枳SPL9和SPL13全长cDNA克隆、亚细胞定位和表达分析

【目的】从枳[Poncirus trifoliata(L.)Raf.]中克隆SBP类[SQUAMOSA(SQUA)promoter-binding-like]转录因子基因SPL9和SPL13全长,构建SPL9和SPL13亚细胞定位表达载体验证其是否具有核定位功能,利用荧光定量PCR研究其在枳不同组织的表达特性,初步确定SPL9和SPL13在枳生长发育过程中的作用。【方法】利用生物信息学结合RACE技术以枳花器官的cDNA为模板,克隆出SPL9和SPL13基因全长,分别命名Pt-SPL9和Pt-SPL13,大小分别是1519bp和1824bp,在GenBank的登录号分别是FJ502237和FJ502238;构建Pt-SPL9和Pt-SPL13亚细胞定位载体35S-GW-FJ502237/FJ502238-GFP,基因枪转化洋葱表皮细胞,暗培养24h后激光共聚焦显微镜下观察;利用SYBR Green I实时定量RT-PCR方法检测Pt-SPL9和Pt-SPL13在根、茎、叶、花序、花和果等不同组织中的表达。【结果】生物信息学分析表明,Pt-SPL9和Pt-SPL13的cDNA序列中都有microRNA156的识别位点,Pt-SPL9与金鱼草、拟南芥和玉米SPL9的同源性分别为48.9%、42.5%和41.7%;Pt-SPL13与拟南芥SPL13、水稻的SPL16和玉米的TGA1同源性分别为40.8%、38.1%和35.8%。Pt-SPL9和Pt-SPL13与其它植物的SBP一样有着高度保守的序列,即SBP结构域和一个双向核定位信号KRXXXRRRK。亚细胞定位结果表明,Pt-SPL9和Pt-SPL13均定位于细胞核中。SYBR Green I实时定量RT-PCR结果表明,Pt-SPL9和Pt-SPL13在各个器官均有表达,但表达量不同,Pt-SPL9在茎中的表达量最高,在花和叶中的表达量次之,在根、花芽和幼果中的表达量最低;Pt-SPL13在幼果中的表达量最高,在茎和花芽中的表达量相当,其次为叶,在花和根中的表达量很低。【结论】转录因子Pt-SPL9和Pt-SPL13均具有核定位功能,Pt-SPL9和Pt-SPL13对枳的茎和果实的发育可能有着重要作用。

白桦BpSPL9基因抑制表达载体的构建及遗传转化研究

为揭示SPL基因在白桦(Betula platyphylla)生长和发育中的功能,在克隆白桦BpSPL9基因的基础上,采用CREST技术构建BpSPL9的抑制表达载体,通过农杆菌(Agrobacterium tumefaciens)介导法进行白桦的遗传转化,对获得的转基因株系进行表型观测,探究BpSPL9基因的功能。结果表明:已成功获得了BpSPL9抑制表达白桦株系。转基因株系的苗高显著低于非转基因对照株系,节间距变短,叶面积变小,转基因株系的细胞分裂素(6-BA)和生长素(IAA)含量均低于对照株系。推测BpSPL9基因通过影响生长素和细胞分裂的合成,从而参与植物的生长发育过程。

草莓miR156靶基因SPL9的克隆与表达分析

【目的】从草莓中克隆SPL(SQUAMOSA promoter binding protein-like)转录因子基因SPL9,并分析其在草莓植株不同生长时期的表达水平及与miR156的表达关系,探讨SPL9在草莓植株生长发育中的作用。【方法】根据苹果SPL基因家族的保守序列设计简并引物,以草莓叶片为试材,利用RT-PCR克隆草莓SPL9基因片段,在此基础上利用RACE方法克隆SPL9的cDNA全长。利用实时定量RT-PCR技术分析草莓叶片中SPL9及miR156的表达水平。【结果】从草莓(Fragaria×ananassa)品种‘全明星’中克隆出SPL9基因的cDNA全长序列,命名为FaSPL9。其CDS长度为1 143 bp,编码381个氨基酸,与拟南芥AtSPL9、葡萄VvSPL9、枳PtSPL9、苹果MdSPL9以及玉米ZmSPL9的氨基酸序列同源性分别为:40.30%、64.57%、56.09%、70.33%,38.35%。FaSPL9有着高度保守的SBP结构域和一个双向核定位信号KRXXXRRRK。FaSPL9基因序列中包含miR156的靶位点,实时定量RT-PCR结果表明,在草莓植株的不同生长时期FaSPL9的表达量不同,且与miR156呈现相反的表达模式。【结论】从草莓中分离出FaSPL9基因,其作为miR156的靶基因,推测FaSPL9对草莓植株的生长发育具有重要的调控作用。

大豆GmSPL3基因家族功能初探

为研究大豆中拟南芥(Arabidopsis thaliana)重要开花调控AtSPL3同源基因的作用,本研究利用生物信息学方法分析大豆的RNA-seq基因表达谱数据,对绥农14根、茎、子叶、初生叶、三出复叶、花、生长点、荚、胚和下胚轴10个组织进行GmSPL3基因的组织特异性表达分析,并利用CRSPR/Cas9的方法构建大豆GmSPL3基因的四突变体并对比其表型。结果显示:GmSPL3基因在大豆的根、子叶、花和种子中有着明显的表达差异;GmSPL3a在各组织中的表达量都非常低,而GmSPL3(b,c,d)在不同组织中存在明显的组织特异性表达;在GmSPL3基因敲除突变体中研究发现,短日照条件下,spl3abcd突变体表现出叶片变小、节数减少、节间距缩短、株高降低的表型,表明GmSPL3在调控大豆植株形态方面发挥重要功能。

葡萄SPL9和SPL10基因全长cDNA克隆、亚细胞定位和表达分析

从‘夏黑’葡萄(Vitisvinifera×V.labrusca ‘Summer Black’)中克隆了SBP(Squamosa promoter binding protein)类重要转录因子基因SPL9和SPL10全长cDNA序列,在GenBank的登录号分别是HM018600和HM018601,序列分析表明二者均具备完全保守的核定位信号序列(KKSR)、SBP结构域以及葡萄microRNA156a(Vv-miR156a)的靶序列。进一步构建Vv-SPL9和Vv-SPL10亚细胞定位表达载体,对其是否具有核定位功能进行了研究,并利用半定量、荧光定量RT-PCR研究了这两个基因与Vv-miR156a在葡萄不同组织的表达情况。结果表明:转录因子SPL9和SPL10均定位于细胞核中,而且两个基因在葡萄各个组织中均有表达,但表达量存在差异,两者均在小果中的表达量最高。Vv-miR156a的积累水平与这两个基因在各个组织中的表达呈现一定的消长关系,并且在小果中最为明显。

Transcriptional Regulation of miR528 by OsSPL9 Orchestrates Antiviral Response in Rice

Many microRNAs (miRNAs) are critical regulators of plant antiviral defense.However,little is known about how these miRNAs respond to virus invasion at the transcriptional level.We previously show that defense against Rice stripe virus (RSV) invasion entailed a reduction of miR528 accumulation in rice,alleviating miR528-mediated degradation of L-Ascorbate Oxidase (AO) mRNA and bolstering the antiviral activity of AO.Here we show that the miR528-A0 defense module is regulated by the transcription factor SPL9.SPL9 displayed high-affinity binding to specific motifs within the promoter region of miR528 and activated the expression of miR528 gene in vivo.Loss-of-function mutations in SPL9 caused a significant reduction in miR528 accumulation but a substantial increase of AO mRNA,resulting in enhanced plant resistance to RSV.Conversely,transgenic overexpression of SPL9 stimulated the expression of miR528 gene,hence lowering the level of AO mRNA and compromising rice defense against RSV.Importantly,gain in RSV susceptibility did not occur when SPL9 was overexpressed in mir528 loss-of-function mutants,or in transgenic rice expressing a miR528-resistant AO.Taken together,the finding of SPL9-mediated transcriptional activation of miR528 expression adds a new regulatory layer to the miR528-A0 antiviral defense pathway.

Gibberellin repression of axillary bud formation in Arabidopsis by modulation of DELLA-SPL9 complex activity

The formation of lateral branches has an important and fundamental contribution to the remarkable developmental plasticity of plants,which allows plants to alter their architecture to adapt to the challenging environment conditions.The Gibberellin(GA)phytohormones have been known to regulate the outgrowth of axillary meristems(AMs),but the specific molecular mechanisms remain unclear.Here we show that DELLA proteins regulate axillary bud formation by interacting and regulating the DNA-binding ability of SQUAMOSA-PROMOTER BINDING PROTEIN LIKE 9(SPL9),a micro RNA156-targeted squamosa promoter binding protein-like transcription factor.SPL9 participates in the initial regulation of axillary buds by repressing the expression of LATERAL SUPPRESSOR(LAS),a key regulator inthe initiation of AMs,and LAS contributes to the specific expression pattern of the GA deactivation enzyme GA2ox4,which is specifically expressed in the axils of leaves to form a low-GA cell niche in this anatomical region.Nevertheless,increasing GA levels in leaf axils by ectopically expressing the GA-biosynthesis enzyme GA20ox2 significantly impaired axillary meristem initiation.Our study demonstrates that DELLA-SPL9-LAS-GA2ox4 defines a core feedback regulatory module that spatially pattern GA content in the leaf axil and precisely control the axillary bud formation in different spatial and temporal.

Overexpression of OsSPL9 enhances accumulation of Cu in rice grain and improves its digestibility and metabolism

Copper （Cu） is an essential trace mineral element for all forms of life, and is an important structural component and co-factor for a variety of metalloenzymes （Pefia et al., 1999; Bertinato and L＇Abbe, 2004）. In humans, Cu deficiency is not common because of the ubiquitous occurrence of Cu and ease of gastrointestinal absorption （Zidar et al., 1977; Uauy et al., 1998）.