ptc-miR801人工microRNA表达载体构建及功能初步研究

在构建盐胁迫下青杨microRNA文库中发现了ptc-miR801,为探索植物在盐胁迫条件下ptc-miR801参与胁迫应答的机制,本实验构建了植物表达载体pCAM2300-ami801,经根癌农杆菌EHA105介导、花序侵染法获得拟南芥转基因植株。RT-PCR半定量结果显示ptc-miR801可以在转基因拟南芥中超表达且NaCl胁迫下ptc-miRNA801转基因植株种子萌发率和根长显著高于野生型,说明ptc-miR801超表达增强了转基因拟南芥耐盐性。该试验为进一步研究miR801在杨树胁迫应答机制中的作用奠定基础。     

盐穗木HcmiR172e和预测的靶基因HcTOE3的克隆及盐胁迫下的靶向关系分析

盐穗木(Halostachys caspica)广泛分布于新疆干旱盐碱地区,属于藜科极端耐盐的盐生灌木。从600 mmol?L-1 Na Cl处理48 h的盐穗木根的小RNA文库中筛选到差异极显著的HcmiR172e做为研究对象,利用盐穗木转录组数据预测其靶基因为HcTOE3,开展HcmiR172e-HcTOE3的分子克隆和胁迫表达相关性工作。通过同源克隆和RACE技术获得盐穗木HcmiR172e前体和HcTOE3全长序列;使用生物信息软件分析前体和靶基因信息及相关性;采用q RT-PCR技术检测了不同盐浓度(200、400和600 mmol?L-1 Na Cl)处理48 h的盐穗木HcmiR172e及预测的靶基因HcTOE3的表达变化。结果显示,克隆得到的HcmiR172e前体序列可形成颈环结构,各项指标符合前体要求。其靶基因HcTOE3 c DNA全长为1 744 bp,开放阅读框1 329 bp,在编码区有一个高度匹配HcmiR172e的结合位点(CTGCAGCATCATCAGGATTC);q RT-PCR分析结果表明HcmiR172e与其预测的靶基因HcTOE3均响应盐的处理,二者呈现一定的负相关性。后续将通过实验逐步阐明盐穗木HcmiR172e对HcTOE3在逆境中的靶向调控作用。     

盐穗木miR166a前体和预测靶基因ATHB8-like的克隆及二者靶向关系的鉴定

以盐生植物盐穗木为实验材料,从前期构建的高盐胁迫下盐穗木根的小RNA文库中候选了差异表达的miR166a,利用生物信息学从盐穗木转录组数据中预测其靶基因;采用5′RLM-RACE技术鉴定盐穗木miR166a对预测靶基因ATHB8-like的靶向性;通过PCR和RACE技术克隆盐穗木miR166a前体和预测靶基因ATHB8-like全长基因序列,并进行相应的生物信息学分析。结果显示:盐穗木miR166a预测的靶基因为ATHB8-like;通过实验鉴定确实存在靶向切割,具体的切割位点位于miR166a成熟体的14~15碱基之间;miR166a成熟体序列在不同植物中高度保守;克隆获得的盐穗木miR166a前体可折叠成完整的颈环结构,符合miRNA的前体特征,候选植物miR166a前体在进化上没有表现出保守性;预测的靶基因ATHB8-like cDNA全长为2 786bp,开放阅读框为2 526bp,编码841个氨基酸,ATHB8-like具有一个HD-ZIPⅢ结构域,在进化上具有保守性。该研究结果为进一步开展盐穗木miR166a和ATHB8-like的生物学功能奠定了基础。     

盐分和水分胁迫对两种盐生植物盐爪爪和盐穗木种子萌发的影响

采用不同浓度NaCl和等渗PEG(分子量为6000)处理2种盐生植物种子(盐爪爪Kalidiumfoliatum、盐穗木Halostachys caspica)。结果表明,(1)NaCl和等渗PEG对种子的萌发均产生抑制作用,且PEG的抑制程度大于等渗NaCl,说明渗透胁迫是影响盐生植物种子萌发的主要因素;(2)盐胁迫和水分胁迫对盐生植物种子的萌发具有明显的抑制作用,降低了种子的萌发率,推迟了种子的初始萌发时间、延长了种子的萌发时间;(3)根据2种盐生植物种子萌发耐盐性比较,两种盐生植物均具有较强的耐盐性,在0~300 mmol.L-1NaCl盐溶液范围内,盐穗木种子相对萌发率达到95%以上,盐爪爪达到80%以上,说明盐穗木比盐爪爪具有更强的耐盐性。盐穗木种子耐盐适宜值为332.5 mmol.L-1,临界值为540 mmo.lL-1,极限值为749 mmo.lL-1;盐爪爪种子耐盐适宜值为246.5 mmol.L-1,临界值为391.8 mmo.lL-1,极限值为537 mmol.L-1;(4)低浓度的NaCl溶液对种子萌发的幼苗生长起着促进作用。在100 mmo.lL-1NaCl处理时,两种盐生植物幼苗的胚...     

胡杨无性系幼苗响应盐胁迫的miRNA表达差异研究

植物miRNA在调控基因表达、细胞周期、生物体发育、抗逆等方面起重要作用。为研究胡杨(Populus euphratica Oliv.)的耐盐机制,以1年生胡杨无性系幼苗为材料,构建具有空间代表性的盐胁迫胡杨cDNA文库,利用二代测序技术测定NaCl胁迫下和正常培养条件下胡杨叶和根miRNA表达情况。结果表明,不同的miRNA之间表达量存在明显差异,表达丰度最高的miRNA有miR156、miR157、miR165、miR166和miR167等,合计占总表达量的90%以上。胡杨根部存在特异表达的miRNA,在整个耐盐调控机制中发挥着生理调节、分子调控和信号传导等极为重要的作用。盐处理样品中发现大量响应盐胁迫的miRNA,对这些转录因子进行靶基因预测和注释后,发现很多盐胁迫响应的miRNA与NAC和SPL等重要转录因子家族相关,与前人的结论一致,另外还发现许多miRNA的调控对象是ATP酶和激素响应因子。     

胡杨PeECT8基因的克隆及功能分析

ECT基因家族已在拟南芥中被发现并报道,然而它们是否参与植物在逆境胁迫下的响应过程却鲜有报道。为研究胡杨PeECT8基因的功能,从胡杨叶片cDNA中克隆出PeECT8基因,构建CaMV 35S::Pe ECT8植物表达载体,利用花序浸染法转化拟南芥,经GUS组织化学染色和PCR检测,获得转基因植株,进而测定不同浓度盐(Na Cl)和甘露醇胁迫下转基因拟南芥种子的萌发率、生长势和根长。测序结果表明,该基因编码区长度为1821 bp,可编码606个氨基酸。蛋白序列比对发现,PeECT8基因与毛果杨PtrECT8同源基因所编码的氨基酸一致性达93.42%,PeECT8蛋白包含一个YT521-B-like保守结构域。相比于野生型,过量表达PeECT8的拟南芥在不同浓度NaCl胁迫下萌发率降低,在D-甘露醇胁迫下萌发率没有明显变化。在NaCl和D-甘露醇胁迫下,转基因拟南芥根的伸长长度小于野生型。这表明PeECT8基因在盐胁迫和渗透胁迫下发挥负调控的作用。     

盐穗木功能未知蛋白 (HcUKPP)的亚细胞定位

藜科的极端盐生植物盐穗木（Halostachys caspica）的高盐胁迫抑制差减文库中有39%的功能未知蛋白（proteins with obscure features, POFs）,利用亚细胞定位分析可以初步判断其可能的功能.将盐穗木的1个POF-cDNA序列HcUKPP的编码区构建至pCAMBIA1301-GFP植物表达载体上,冻融法将重组质粒pCAMBIA1301-HcUKPP-GFP转化农杆菌EH105A,利用花序浸染法将基因导入拟南芥,经潮霉素筛选获得T1代阳性幼苗.通过激光扫描共聚焦显微镜观察转基因拟南芥植株的根部细胞. 结果显示,表达GFP蛋白的对照转基因植株中,绿色荧光在细胞核、细胞膜以及细胞质中均能检测到,而表达HcUKPP-GFP融合蛋白的转基因植株中,绿色荧光只在细胞质膜上表达,说明HcUKPP蛋白为细胞质膜相关蛋白.本研究为深入探讨盐穗木未知蛋白的功能奠定了基础.     

盐胁迫下过表达BoRACK1基因对羽衣甘蓝种子萌发的影响

蛋白激酶C1受体(RACK1)是一个高度保守的蛋白,它具有多种功能,对植物生长发育有重要的调控作用。本研究构建过表达BoRACK1转基因羽衣甘蓝植株,以阐明该基因在羽衣甘蓝中对盐胁迫的可能功能。转基因羽衣甘蓝对盐胁迫的耐受力比野生型高,表现在其种子发芽率显著高于野生型;活性氧产生比野生型活跃,H_2O_2也可能参与BoRACK1调控的种子萌发过程。实时定量PCR分析表明,在过表达BoRACK1转基因植株中,BoRbohs基因的转录水平显著增强。这些结果表明,在种子萌发过程中,过表达BoRACK1正调控植物耐盐胁迫。     

响应盐胁迫调控的露地菊miR398a的克隆及功能研究

miRNAs在非生物胁迫中起着重要的作用。通过前期对露地菊Small RNA高通量测序数据测得到miR398a成熟体和前体序列,命名为cgr-miR398a和cgr-MIR398a。序列对比显示,cgr-miR398a与其他植物中已经鉴定的miR398a序列高度保守;利用前期露地菊降解组数据获得miR398a预测的靶基因,cgr-miR398a根和叶中的靶基因共有18个,其中有铜/锌超氧化物歧化酶（CSD2）、铜伴侣蛋白（CCS）、类A20/AN1-l锌指家族蛋白（SAP8）等与抗性相关的基因。qPCR结果显示盐胁迫下露地菊miR398a及靶基因在不同组织部位的表达水平存在显著的负相关性。为探究cgr-miR398a响应盐胁迫的功能,克隆cgr-MIR398a并构建过表达载体转化拟南芥。结果表明,拟南芥中过表达cgr-MIR398a降低了盐胁迫下种子发芽率以及成苗期的抗盐性,说明cgr-miR398a在拟南芥响应盐胁迫中起着负调控作用。这为进一步研究露地菊mi398a的功能和露地菊的抗盐机理奠定了基础。     

一个种子特异的大豆AP2类转录因子参与调控种子萌发

植物种子的发育和萌发受严格的时空调控,其中转录因子起着重要的作用．本研究发现了一条在发育过程中的大豆种子里特异表达的表达序列标签（expressed sequence tag,EST）．通过末端快速扩增技术（rapid amplification of cDNA end,RACE）得到该基因的全长序列并命名为GmSGR．序列分析表明该基因属于AP2／ERF类转录因子家族,其AP2结构域与拟南芥（Arabidopsis thaliana）中的AP2／ERF基因家族DREB亚家族中A-3组的AtABI4的AP2结构域同源性极高．在酵母系统中,未检测到GmSGR具有转录激活活性．将该基因在拟南芥中过量表达,在高浓度的脱落酸（abscisicacid,ABA）及葡萄糖的条件下,转基因植物种子的萌发率均高于对照组;而在高浓度的NaCl条件下,转基因植物种子的萌发率低于对照组．在转基因植物的幼苗中,AtEm6和AtRD29B的表达较野生型幼苗高．这些结果表明GmSGR可能通过调控AtEm6和AtRD29B的表达,从而导致转基因种子对ABA敏感度降低,对盐胁迫更敏感．     

A Role for Arabidopsis miR399f in Salt,Drought, and ABA Signaling

MiR399f plays a crucial role in maintaining phosphate homeostasis in Arabidopsis thaliana. Under phosphate starvation conditions, AtMYB2, which plays a role in plant salt and drought stress responses, directly regulates the expression of miR399f. In this study, we found that miR399f also participates in plant responses to abscisic acid (ABA), and to abiotic stresses including salt and drought. Salt and ABA treatment induced the expression of miR399f, as confirmed by histochemical analysis of promoter-GUS fusions. Transgenic Arabidopsis plants overexpressing miR399f (miR399f-OE) exhibited enhanced tolerance to salt stress and exogenous ABA, but hypersensitivity to drought. Our in silico analysis identified ABF3 and CSP41b as putative target genes of miR399f, and expression analysis revealed that mRNA levels of ABF3 and CSP41b decreased remarkably in miR399f-OE plants under salt stress and in response to treatment with ABA. Moreover, we showed that activation of stress-responsive gene expression in response to salt stress and ABA treatment was impaired in miR399f-OE plants. Thus, these results suggested that in addition to phosphate starvation signaling, miR399f might also modulates plant responses to salt, ABA, and drought, by regulating the expression of newly discovered target genes such as ABF3 and CSP41b.     

Differential regulation of microRNAs in response to osmotic,salt and cold stresses in wheat

MicroRNAs (miRNAs) are tiny non-coding regulatory molecules that modulate plant’s gene expression either by cleaving or repressing their mRNA targets. To unravel the plant actions in response to various environmental factors, identification of stress related miRNAs is essential. For understanding the regulatory behaviour of various abiotic stresses and miRNAs in wheat genotype C-306, we examined expression profile of selected conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR397, miR529 and miR1029 tangled in adapting osmotic, salt and cold stresses. The investigation revealed that two miRNAs (miR168, miR397) were down-regulated and miR172 was up-regulated under all the stress conditions. However, miR164 and miR1029 were up-regulated under cold and osmotic stresses in contrast to salt stress. miR529 responded to cold alone and does not change under osmotic and salt stress. miR393 showed up-regulation under osmotic and salt, and down-regulation under cold stress indicating auxin based differential cold response. Variation in expression level of studied miRNAs in presence of target genes delivers a likely elucidation of miRNAs based abiotic stress regulation. In addition, we reported new stress induced miRNAs Ta-miR855 using computational approach. Results revealed first documentation that miR855 is regulated by salinity stress in wheat. These findings indicate that diverse miRNAs were responsive to osmotic, salt and cold stress and could function in wheat response to abiotic stresses.     

The expanding role of miR-302–367 in pluripotency and reprogramming

MicroRNA (miRNA) has been shown to be essential for regulating cell fate and pluripotency; however, our knowledge of miRNA function in stem cells is incomplete due to experimental limitations and difficulties in identifying their physiological targets. Recent studies implicated hESC-expressed miRNAs (miR?302–367 and miR?371–373 clusters) in regulating BMP signaling and promoting pluripotency, suggesting that low levels of BMP signaling may promote pluripotency by preventing neural induction. A comprehensive list of miR?302–367 targets recently identified by genome-wide approaches suggests a number of additional cellular processes and signaling pathways whose regulation by miR?302–367 may promote pluripotency and reprogramming, such as cell cycle, epigenetic changes, metabolism and vesicular transfer.