番茄Trihelix家族SIP1亚家族基因(SlGT-33)克隆表达分析及功能研究

【目的】对番茄三螺旋（Trihelix）家族SIP1亚家族基因（SlGT-33）进行克隆表达及功能研究,为深入解析SIP1亚家族成员调控植物生长发育机制及选育矮化番茄品种提供理论依据。【方法】以番茄品种AC++为材料,PCR扩增SlGT-33基因,对其进行序列分析,采用实时荧光定量PCR（qRT-PCR）检测SlGT-33基因在不同组织及外源激素和非生物胁迫下的表达模式,并利用RNAi技术鉴定SlGT-33基因的生物学功能。【结果】扩增获得的SlGT-33基因（GenBank登录号Solyc12g043090）开放阅读框（ORF）为1125 bp,编码374个氨基酸残基,与已知同源基因序列（GenBank登录号XP004252336.1）仅缺少3个碱基。SlGT-33与SlGT-17（GenBank登录号Solyc05g018350）位于同一分支,虽然二者的氨基酸序列相似性最高,但仅为45.8%。SlGT-33基因在茎和成熟叶中的相对表达量较高,显著高于其他组织（P<0.05,下同）;经IAA、GA₃和MeJA处理后,SlGT-33基因的相对表达量均与对照无显著差异（P>0.05,下同）;SlGT-33基因经ABA处理2～24 h时相对表达量均显著高于对照。SlGT-33基因在高盐胁迫和机械损伤下的相对表达量与对照无显著差异;高温胁迫和低温胁迫下SlGT-33基因表达整体上均呈逐渐降低的变化趋势;在脱水胁迫下SlGT-33基因表达整体上均呈逐渐升高的变化趋势。通过农杆菌介导转化法获得6个SlGT-33-RNAi沉默株系,沉默效率为64%～83%。生长70 d的SlGT-33-RNAi沉默株系RNAi-4和RNAi-5幼苗株高和节间长度均为野生型的60%左右,且复叶结构尺寸明显变小,花序提前形成。茎尖组织中顶端分生组织关键转录因子基因KNOX2和WUS基因在SlGT-33-RNAi沉默株系的相对表达量均极显著（P<0.01）或显著高于野生型,腺苷酸异戊烯转移酶（CTK合成的关键酶）编码基因IPT2和茎尖生长点调控基因PHAN基因在2个SlGT-33-RNAi沉默株系的相对表达量均显著低于野生型。顶端分生组织关键转录因子基因KNOX1基因在2个SlGT-33-RNAi沉默株系的相对表达量与野生型无显著差异。SlGT-33-RNAi沉默株系茎尖组织的细胞分裂素（CTK）含量显著低于野生型。【结论】SlGT-33基因属于环境敏感型基因,其表达受ABA和脱水胁迫诱导,但受极端温度的抑制。抑制SlGT-33基因表达会导致了番茄植株矮化和生殖生长加速,其作用机制与顶端分生组织的CTK合成受抑制及茎尖组织调控基因KNOX2、PHAN和WUS的异常表达密切相关。     

Arabidopsis CLV3 peptide directly binds CLV1 ectodomain

CLV1, which encodes a leucine-rich repeat receptor kinase, and CLV3, which encodes a secreted peptide, function in the same genetic pathway to maintain stem cell populations in Arabidopsis shoot apical meristem. Here, we show biochemical evidence, by ligand binding assay and photoaffinity labeling, that the CLV3 peptide directly binds the CLV1 ectodomain with a dissociation constant of 17.5 nM. The CLV1 ectodomain also interacts with the structurally related CLE peptides, with distinct affinities depending on the specific amino acid sequence. Our results provide direct evidence that CLV3 and CLV1 function as a ligand-receptor pair involved in stem cell maintenance.     

青杄分子伴侣PwTCP-1基因的克隆及表达分析

分子伴侣TCP-1在调节植物生长发育过程中具有重要作用,但在林木生长发育及对非生物逆境胁迫响应机制方面的研究较少。从青杄cDNA文库中筛选出1个分子伴侣PwTCP-1(T-complex polypeptide 1)片段,通过RACE-PCR技术获得PwTCP-1的末端序列,经过与EST序列拼接,获得PwTCP-1 cDNA全长序列。使用clustalx、Mega5.0、DNAMAN和Expasy等工具对PwTCP-1的理化性质、二级结构和三级结构进行生物信息学分析。结果显示,PwTCP-1 cDNA全长为2 024 bp,开放阅读框1 605 bp,编码534个氨基酸残基的蛋白质,其理论分子质量为59.2 kDa,理论等电点为6.12,富含α-螺旋。通过RT-qPCR技术检测了PwTCP-1组织特异性表达、激素响应模式及在种子萌发过程中表达量变化。结果表明:PwTCP-1在青杄各个组织中均有表达,在花粉中表达量最高,在茎中表达量最低;PwTCP-1表达量随着种子萌发先上升,在第4天达到最高,之后缓慢下降。PwTCP-1能响应多种激素处理。油菜素内酯(brassinolide,BR)和赤霉素(gibberellin,GA)分别处理后,PwTCP-1的表达量均显著降低,而茉莉酸甲酯(methyl jasmonate,MeJA)处理后该基因表达量极显著升高。这些结果显示,PwTCP-1可能参与植物种子萌发过程并响应了多种激素反应过程。      

肌肉发育相关LncRNA的研究进展

生长发育性状是受遗传和环境因素共同作用和/或相互作用的复杂性状,尽管利用全基因组关联研究可分析基因组上全部基因,筛选出与某类性状关联的SNP,但很难综合评价某个基因对其确切的作用。寻找与生长发育相关的精准基因是育种研究的目标之一。长链非编码RNA(long noncoding RNA,LncRNA)在细胞增殖分化、个体发育、信号转导、干细胞维持、代谢等几乎所有重要生命活动中发挥关键的调控作用,在表观遗传水平、转录水平及转录后水平等方面具有控制基因表达的作用,与多种重大疾病的发生密切相关。LncRNA是一类长度大于200个nt,且不表现出蛋白质编码潜能的RNAs,通过多种机制发挥生物学功能,参与染色质修饰、X染色体沉默以及基因组印记、转录干扰、转录激活、核内运输等多种重要调控过程,涉及表观遗传调控、转录调控及转录后调控等多个层面。深入探讨LncRNA调控生肌因子进而调节肌肉发育分化的新路径,阐释哺乳动物生肌分子时间,寻找肌肉组织中与生长发育相关的新LncRNA分子,深入研究与生长发育密切相关的LncRNA分子及其靶基因的生物学功能,阐明 LncRNA 在肌肉生长发育的调控机制,是肌肉发育遗传育种的主要研究内容。本文就LncRNA在哺乳动物肌肉生长发育、细胞生长、分化、增殖中的作用进行综述。     

Induced pluripotent stem cell lines derived from human somatic cells

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.     

Aconitase couples metabolic regulation to mitochondrial DNA maintenance

Mitochondrial DNA (mtDNA) is essential for cells to maintain respiratory competency and is inherited as a protein-DNA complex called the nucleoid. We have identified 22 mtDNA-associated proteins in yeast, among which is mitochondrial aconitase (Aco1p). We show that this Krebs-cycle enzyme is essential for mtDNA maintenance independent of its catalytic activity. Regulation of ACO1 expression by the HAP and retrograde metabolic signaling pathways directly affects mtDNA maintenance. When constitutively expressed, Aco1p can replace the mtDNA packaging function of the high-mobility-group protein Abf2p. Thus, Aco1p may integrate metabolic signals and mtDNA maintenance.     

Nanos maintains germline stem cell self-renewal by preventing differentiation

Despite much progress in understanding how extrinsic signaling regulates stem cell self-renewal, little is known about how cell-autonomous gene regulation controls this process. In Drosophila ovaries, germline stem cells (GSCs) divide asymmetrically to produce daughter GSCs and cystoblasts, the latter of which develop into germline cysts. Here, we show that removing the translational repressor Nanos from either GSCs or their precursors, the primordial germ cells (PGCs), causes both cell types to differentiate into germline cysts. Thus, Nanos is essential for both establishing and maintaining GSCs by preventing their precocious entry into oogenesis. These functions are likely achieved by repressing the translation of differentiation factors in PGCs and GSCs.     

四种水稻蛋白与水稻黑条矮缩病毒编码非结构蛋白P7-2的互作分析

水稻黑条矮缩病毒(RBSDV)是斐济病毒属的成员之一,可侵染玉米和水稻等作物,给亚洲地区的田间生产带来严重的损失。RBSDV有10条双链RNA(double strand RNA, dsRNA)基因组,编码12个蛋白。其中6个蛋白是病毒粒子的结构成分(P1,P2,P3,P4,P8,P10),6个非结构蛋白分别为P5,P6,P7\|1,P7\|2,P9\|1,P9\|2。在非结构蛋白中,P5,P6和P9\|1已被证实参与形成毒质结构,P7\|1被认为可在细胞质中形成类似管状的结构作为病毒胞间扩散的通道,P7\|2和P9\|2的功能目前尚不明确。文章采用Pull\|Down和液相色谱—质谱联用(LC\|MS/MS)等技术来鉴定水稻蛋白(日本晴)与P7\|2蛋白间可能存在的互作关系。通过Pull\|Down实验分析,发现有4种水稻蛋白可与P7\|2相结合,其中2个蛋白为转录相关蛋白,1个为氨基转移酶,还有1个为假定的分子伴侣60前体。实时荧光定量PCR分析显示,感病寄主中的转录相关蛋白和假定的分子伴侣60前体的表达量上调,而氨基转移酶的表达量降低。