番茄茉莉酸缺失突变体灰霉菌侵染响应miRNA及其表达分析

为揭示miRNA对番茄灰霉菌胁迫的响应机制，以番茄茉莉酸（JA）缺失突变体def1、spr2及其野生型（CM）为试验材料，构建了3个材料接种灰霉菌前后（0 h、48 h）2个时期的miRNA文库，并采用Illumina平台测序，对测序数据进行生物信息分析，结合实时荧光定量检测目的miRNA及其预测的靶基因表达情况。结果表明灰霉菌侵染番茄后，JA缺失突变体def1和spr2的病情指数显著高于CM，H2O2含量低于CM。高通量测序鉴定了130个已知miRNA和811个新miRNA。进一步筛选出8个保守miRNA（Sly-miR156e-3p、Sly-miR166c-5p、Sly-miR171f、Sly-miR172b、Sly-miR319a、Sly-miR390b-5p、Sly-miR399b、Sly-miR482d-5p），其在6个样本中表达模式各异。预测差异miRNA靶基因122个，结合qRT-PCR技术分析了8个miRNA和8个靶基因的表达情况，与高通量测序结果基本一致。推测Sly-miR156e-3p、Sly-miR390b-5p、Sly-miR399b、Sly-miR482d-5p通过依赖JA信号途径参与番茄对灰霉病的抗性。     

辣椒GMS育性相关miRNA的鉴定和表达分析

研究辣椒花药中sRNA分布,筛选育性相关miRNA,通过miRNA及其靶基因的表达分析探讨miRNA对雄性育性的调控。利用高通量测序技术对辣椒细胞核雄性不育两用系处于小孢子单核靠边期的花药进行sRNA测序,同时分析两材料间miRNA的表达差异。通过qRT-PCR技术验证差异分析结果,并对miRNA及其靶基因在小孢子发育不同时期的表达模式进行分析。在构建的不育株和可育株两个文库中共发掘出857个可信度高的miRNA;筛选得到42个表达差异显著的miRNA;通过靶基因预测与注释,预测出的差异表达miRNA的靶基因中与繁殖有关的基因有152个,与生殖过程有关的基因有151个。通过qRT-PCR验证了选出的9条miRNA的存在,其表达差异与测序分析结果基本一致;单核靠边期多数miRNA负调控其靶基因,不同发育时期其调控模式会发生变化。本研究为揭示辣椒miRNA与雄性育性的关系提供了重要信息。     

基于microRNA深度测序的猕猴桃性别分化初探

利用新一代高通量测序技术,对猕猴桃雌花和雄花中表达的小RNA进行了测序,分别得到雌花18 408 610条序列和雄花11 191 469条序列。通过生物信息学分析,共鉴定和预测得到39个保守miRNA家族和400个新的miRNA家族,其中有170个miRNA家族在雌、雄花样品间显著差异表达。对差异表达miRNA进行靶基因的预测及注释,结果显示,靶基因产物具有包含核苷三磷酸水解酶的磷酸环结构域的miRNA数量最多。在猕猴桃25号连锁群（Chr25）上共预测得到3个miRNA,其中novel-ach-miR362的靶基因Achn298021可能与猕猴桃花的性别发育有关。     

利用转录组测序分析外源GA_3解除大蒜气生鳞茎休眠相关基因

利用外源GA_3打破'金乡'大蒜气生鳞茎休眠,采用高通量测序技术对休眠气生鳞茎和GA_3处理气生鳞茎进行转录组测序,分析筛选响应外源GA_3的差异表达基因,探究差异表达基因与GA_3解除气生鳞茎休眠的关系。结果表明:外源GA_3可有效打破气生鳞茎休眠;通过转录组测序得到上调差异基因4 588个,下调差异基因55 857个;28 107个差异表达基因在KEGG中获得注释,其中在代谢途径中获得注释的差异基因最多,其次为遗传信息处理途径。差异表达基因较多的富集于植物激素信号转导途径且富集结果显著;筛选出参与GA、ABA、ZR信号转导的显著差异表达基因GAI、SLR1、PP2C等,其表达情况与气生鳞茎内源激素含量的变化情况一致,推测外源GA_3可通过抑制GA信号途径负调控因子DELLA基因的表达,激活赤霉素的生物合成及信号转导过程,使气生鳞茎休眠解除,同时PYL的表达量显著降低,从而使得脱落酸含量相对降低,有利于休眠解除;qRT-PCR验证结果显示测序结果和实际结果一致。     

辣椒(Capsicum annuum L.)果实发育及品质相关新microRNA的鉴定与分析

MicroRNA(miRNA)是一种非编码的小RNA,在各种生物过程中起着重要的调节作用。前人研究表明,模式植物中miRNA与果实发育相关。然而,人们对辣椒(Capsicum annuum L.)果实品质及发育相关的miRNA了解尚不清楚。本研究利用二代测序技术(Next-generation sequencing technology)比较了不同品种辣椒果实在不同发育时期的小RNA。利用miRDeep2软件从4个样本中共鉴定出了59个已知miRNA和310个新miRNA。预测了新miRNA的靶基因,共656个,并对其中402个靶基因进行了注释。GO分析和KEGG途径表明,某些靶基因与淀粉糖、氨基糖以及核糖代谢相关。qPCR表明一些miRNA的表达模式与其靶向基因相反,该结果为今后研究miRNA调控果实发育和品质形成提供了重要基础。     

库尔勒香梨9个新miRNA克隆鉴定

利用在小RNA高通量测序试验中筛选出的脱萼组与宿萼组差异表达新miRNA基因,采用 Stem-loop法对总体表达量居前20位、在脱萼组和宿萼组中具有显著性差异表达、在子房和萼片组织中具有显著性差异表达的新miRNA进行成熟体的克隆鉴定、前体序列二级结构分析、qRT-PCR试验以及靶基因预测。结果显示,在不同的样本中有9个新miRNA（novel_miRNA）的成熟体序列以及4个novel_miRNA的表达量与高通量测序结果完全一致,并且预测得到大量具有生物学功能的靶基因。新发现的miRNA可能与‘库尔勒香梨’萼片脱落和宿存有密切关系。     

基于高通量测序发掘番木瓜果实成熟相关miRNA

【目的】番木瓜是典型的呼吸跃变型果实,外源乙烯处理使番木瓜呼吸跃变提前,促进果实成熟。分离番木瓜果实成熟相关miRNA,为深入了解呼吸跃变型果实的成熟分子机制奠定基础。【方法】利用高通量测序技术对乙烯(ETH)、1-MCP和清水对照(CG)处理的番木瓜果实进行miRNA和转录组高通量测序,然后对测序获得数据进行生物信息学分析,进行miRNA鉴定和靶基因预测,并与转录组测序结果进行关联分析。【结果】乙烯、1-MCP和对照处理分别获得10 734 196、16 486 803和16 067 290条纯净序列,共鉴定出523个miRNA。其中,已知miRNA个数为1-MCP(303)、CG(214)和ETH(239),新miRNA个数为1-MCP(184)、CG(188)和ETH(114)。与对照相比,在乙烯处理中上调和下调表达的miRNA分别是123和72条。靶基因预测共获得5 053个靶基因,KEGG功能富集分析显示它们参与了戊糖、葡萄糖醛酸转换、淀粉和蔗糖代谢、卟啉和叶绿素代谢、类胡萝卜素合成等代谢途径。筛选出的番木瓜果实成熟衰老相关候选miRNA,包含4个果实软化调控相关miRNA(miR167-y、miR4993-x、miR3946-x和miR5059-x)、3个果实颜色调控相关miRNA(miR4993-x、miR815-y和miR7810-x)、3个激素调控相关miRNA(miR4993-x、miR8004-x和miR9722-x)和4个转录因子调控相关miRNA(miR5641-y、miR9722-x、miR838-y和miR319-y)。【结论】筛选的番木瓜果实成熟衰老相关miRNA为今后果实成熟衰老调控网络研究提供了可能的线索。     

梨两个休眠相关MADS-box 基因特征及在其休眠过程中的表达分析

 PpMADS1 和PpMADS2 是从‘酥梨’（Pyrus pyrifolia white pear group‘Suli’）休眠芽转录组 文库筛选的两个与休眠相关的MADS-box 基因序列。为了解序列的特征,对其进行了相关生物信息学分 析,并以‘翠冠’和‘圆黄’梨为试材,用实时定量PCR 技术分析其花芽休眠不同阶段的表达变化。结 果表明：两个基因都具有MADS-box 家族的特征序列MIKC-基序,PpMADS1 和PpMADS2 分别与日本梨 （P. pyrifolia‘Kosui’）休眠相关的两个MADS-box 基因PpMADS13-1 和PpMADS13-2 聚在一起,且单独 聚为一支,与李属植物休眠相关的MADS-box 基因关系最近。在两个品种的休眠过程中,PpMADS1 和 PpMADS2 的表达呈现相似的变化趋势,都有一个表达高峰,但‘翠冠’梨PpMADS1 和PpMADS2 的表 达高峰均出现在11 月15 日,而‘圆黄’梨PpMADS1 的表达高峰延后到12 月30 日,PpMADS2 的表达 高峰出现在12 月15 日。两个品种PpMADS1 和PpMADS2 的表达高峰都出现在内休眠解除之前,随内休 眠解除其表达量下调,在生态休眠阶段表达量维持在较低的水平。据此推测PpMADS1 和PpMADS2 的表 达对梨芽内休眠的解除具有调控作用     

油桃开花时间调控基因在花芽休眠过程中的表达分析

对拟南芥中21个调控开花时间的基因进行NCBI blast比对后找到桃中所对应的同源基因,以拟南芥的突变体表型及基因注释功能为依据分为促进开花和抑制开花两类。以7年生‘曙光’油桃花芽为试材,利用水培法测定萌芽率,界定休眠分为诱导期、自然休眠期和休眠解除期3个阶段。研究结果表明诱导期和自然休眠期内花芽单芽质量上升平缓,休眠解除后突然升高到0.12 g,翌年2月9日达到0.15 g;而花芽含水量从诱导期开始缓慢下降,在休眠解除时达到最低水平（39%）,之后平稳上升到53%。聚类分析发现,两类基因对开花的影响与其在休眠进程中的表达趋势不存在必然的相关性。利用荧光定量PCR（RT-PCR）技术测定目的基因在休眠过程中的表达特性,发现在休眠进程（自然休眠和休眠解除期）中PpFT-like和PpCO-like表达量逐渐升高,推测其可能与需冷量积累相关。PpDDL-like、PpCCT-like、PpELF8-like、PpAMP1-like、PpHUA2-like和PpHUB1-like表达量先升高后降低,在休眠解除时达到最高水平之后随休眠解除下降,与花芽休眠解除进程一致,推测其可能参与调控花芽休眠解除过程。     

Simultaneous down-regulation of DORMANCY-ASSOCIATED MADS-box6 and SOC1 during dormancy release in Japanese apricot (Prunus mume) flower buds

In temperate deciduous fruit crops such as Prunus spp., bud endodormancy is an important physiological phase affecting the timing of blooming and subsequent fruit development. Japanese apricot (Prunus mume) bears unmixed flower buds, separate from vegetative buds, that bloom slightly more than a month before vegetative bud burst. Seasonal expression of Prunus mume DORMANCY ASSOCIATED MADS-box genes (PmDAMs) has previously been analyzed only in vegetative buds, with an association between these genes and flower bud endodormancy release not yet confirmed. In this study, we performed a seasonal expression analysis of PmDAM1–6 genes in flower buds of two Japanese apricot genotypes – namely, high-chill and low-chill cultivars. The analysis revealed that PmDAM3, PmDAM5, and PmDAM6 expressions are closely associated with dormancy release in both flower and vegetative buds. In addition, a yeast two-hybrid screening demonstrated that PmDAM6 can interact in yeast with the homolog of Arabidopsis SOC1 (PmSOC1). Synchronized expression patterns were detected in PmDAM6 and PmSOC1 during dormancy release in flower buds of the two genotypes. Taken together, these results suggest that the dimer of PmDAM6 and PmSOC1 may play a role in the regulation of dormancy transition and blooming time in Japanese apricot flower buds.     

Differentially expressed microRNAs during solid endosperm development in coconut (Cocos nucifera L.)

MicroRNAs (miRNAs) are a class of 20–24 nt, endogenously expressed, non-coding RNAs that play important regulatory roles in plants and animals. To identify miRNAs potentially involved in tissue development and compound anabolism, we studied miRNA expression profiles in endosperm of coconut at different developmental stages. Based on the annotation in miRBase (release 10.1), we measured a total of 179 miRNAs in immature (95 expressed miRNAs) and mature tissues (176 expressed miRNAs) using microarrays, respectively. The comparative analyses on miRNA expression profiles between these two groups of tissues showed that 23 miRNAs were up-regulated and nine miRNAs were down-regulated in matured endosperm. We further confirmed the increased expression of four miRNAs and decreased expression of a miRNA in immature endosperm using real-time PCR. Moreover, we computationally predicted the target genes of 32 miRNAs with differential expression (p < 0.01), and identified the lowest-score targets of six miRNAs. Finally, we discussed the potential functional relevance of several differentially expressed miRNAs.     

Transcriptome profiling reveals differentially expressed genes associated with wizened flower bud formation in Chinese pear (Pyrus bretschneideri Rehd.)

Flower buds are very important for pear yield; non-germinated flower buds become wizened and drop from the branch, reducing pear production. However, little research has focused on the study of wizened flower bud (WB) formation in spring. In order to elucidate the mechanism of WB formation in pear, physiological indices relating to plant hormones and antioxidases were measured. We found that activities of peroxidase (POD) and superoxide dismutase (SOD) were higher and lower, respectively, in WBs than in normal flower buds (NBs). The contents of indole acetic acid (IAA), gibberellin (GA), and cytokinin (CTK) were lower in WBs, while the level of addition of abscisic acid (ABA) was higher in WBs than in NBs. Differentially expressed genes (DEGs) were surveyed between WBs and NBs. In total, 23 DEGs relating to POD and SOD were detected from GO (Gene Ontology) enrichment, and 29 DEGs associated with plant hormone biosynthesis were found from the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway. Notably, the expression patterns of these 52 genes were consistent with variations of antioxidase activities or hormone contents. Because POD and SOD are stress-response enzymes, the differences in POD and SOD activities between NBs and WBs indicated that WB formation in pear could result from ambient environmental stresses that influence expression levels of hormone biosynthesis genes.     

剥鳞和化学药剂处理对甜樱桃花芽休眠及酚类物质的影响

  以7年生甜樱桃‘红灯’和‘早红宝石’为试材, 分析了自然休眠期间剥鳞和化学药剂处理对酚类物质含量及对休眠解除的影响。结果发现, 休眠花芽中的酚类物质主要分布于鳞片中, 剥鳞后, 花芽中酚类物质含量锐减。自然休眠的不同时期剥鳞对打破休眠的效果不同, 前期效果较为明显, 中期处于休眠的最深时期, 剥鳞不能打破休眠, 后期剥鳞也能打破休眠促进萌发。不同化学药剂在休眠的不同时期对酚类物质含量的影响不同: 自然休眠前期, KNO₃ 和硫脲减缓了酚类物质的积累速度, 相反H₂O₂ 加速了酚类物质的积累; 中期上述3种化学药剂对酚类物质含量的影响与早期相似; 后期KNO₃ 处理降低了酚类物质含量, 硫脲使酚类物质含量略有增加, 而H₂O₂ 使之显著增加。在打破休眠方面, KNO₃ 可提前2～3 d打破休眠, 硫脲没有明显效果, H₂O₂ 反而抑制休眠的解除。     

Preliminary study on miRNA expression spectrum related to chemotherapy resistance in colon cancer

AIM: To screen the chemotherapy resistance-related microRNAs (miRNAs) of colon cancer using gene chip technique, and to explore the mechanism of miRNAs regulating chemotherapy resistance. METHODS: Gene chip technique was used to analyze the expression of miRNAs in colon cancer cell line HCT8 and vincristine-resistant cell line HCT8/v, and screen the miRNAs with significantly different expression. The results were verified by RT-qPCR. The target genes of these miRNAs were predicted, and the Gene Ontology (GO) analysis and the signaling pathway analysis of the predicted genes were carried out. RESULTS: Altogether 342 miRNAs with significantly differential expression were selected, in which 190 were up-regulated, and 152 were down-regulated. The verification results of RT-qPCR showed that the expression of miR-125-5p, miR-181c-5p and miR-153-3 was consistent with the results of chip detection. The expression of miR-130a-3p and miR-149-3p was not consistent with the results of chip detection. The results of GO analysis showed that the main pathway of chemotherapy resistance-related genes was RNA polymerase II regulatory region sequence-specific DNA binding. The chemotherapy resistance-related genes played roles mainly through positive regulation and are mainly located in intracellular membrane-bound organelles. The results of KEGG analysis showed that the pathways associated with the most enriched chemotherapy resistance-related genes were axon guidance pathway, insulin signaling pathway, and phospholipase D signaling pathway.CONCLUSION: miRNAs are closely related to chemotherapy resistance in colon cancer. Through the researches on miRNAs, we can have a deeper understanding of the mechanism of chemotherapy resistance and provide new ideas for reversing chemotherapy resistance in colon cancer.     

‘翠冠’梨花芽休眠期碳水化合物变化及其相关基因表达研究

 以‘翠冠’梨（Pyrus pyrifolia Nakai）花芽为试材,连续两年研究了其在休眠过程中的碳水 化合物含量变化及编码α–淀粉酶、β–淀粉酶和α–葡聚糖磷酸化酶等碳水化合物代谢相关基因的表达 模式。两年的结果显示,随着‘翠冠’梨花芽休眠的加深,可溶性糖含量逐渐下降,11 月15 日降到最低。 此后,在内休眠及其解除过程中可溶性糖含量呈上升趋势。花芽中淀粉含量随着休眠的加深逐渐下降, 最低含量出现的时间在年度间有所不同。随着休眠开始解除,淀粉含量逐渐增加,在完全解除前达到最 大值,其后则逐渐下降。‘翠冠’梨花芽的PpAMY、PpBAM1、PpBAM2 和PpPHS 在内休眠阶段均出现一 个表达高峰,然后逐渐下降直到内休眠完全解除前。之后,除PpBAM2 外,其余3 个基因再次上调表达。 ‘翠冠’梨花芽休眠期间,淀粉含量的变化与PpAMY、PpBAM1、PpBAM2 和PpPHS 的表达变化之间存 在联系,由此推测这些基因可能参与了‘翠冠’梨花芽休眠期碳水化合物代谢的调控。