops plays a crucial role for their yields. The large scale utilization of semi-dwarf varieties has greatly improved crop yield, providing an effective support for world food security. In rice, a main food for over half of the world’s population, a number of dwarf loci have been identiifed. However, most of them are recessive, such as the ‘green revolution’ genesd1. To gain more beneifcial loci for rice breeding programs, exploring new mutations is needed, especialy the dominant loci which can be used broadly for hybrid breeding. Here, we isolated a novel dominant dwarf rice mutant, slr1-d5. Al of the internodes ofslr1-d5are reduced. We ifnd that the responsiveness ofslr1-d5to gibberelin （GA）, GA3, was signiifcantly reduced. Map-based cloning revealed that the dominant dwarifsm ofslr1-d5was caused by an amino acid substitution in the N-terminal TVHYNP domain of rice DELLA protein, SLR1, where the conserved amino acid Pro （P） was substituted to His （H）. Our ifndings not only further prove the pivotal role of TVHYNP motif in regulating SLR1 stability, but also provide a new dwarf source for improvement of rice germplasms.

楸树DELLA基因家族生信分析及CbuGRAS9的功能分析

通过鉴定楸树(Catalpa bungei)DELLA家族基因并分析CbuGRAS9的基因功能,为楸树生殖调控性状的遗传改良提供理论依据。基于楸树基因组数据,鉴定并克隆了5个与拟南芥(Arabidopsis thaliana)同源的CbuDELLAs基因;利用ExPASy、SWISS-MODEL、Plant-mPloc、PlantCare等在线工具对CbuDELLAs蛋白进行等电点、蛋白结构、亚细胞定位及启动子顺式作用元件预测;以9-1(Catalpa bungei‘Luoqiu No.1’)和‘百日花’楸树(Catalpa‘Bairihua’)为材料,分析了CbuDELLAs基因的表达量差异,并通过异源转化拟南芥证实了CbuGRAS9的分子功能,利用酵母双杂交文库筛选了与CbuGRAS9互作的蛋白。结果表明:5个CbuDELLAs蛋白的氨基酸数目为455~588,蛋白相对分子质量为5.04~6.43 kDa,等电点为4.81~5.14;CbuDELLAs蛋白均含有DELLA和GRAS保守结构域,全部为亲水性蛋白。亚细胞定位预测结果显示CbuDELLAs蛋白均定位于细胞核中。启动子顺式作用元件分析发现,这5个DELLAs基因启动子区均含有参与赤霉素反应的顺式作用元件。qRT-PCR结果显示‘,百日花’楸中CbuDELLAs基因表达量显著高于对照9-1楸,其中CbuGRAS9为差异最显著的基因,CbuGRAS9转基因株系的开花时间被明显推迟。鉴定到与CbuGRAS9互作的蛋白质主要富集在核糖体、氨基酸合成、次级代谢、光合作用、TCA循环等代谢通路。

Arabidopsis WRKY45 Interacts with the DELLA Protein RGL1 to Positively Regulate Age- Triaaered Leaf Senescence

Leaf senescence can be triggered and promoted by various environmental stressors, developmental cues, and endogenous hormone signals. Several lines of evidence have suggested the involvement of WRKY transcription factors in regulating leaf senescence, but the underlying mechanisms and signaling pathways involved remain elusive. In this study, we identified Arabidopsis thaliana WRKY DNA-binding protein 45 （WRKY45） as a positive regulator of age-triggered leaf senescence. Loss of W^KY45 function resulted in increased leaf longevity in age-triggered senescence, whereas overexpression of WRKY45 significantly accelerated age-triggered leaf senescence. Consistently, expression of SENESCENCE- ASSOCIATED GENEs （SAGs） was significantly reduced in wrky45 mutants but markedly enhanced in transgenic plants overexpressing WRKY45. Chromatin immunoprecipitation assays revealed that WRKY45 directly binds the promoters of several SAGs such as SAG12, SAG13, SAG113, and SEN4. Both in vivo and in vitro biochemical analyses demonstrated that WRKY45 interacts with the DELLA pro- tein RGA-LIKE1 （RGL1）, a repressor of the gibberellin （GA） signaling pathway. We found that RGL1 repressed the transcription activation function of WRKY45, thereby attenuating the expression of its reg- ulon. Consistent with this finding, overexpression of RGL1 resulted in significantly increased leaf longevity in age-triggered senescence. Taken together, our results provide compelling evidence that WRKY45 functions as a critical component of the GA-mediated signaling pathway to positively regulate age-triggered leaf senescence.

The blue light receptor CRY1 interacts with GID1 and DELLA proteins to repress GA signaling during photomorphogenesis in Arabidopsis

Light is a critical environmental cue that regulates a variety of diverse plant developmental processes.Cryptochrome 1(CRY1)is the major photoreceptor that mediates blue light-dependent photomorphogenic responses such as the inhibition of hypocotyl elongation.Gibberellin(GA)participates in the repression of photomorphogenesis and promotes hypocotyl elongation.However,the antagonistic interaction between blue light and GA is not well understood.Here,we report that blue light represses GA-induced degradation of the DELLA proteins(DELLAs),which are key negative regulators in the GA signaling pathway,via CRY1,thereby inhibiting the GA response during hypocotyl elongation.Both in vitro and in vivo biochemical analyses demonstrated that CRY1 physically interacts with GA receptors-GA-INSENSITIVE DWARF 1 proteins(GID1s)-and DELLAs in a blue light-dependent manner.Furthermore,we showed that CRY1 inhibits the association between GID1s and DELLAs.Genetically,CRY1 antagonizes the function of GID1s to repress the expression of cell elongation-related genes and thus hypocotyl elongation.Taken together,our findings demonstrate that CRY1 coordinates blue light and GA signali ng for plant photomorphogenesis by stabilizing DELLAs through the binding and in activation of GID1s,providing new in sights into the mechanism by which blue light antagonizes the function of GA in photomorphogenesis.

赤霉素关键蛋白DELLA对植物雄蕊发育研究进展

在植物生长发育过程中,DELLA蛋白作为响应赤霉素(GA)信号通路负调控因子,其家族成员在植物雄蕊组织中均有表达,对植物生长发育起着重要作用.通过近几年研究发现,无论是拟南芥还是水稻,DELLA基因的突变都会导致植株的雄性不育.该文综述了DELLA蛋白基本特征、参与赤霉素信号通路的调控,以及在雄蕊发育中的作用,为今后的研究提供一定的理论基础.

燕麦AsGRAS36基因的克隆与表达分析

非生物胁迫在一定程度上制约了燕麦(Avena sativa L.)的生长和发育,其关键基因的克隆和表达分析可为提高植物逆境耐受性奠定理论基础。本研究从燕麦叶片中克隆到AsGRAS36基因,该蛋白N端含有DELLA结构域和TVHYNP结构域,确定其属于DELLA蛋白。蛋白理化性质分析表明AsGRAS36编码611个氨基酸,蛋白质相对分子质量为64.81 kD,理论等电点为4.97,为亲水性蛋白,主要由α-螺旋和无规则卷曲组成。系统进化分析显示AsGRAS36与大麦GRAS28和小麦GRAS115的亲缘关系最近。顺式作用元件分析揭示AsGRAS36基因启动子含有脱落酸和水杨酸等激素响应元件和干旱、低温等胁迫响应元件。蛋白互作预测显示与AsGRAS36互作的蛋白参与到植物的胁迫响应过程。基因表达分析表明AsGRAS36在燕麦受到非生物胁迫后迅速表达,对高温、干旱等胁迫和激素诱导响应敏感。该研究结果为AsGRAS36基因及DELLA蛋白在燕麦非生物胁迫调控中的作用提供了科学依据。

棉花DELLA蛋白基因GhGAI3和GhGAI4启动子的克隆及序列分析

棉纤维是纺织行业的重要原材料。赤霉素能够促进棉纤维的生长发育,而DELLA蛋白又是赤霉素信号转导通路中的关键调控因子,因此研究棉花DELLA蛋白基因表达的调控模式,对阐明棉纤维生长发育的分子生物学机理具有重要意义。本研究根据两个棉花DELLA蛋白基因GhGAI3和GhGAI4序列设计特异引物,以陆地棉新陆早13号的基因组DNA为模板,用基因组步移法克隆了棉花DELLA蛋白基因GhGAI3和GhGAI4的启动子。对两个启动子进行序列分析表明,这两个启动子均具有真核生物典型的核心启动子区,同时也都具有一个或多个光响应元件和赤霉素响应元件,说明这两个基因可能受到光信号和赤霉素信号的调控,这与其它植物中DELLA蛋白的表达模式是一致的。此外,这两个启动子还具有各种转录调控相关的顺式作用元件,比如其它激素类的响应元件和逆境胁迫响应元件,说明棉花中DELLA蛋白基因可能在响应其它激素信号以及逆境胁迫中也起到一定的作用。

棉花DELLA蛋白GhGAI2b基因的克隆和功能初步分析

DELLA蛋白是赤霉素信号途径的负调节因子,在棉花纤维发育中有着重要作用。本研究采用同源克隆方法,从棉花中克隆DELLA蛋白Gh GAI2b基因,构建p BP35S:Gh GAI2b和p BP35S:Ghgai2b植物过表达载体,通过农杆菌介导的滴花法转化Col野生型拟南芥。结果表明,棉花DELLA蛋白Gh GAI2b包含了DELLA蛋白家族中所有的典型保守区域;转基因拟南芥表现出莲座叶半径变短,植株矮化,花序紧凑,花器官发育迟缓等生长发育受抑制表型。说明棉花DELLA蛋白Gh GAI2b基因可能参与GA信号途径抑制植物生长发育。

海岛棉DELLA蛋白编码基因GbGAI的克隆与功能初步分析

赤霉素在棉花纤维发育过程中起着重要作用。DELLA蛋白是赤霉素信号传导通路中的一类重要负调节因子。通过同源克隆的方法,克隆了新疆特有棉花栽培种海岛棉中DELLA蛋白的同源基因GbGAI。序列比对分析表明GbGAI编码的氨基酸序列具有DELLA蛋白的典型结构域。构建其过量表达载体,通过农杆菌侵染花序的方法转入野生型拟南芥中,观察表型。转基因株系表现出晚花,株高变矮等赤霉素不敏感突变体的表型。

棉花DELLA蛋白GhGAI4b基因的克隆及功能初步分析

【目的】DELLA蛋白已在不同的物种中被广泛鉴定,并且功能区高度保守,是GA信号通路中的负调控因子,在棉花纤维细胞起始和发育中起重要的作用。研究棉花中DELLA基因的功能。【方法】从棉花中克隆DELLA蛋白的同源基因GhGAI4b,构建35S:GhGAI4b植物过量表达载体,通过农杆菌侵染花序的方法转入野生型拟南芥中,观察转基因植株的表型。【结果】序列分析表明GhGAI4b全长开放阅读框1 617 bp,编码538个氨基酸,具有DELLA蛋白的典型结构域。采用Clustal X将GhGAI4b的氨基酸序列与其他已知的DELLA蛋白氨基酸序列进行相似性比对,发现GhGAI4b氨基酸序列与陆地棉、拟南芥的相似性较高,达到47%以上。转基因株系表现出叶片颜色深绿,叶片缩小且肥厚,叶片细长呈锯齿状;有些株系会出现多重莲座叶,雄蕊发育不全,晚花,株高变矮等赤霉素不敏感突变体的表型。【结论】过表达GhGAI4b能够抑制拟南芥莲座叶的生长和发育,影响转基因拟南芥花器官的发育,严重时引起转基因拟南芥的不育。

‘辽宁2号’DELLA蛋白编码基因的克隆及表达分析

以‘辽宁2号’核桃的叶片为试材,通过同源克隆和RACE技术克隆DELLA家族蛋白的编码基因,并进行了同源性分析和氨基酸序列的多重比对;通过实时荧光定量PCR分析了该基因在‘辽宁2号’核桃中的组织特异性表达情况。主要研究结果如下:获得了DELLA蛋白编码基因的cDNA全长,命名为JrGAI(Genebank:JF766606),cDNA全长为2 361bp,包含1 842bp编码区序列,推测其编码的蛋白包含613个氨基酸,分子量为66.78kDa。同源性分析及氨基酸序列的多重比对结果表明该蛋白与其他物种的DELLA蛋白高度相似,并且具有完整的DELLA、TVHYN、VHIID、RVER和SAW等结构域;实时荧光定量PCR结果显示JrGAI基因在‘辽宁2号’核桃的叶芽、混合花芽、叶片、雄花、茎段和果实中普遍表达,并且在休眠期组织中具有较高的表达量,其中以休眠期的混合花芽中的表达量最高。

蔷薇科植物DELLA蛋白的生物信息学分析

为探索蔷薇科植物DELLA蛋白的结构特征和亲缘进化关系,以蔷薇科植物苹果、梨和玫瑰等19个DELLA蛋白为试材,利用expasy、PSORT和PROSITE数据库、TM-HMM方法、SignalP4.0Server、CDD、DNAMAN和MEGA version5.3等软件对蛋白质进行了生物信息学分析。结果表明:蔷薇科植物中19个DELLA蛋白氨基酸序列组成成分和理化性质差异不明显,均为非跨膜类亲水性蛋白,且不含信号肽;不同DELLA蛋白之间同源性较高,达到74.38%以上,具有DELLA和GRAS2个保守结构域,功能位点是GRAS;19个DELLA蛋白N端同源性较低,但是存在TVHYNP、VHIID和RVER等DELLA蛋白典型结构域;进化树显示梨属和蔷薇属属内植物DELLA蛋白亲缘关系较近,苹果属内植物亲缘关系相差较远。本研究的开展为蔷薇科植物遗传演化研究提供理论依据。

棉花GhGAI3a DELLA功能域缺失过表达载体的构建及其功能初步分析

本研究对棉花GhGAI3aDELLA功能域缺失片段的克隆及对其功能进行了初步分析。本文以质粒p121GhGAI3aDNA为模板,通过PCR方法获得基因GhGAI3aN端赤霉素响应关键区域DELLA缺失的片段Gh-gai3a,构建出过量表达载体pBP35S:gai3a。利用农杆菌介导滴花法转化拟南芥获得T1代转基因植株。DNA检测结果显示,获得了24个转基因株系,RT-PCR表明了该基因在部分株系中已有表达。初步的表型观察发现该DELLA区缺失基因严重影响花器官、侧枝的发育,抑制植物的生长。

棉花DELLA蛋白GhGAI4a基因在拟南芥中功能初步分析

为了探究棉花DELLA蛋白基因GhGAI4a在拟南芥中的功能,构建植物表达载体pBP35S∶GhGAI4a和pBP35S∶Ghgai4a,利用农杆菌介导的花滴法将其转入Col野生型拟南芥。结果显示,2个载体各自获得6个独立的转基因拟南芥纯合株系。分别统计48-96h种子萌发率,测量生长7d后幼苗的主根长度,与非转基因植株相比,过量表达GhGAI4a、Ghgai4a对拟南芥种子萌发及主根生长具有明显的抑制作用。1μmol/L GA处理转基因植株,GhGAI4a转基因植株主根长和萌发率均增大,而Ghgai4a转基因植株主根长度和萌发率均无显著变化。

海岛棉DELLA蛋白基因GbGAI2的克隆及其在胚珠中表达分析

赤霉素(Gibberellins,GA)在棉花纤维的发育过程中起重要作用,DELLA蛋白是GA信号通路中的起关键作用的负调控因子。本研究采用同源克隆方法,获得了海岛棉的DELLA蛋白同源基因GbGAI2。序列比对分析结果表明,GbGAI2编码的氨基酸序列具有DELLA蛋白的典型结构域,并与陆地棉DELLA蛋白GhGAI2氨基酸有90.13%相同。半定量RT-PCR分析结果表明,GbGAI2在1-5DPA和23DPA棉胚中的表达量较高,说明该基因可能在海岛棉棉纤维发育的起始阶段和棉纤维次生壁加厚阶段起作用。

拟南芥DELLA蛋白编码基因RGA和GAI的原核表达和多克隆抗体制备

通过聚合酶链反应方法扩增了RGA和GAI两个拟南芥DELLA蛋白基因,并将其克隆到pGEX-4T-3原核表达载体上,转化大肠杆菌Rosetta菌株,利用异丙基硫代半乳糖苷诱导表达了分子量为90 kD的GST-RGA和分子量为85 kD的GST-GAI融合蛋白,这两种融合蛋白均以包涵体的形式存在.将包涵体直接作为抗原免疫新西兰白兔,制备出多克隆抗体血清,血清效价超过1:400 000.WB分析结果表明这两种抗体可以特异性识别拟南芥RGA和GAI蛋白,为进一步研究DELLA蛋白的调控机制奠定了基础.

DELLA家族蛋白研究进展

在已发现的植物GA信号传递分子中,有一类N端具有高度保守的DELLA结构域,称之为DELLA家族蛋白。介绍了DELLA蛋白的保守结构域和降解途径,并综述了其对多种植物激素信号的调控作用,以期提示植物生长发育和植物激素信号转导的奥秘。