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。     

梨花芽休眠相关miRNA的鉴定和差异表达分析

为了探究梨花芽休眠进程中miRNA的表达模式和靶基因,利用Solexa测序技术、生物信息学分析和实时荧光定量PCR（qPCR）技术,对内休眠、内休眠解除和生态休眠解除3个时期梨花芽的miRNA进行高通量测序、筛选和鉴定。结果表明,内休眠、内休眠解除和生态休眠解除时期3个样本文库中分别有12 276 226、10 135 952、11 453 981条Unique序列。miRNA主要分布在21 ~ 24 nt之间,其中长度为24 nt的数量最多。共检测到151个已知的miRNA,属于39个不同的家族,并利用生物信息学软件预测到了209个新的miRNAs。比较分析从内休眠进入到生态休眠解除的整个休眠转换时期差异表达的miRNA,筛选出8个miRNA（ahy-miR156b-5p、cpa-miR319、aly-miR172c-3p、aau-miR396、mdm-miR858、aly-miR171b-3p、bdi-miR160f和hbr-miR166a）,其靶基因主要参与转录调控、信号传导等过程。利用qPCR验证了8个miRNA及其8个靶基因的表达。     

牡丹LINE 类反转录转座子RT 序列的克隆及分析

 利用LINE 简并引物从中原牡丹品种‘洛阳红’中扩增出580 bp 左右的目的条带,对其回 收、克隆、测序及相关生物信息学软件分析后获得32 条LINE 类牡丹反转录转座子RT（反转录酶）序列。 这些序列长度为547 ~ 593 bp,主要表现为点突变,经Clustal W 比对其同源性为25.7% ~ 94.2%。将其核 苷酸序列经聚类后可分为4 个家族,其中家族Ⅰ和 Ⅲ 分别占总序列数的50%和28%。将32 条RT 序列翻 译成氨基酸序列,在第18 氨基酸序列处有1 个非常保守的的甘氨酸（Gly）,在138 氨基酸序列处有1 个 半保守的赖氨酸（Lys）位点。32 条序列均发生较多的终止密码子突变和移码突变。与其他物种的系统进 化树分析,表明牡丹LINE 类反转录转座子RT 序列既有一定的保守性,同时也与其他物种间存在一定的 同源性。     

miR168家族进化特性及其在砂梨休眠期的表达模式分析

为了探究miR168家族的进化特性及其在梨休眠进程中的表达模式,通过检索实验室前期构建的砂梨（‘黄花’梨）miRNA文库、转录组数据库和miRBase数据库,得到植物miR168家族成员的成熟体和前体序列。从‘黄花’梨花芽中克隆了两条miR168前体,发现梨miR168家族包含2条前体序列和4条成熟体序列。采用生物信息分析和实时荧光定量PCR(qRT-PCR),对梨miR168家族的成熟体序列及前体（pre-miR168）序列进行分析,与已知植物miR168构建系统发育进化树,预测其靶基因,分析其在梨休眠发生进程中的表达情况。结果表明,梨pre-miR168序列均能形成稳定的发夹结构;植物pre-miR168两端序列较保守,而中间序列不保守;mi168成熟体保守性较高,进化树分析表明梨pre-miR168与苹果的亲缘关系最近,miR168成熟体主要分为两大类。梨miR168调控的潜在靶基因主要包含Argonaute1蛋白基因（protein argonaute 1,AGO1）和胼胝质合成酶3基因（callose synthase 3,CalS3）共21条mRNA,都是以裂解靶标的方式进...     

西瓜bHLH转录因子家族基因的鉴定及其在非生物胁迫下的表达分析

利用生物信息学方法,在西瓜测序基因组97103中共鉴定出96个bHLH家族成员,其中有94个可以被定位到西瓜的11条染色体上。通过基因结构和结构域序列保守性的预测,发现这些基因的序列长度和内含子数量变化很大,但bHLH结构域序列比较保守。用拟南芥中39条已知的bHLH蛋白序列和西瓜的96条bHLH蛋白序列构建系统发育树,结果显示西瓜的bHLH家族可以进一步被分为11个亚族。运用荧光定量实时PCR技术,分析了该家族中21个基因在西瓜响应非生物胁迫时的表达水平,结果表明,8个基因受低温胁迫诱导表达,13个基因受ABA胁迫诱导表达,14个基因受盐胁迫诱导表达。ClabHLH41在3种胁迫下表达量均显著增加,说明其在低温、ABA和盐胁迫应答中可能发挥着重要作用。     

果树miRNAs研究进展

模式植物的研究表明,miRNA作为一种转录后调控因子,在植物的生长发育、逆境胁迫应答等生物学过程中发挥着重要的调节作用。截至目前,虽然已经从多种果树中鉴定了大量的miRNAs,但大多数miRNAs的靶基因和功能特性还不清楚。笔者总结了目前果树中miRNAs的研究进展,特别是miRNAs在葡萄(Vitis vinifera)、桃(Prunus per-sica)、梨(Pyrus spp.)、苹果(Malus domestica)和柑橘(Citrus spp.)等具有重要经济价值的果树等方面的作用,比如调控果树生长发育与果实品质、激素信号转导和环境胁迫应答。探讨了果树miRNAs的前景和研究方向。     

枣AP2/ERF转录因子鉴定及其响应枣疯病植原体表达分析

利用生物信息学方法,从植物转录因子数据库和NCBI数据库中分别得到175和164个候选的枣AP2/ERF转录因子序列,使用DNAMAN软件进行序列比对、去除重复序列,采用SMART软件预测蛋白结构域发现,枣基因组中包含有145个AP2/ERF基因,其中ERF、AP2、RAV亚家族分别含有116个、23个、5个,另有1个独立基因。预测枣AP2/ERF转录因子氨基酸数量在111～692之间,分子量在12 446.87～76 154.10之间,pI在4.31～10.11之间。鉴定出的145个AP2/ERF转录因子,105个分别定位到12条染色体上,40个未能定位。在此基础上,利用嫁接病芽方法将枣疯病植原体转至健康枣植株,通过转录组测序和qRT-PCR手段,分析了AP2/ERF转录因子对枣植原体侵染的响应,在植原体侵染枣的6个不同时期,枣AP2/ERF表达数量和表达量均不相同,共有48个差异表达基因,其中ZjAP2*9、ZjERF49和ZjERF91是响应枣疯病植原体最为重要的AP2/ERF转录因子。     

苹果WRKY转录因子家族基因生物信息学分析

 利用生物信息学方法对苹果MdWRKY转录因子家族成员、基因分类、染色体定位、系统进化关系和结构域序列保守性进行了预测,并分析基因在果实成熟期和砧穗互作中的表达差异。苹果MdWRKY家族包含116个基因,分为GroupⅠ、GroupⅡ和GroupⅢ,其中GroupⅡ又可细分为GroupⅡa、GroupⅡb、GroupⅡc、GroupⅡd和GroupⅡe亚类;苹果的17条染色体均有WRKY转录因子分布,其中第1条染色体上的分布最多,有12个WRKY基因分布。MdWRKY编码的蛋白在118 ~ 965个氨基酸范围内,等电点位于4.81 ~ 10.16之间。Microarray分析发现,在苹果果实成熟时期和砧木接穗互作过程中,多数MdWRKY基因的表达都有不同程度的变化。     

胡萝卜类甜蛋白家族鉴定与生物信息学分析

为了全面了解胡萝卜基因组中类甜蛋白家族基因结构和蛋白功能,利用胡萝卜基因组数据库,通过生物信息学方法对筛选的32个胡萝卜类甜蛋白家族成员进行鉴定、聚类及结构功能分析。结果表明:胡萝卜类甜蛋白家族基因分布在8条染色体上,该家族蛋白保守性较强。系统发育分析显示,胡萝卜类甜蛋白家族属于10个进化组,其中进化组5中的基因主要来自1号染色体,该组成员具有抑菌潜力,值得深入研究。     

矮丛蓝莓品种“美登”叶片转录组测序和生物信息学分析

以矮丛蓝莓品种"美登"为试材,采用第二代测序技术对其嫩叶转录组进行了测序及生物信息学分析,研究了矮丛蓝莓功能基因组信息,以期增加对矮丛蓝莓生长发育分子机理的了解,并为矮丛蓝莓的分子标记辅助育种奠定基础。结果表明:在获得的约1G纯净数据,共拼装了41 929条Unigenes,其中被GO数据库成功注释Unigene分别涉及到生物学过程、细胞成分及分子功能相关的共45类生理代谢功能;KOG数据库注释到19 149条Unigenes,共涉及25条代谢通路;KEGG数据库成功注释13 072条Unigenes,共涉及到5个大类、18个中类、129条代谢通路;Nr数据库成功注释30 738条Unigenes,这些Unigenes共对应32 949条蛋白质的编码区。同时,生物信息学分析还显示,全部Unigenes中,有884条编码转录因子,涉及到55个家族;2 261条编码抗性基因,涉及18个家族;检测到6 591个SSR位点,其中2碱基重复的SSR数目最多,占总SSR位点数的70%。     

Cloning and identification of novel miRNAs in the flower organs of Korla fragrant pear at anthesis

The objective of the study was to learn more about some newly identified miRNAs related to calyx persistence in Korla fragrant pear (Pyrus sinkiangensis Yu). Small RNAs were subjected to high-throughput sequencing after extraction from the ovaries and sepals of flowers with either a deciduous or a persistent calyx. Differentially expressed miRNAs were screened, and 73 new miRNAs were obtained. Twenty of these new miRNAs were selected to further validate all of the new miRNAs. Their mature miRNAs were cloned and identified, the secondary structures of the precursor miRNAs (pre-miRNAs) were analysed, and then qRT-PCR analysis was conducted. The results showed that the mature sequences of nine new miRNAs (novel_miRNA) in different samples were consistent with the results of high-throughput sequencing. Overall, this study improved current methods for the molecular cloning and identification of fruit tree miRNA. Nine new miRNA types were identified. This study laid a good foundation for elucidating the biological functions of these new miRNAs.     

枣树阶段转变相关microRNA家族的鉴定及其表达分析

与其他果树相比,枣树具有童期短、成花快的特征.已有研究表明,多个microRNA(miRNA)家族参与植物阶段转变和开花时间调控等过程.研究枣树阶段转变相关的miRNA家族对果树童期调控具有重要意义.以枣实生后代植株不同发育阶段(节位)的当年生枝(枣吊)为材料,通过Small RNA测序,在童期、过渡期和成年期等3个时...     

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。     

蕹菜耐受长时间高温后的miRNA分析

miRNA是广泛存在于真核生物体内的一类长度为20～24 nt的小分子非编码RNA,在植物生长发育和逆境适应过程中发挥重要作用。蕹菜(Ipomoea aquatica)可以耐受长时间高温,而其miRNA还未被鉴定,其在耐受长时间高温后的作用还未知。以蕹菜耐热性强的品种‘泰国三叉’和耐热性差的品种‘柳叶’为材料,经42℃高温处理15d后分别构建两个高质量sRNA库,经检测两个库中碱基质量值大于或等于30的序列数量均超过17.3Mb,且‘泰国三叉’中24nt的序列数量远远高于‘柳叶’。与蕹菜转录组比对分析后,分别得到1 363 258和1 629 209条序列,两个库共有的序列仅有1 047 133条(11.36%),说明耐热性不同的蕹菜中s RNA有很大差别。共鉴定出蕹菜中存在71个miRNA,其中差异表达的有22个,通过TargetFinder预测其靶基因,共得到233个靶基因。进一步以耐热性强的品种‘本地三叉’和耐热性弱的品种‘竹叶’为材料研究其在长时间高温后miR160、miR166、miR172、miR393和miR3627前体的差异表达量,结果表明,高温后两品种中pre-miR160-2的表达都显著上调,但是不耐热的‘竹叶’中上调的倍数更多;mi R166在耐热性强的‘本地三叉’中表达量下降,在耐热性较差的‘竹叶’中表达量显著上升;miR172可以被长时间高温诱导,但在耐热性不同的两个品种蕹菜中并没有差异;miR3627的表达量在耐热蕹菜‘本地三叉’中下降,在不耐热蕹菜‘竹叶’中没有明显变化。miRNA前体表达模式的差异说明其在蕹菜耐受长时间高温过程中的作用机制不同。     

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.     

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、Sl...     

Microarray-based miRNAs profiling in lower limb arteriosclerosis of diabetic rats

AIM: To establish the profiling of microRNAs (miRNAs) in the lower extremity arterial tissue between diabetic rats with lower limb arteriosclerosis (DAS) and diabetic rats with normal lower limb (DN), and to explore the possible molecular mechanisms involved in aberrant miRNA expression in DAS. METHODS: The rat models of DAS and DN were successfully established. The respective lower extremity arterial tissue was isolated. The total miRNAs were purified for a hybridization detection by miRNA microarray. The results of chip scanning and data were analyzed and verified by RT-qPCR. RESULTS: Ten miRNAs related to DAS, including rno-miR-206-3p, rno-miR-133a-5p, rno-miR-133b-3p, rno-miR-133a-3p, rno-miR-325-5p, rno-miR-675-3p, rno-miR-411-5p, rno-miR-329-3p, rno-miR-335 and rno-miR-126a-3p, were determined. All 10 abnormally expressed miRNAs were up-regulated. The validating results of RT-qPCR confirmed 9 of the miRNAs in line with chip expression. Just rno-miR-335 showed the opposite between PCR detection and microarray result. CONCLUSION: A group of miRNAs in diabetic rats suffering from lower limb arteriosclerosis plays an important role in the vascular atherosclerosis process. The abnormal expression of miRNAs is likely to affect the vascular atherosclerosis process.     

MicroRNA profile analysis of ischemic myocardial tissues from rats with acute myocardial infarction

AIM：To identify differentially expressed microRNAs (miRNAs) in ischemic myocardial tissues from the rats with acute myocardial infarction (AMI) by miRNA array technique, and to predict their targets and analyze their functions using bioinformatics. METHODS：The rat models of AMI (n=3) were prepared by ligaturing the left anterior descending coronary artery (LAD) of Wistar rats. Electrocardiogram and blood pressure were detected during the operation, and the myocardial infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Ischemic myocardial tissues were isolated from the infarct area 4 h after ischemia. The same procedure in sham group (n=3) was performed except for ligaturing LAD. Total RNA was extracted from ischemic and normal myocardial tissues. miRNA was isolated from total RNA, labeled with Cy3 and hybridized on miRNA array. Real-time PCR was applied to verify the reliability of miRNA array results. The targets of differentially expressed miRNAs were predicted and their functions were analyzed by bioinformatics. RESULTS：Rat model of AMI was successfully prepared and verified by electrocardiogram detection, blood pressure measurement and pathological observation. Compared with sham group, microarray screening showed that total 11 AMI-related miRNAs were selected, including 6 up-regulated and 5 down-regulated. Three of them (rno-miR-181c, rno-miR-146b and rno-miR-208) were related to the cardiovascular functions, while the functions of the others (rno-miR-672*, rno-miR-743b, rno-miR-128, rno-miR-138-1*, rno-miR-336, rno-miR-138-2*, rno-miR-325-3p and rno-miR-3572) were unknown and might be novel AMI-related biomarkers. Parts of the miRNA targets were also related to the cardiovascular functions. CONCLUSION：Differentially expressed miRNAs in AMI rats may serve as novel biomarkers for diagnosis of AMI and potential targets for treatment of AMI.     

Effect of Ad-14-3-3σ on microRNA expression in different radioresistant nasopharyngeal carcinoma cells

AIM: To discuss the effect of Ad-14-3-3σ to microRNA (miRNA) in different radioresistant nasopharyngeal carcinoma (NPC) cells, CNE-1 and CNE-2, and study the relationship between the discrepancy of miRNA and radiosensitivity of NPC.METHODS: Ad-14-3-3σ was transfected to CNE-1 and CNE-2 cells, and then miRNAs were detected by Paraflo microfluidic microRNA chip. Hybridization images were collected using a laser scanner and the signals were normalized using a LOWESS filter. The effect of Ad-14-3-3σ to miRNAs and the relationship between the discrepancy of miRNA and radiosensitivity of NPC were studied according to Targetscan3.1 database (http://www.targetsan.org) after analyzing data.RESULTS: After treated by Ad-14-3-3σ, comparing to CNE-2 cells, there are 37 miRNAs changed remarkably, including 17 over-expression microRNAs and 20 under-expression microRNAs in CNE-1 cells. 6 miRNAs that one detective value was more than 1 000 and 3 folds than the other were hsa-miR-152,hsa-miR-205,hsa-miR-203,hsa-miR-7,hsa-miR-636 and hsa-miR-100.CONCLUSION: Ad-14-3-3σ can change the expression of miRNAs in different radioresistant nasopharyngeal carcinoma, and some miRNAs have relevance to carcinoma and radiosensitivity.