Genome-wide analysis of the calcium-dependent protein kinase gene family inGossypium raimondii

Plant calcium-dependent protein kinases(CDPKs) play important roles in diverse physiological processes by regulating the downstream components of calcium signaling. To date, only a few species of the plant CDPK gene family have been functionally identified. In addition, there has been no systematic analysis of the CDPK family in cotton. Here, 41 putative cotton CDPK(Gr CDPK) genes were identified via bioinformatics analysis of the entire genome of Gossypium raimondii and were classified into four groups based on evolutionary relatedness. Gene structure analysis indicated that most of these Gr CDPK genes share a similar intron-exon structure(7 or 8 exons), strongly supporting their close evolutionary relationships. Chromosomal distributions and phylogenetics analysis showed that 13 pairs of Gr CDPK genes arose via segmental duplication events. Furthermore, using microarray data of upland cotton(G. hirsutum L.), comparative profiles analysis of these Gh CDPKs indicated that some of the encoding genes might be involved in the responses to multiple abiotic stresses and play important regulatory roles during cotton fiber development. This study is the first genome-wide analysis of the CDPK family in cotton, and it will provide valuable information for the further functional characterization of cotton CDPK genes.     

Generation and Analysis of Expressed Sequence Tags （ESTs） from Muscle Full-Length cDNA Library of Wujin Pig

Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.     

Differential Gene Expression Between Hybrids and Their Parents During the Four Crucial Stages of Cotton Growth and Development

The study aims to clarify the differential gene expression between cotton hybrids and their parents in order to better understand the molecular basis of cotton heterosis. The research focused on cotton heterotic and lower heterotic hybrids and their parents during the four crucial stages, which were analyzed using a differential display technique. The results indicated that there were both quantitative and qualitative differences in gene expression amongst them. The quantitative differences include over- and under-expression of parental genes and the dominant expression of highly-expressed parental genes in hybrids. In contrast, the qualitative differences are the following： （i） Bands were observed in both parents but not in the F1 hybrid （BPnF1）; （ii） bands occurred in either of the parents but not in the F1 hybrid （UPnF1）; （iii） bands presented only in the F1 hybrid but not in either of the parents （UF1nP）; and （iv） bands were detected in either of the parents and the F1 hybrid （UPF1）. Overall, the major differences of gene expression occurred in the qualitative level and four related differential patterns were observed. Furthermore, the amount of differential patterns during the flowering stage was relatively higher than those of other stages. At this juncture, both the amount of hybrid-specific expression patterns at flowering stage and the silenced expression patterns at boll-forming stage in highly heterotic hybrids were found higher than those in the lower heterotic ones. It was concluded that significant differences of gene expression in leaves were present between cotton hybrid and its parents during the whole growing stages. Hence, these differences might be responsible for the observed cotton heterosis.     

Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

The normalization of quantitative real-time PCR （qPCR） is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions, qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes （LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9） in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were fiarther confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.     

Differential Gene Expression in Response to Drought Stress in Maize Seedling

To provide the useful information for the choice of molecular marker used in marker-assisted selection of drought tolerance, it is necessary to find out more candidate genes and fulfill the information gaps in gene expression regulation under drought stress. In this study, we isolated four differentially expressed cDNA fragments from leaves of a droughttolerant inbred line by suppression subtractive hybridization and reverse Northern hybridization, and validated their differential expression patterns among six inbred lines with different drought tolerance in response to drought stress by quantitative real-time PCR. Sequence similarity analysis indicated that two of four differentially expressed cDNA showed homology to gene DegP encoding trypsin-like serine protease, and gene PGAM-i encoding cofactor-independent phosphoglyceromutase, respectively. Expressions of the genes corresponding to four cDNA fragments was decreased at 6 h after drought stress treatment in most of the six inbred lines, and then returned to the control level with further stress in three of the tolerant inbred lines. The expression of the gene PGAM-i and the genes corresponding to fragments E4 and F4 were increased to a high level in tolerant inbred line 81565. In the two drought-sensitive inbred lines （Dan340 and ES40）, the expression of these genes was still down-regulated. The probable mechanisms of these genes in response to drought stress were discussed. These results indicated that the drought-tolerant inbred lines upregulated the expression of the drought-tolerant candidate genes, in contrast, drought-sensitive inbred lines downregulated the expression of the genes.     

Alterations of Alternative Splicing Patterns of Ser/Arg-Rich （SR） Genes in Response to Hormones and Stresses Treatments in Different Ecotypes of Rice （Oryza sativa）

Ser/Arg-rich (SR) genes encode proteins that play pivotal roles in both constitutive and alternative splicing of pre-mRNA. However, not much effort has been made to investigate the alternative splicing of their own pre-mRNA. In this study, we conducted comprehensive analyses of pre-mRNA splicing for 22 SR genes in three rice (Oryza sativa L.) ecotypes indica, japonica and javanica. Using different ecotypes we characterized the variations in expression and splicing patterns of rice SR genes in different tissues and at different developmental stages. In addition, we compared the divergence in expression and splicing patterns of SR genes from seedlings of different rice ecotypes in response to hormones application and environmental stresses. Our results revealed the complexity of alternative splicing of SR genes in rice. The splicing varies in different tissues, in different ecotypes, in response to stresses and hormones. Thus, our study suggested that SR genes were subjected to sophisticated alternative splicing although their encoding proteins were involved in the splicing process.     

Evolution Analysis About Soybean MIR166 Family

MicroRNA(miRNA) is a class of important regulating non-coding small molecular RNA. The gma-MIR166 gene family consists of 21 members and their expression patterns diversify widely. It is important to analyze the evolution of gma-MIR166 gene family in order to understand the evolutionary mechanisms of miRNAs in soybean. In this study, we implemented soybean wide genome block analysis, the molecular phylogeny of gma-MIR166 and block analysis of gma-MIR166 family. The results showed that both chromosome big segmental duplications and tandem duplications were main reasons contributed to the expanding of gmaMIR166 gene family. These findings suggested that gma-MIR166 gene family might originate from one or two ancient miRNA genes. The results of research provided a support for evolutionary study of miRNAs in soybean and related species in Fabaceae.     

Identification and expression of the CEP gene family in apple (Malus×domestica)

Plant peptide hormones play important roles in plant growth and development. Among these hormones, the C-TERMINALLYENCODED PEPTIDE(CEP) belongs to a newly found peptide family that regulates root development in Arabidopsis as well as in other species. However, nothing is known about the CEP genes in apple(Malus×domestica, MdCEP). In this study, a total of 27 apple CEP genes were identified through a genome-wide analysis and were phylogenetically divided into three classes(Ⅰ, Ⅱ and Ⅲ). The predicted MdCEP genes were distributed across 10 of 17 chromosomes with different densities. Next, the gene structures and motif compositions of the MdCEP genes were analyzed. Subsequently, the expression analysis suggested that the MdCEP genes were highly activated in roots and that MdCEP23 may play an important role in regulating the growth and development of roots. Moreover, all of the MdCEP genes were responsive to multiple abiotic stresses, indicating that MdCEP genes may be involved with various aspects of physiological processes in apple. Nearly one-third of MdCEP genes had a significant response to low nitrogen treatment. Most of the MdCEP genes were up-regulated under stress, including mannitol, polyethylene glycol(PEG) and abscisic acid(ABA), suggesting that MdCEP genes may be involved in the drought stress response. This study provides insight into the putative functions of the MdCEP genes using a genome-wide analysis of the CEP gene family.     

普通烟草CDPK基因家族的克隆及表达分析

 【目的】从烟草中分离更多CDPK基因,分析该基因家族的序列特征、进化关系和表达谱,为全面揭示其功能奠定基础。【方法】采用RT-PCR、RACE和生物信息学的方法,分离普通烟草CDPK基因;采用MEGA4.0软件构建系统进化树;利用RT-PCR研究普通烟草全长基因的表达谱。【结果】分离到普通烟草CDPK基因8个,其中3个可以找到完整的开放读码框;系统进化树分析显示,烟草属11个全长CDPK基因位于4个亚家族,预测的2对普通烟草和拟南芥的直系同源基因在功能上可能非常保守;3个普通烟草全长基因虽然在所有的组织中都能检测到,但是它们的表达谱在不同的组织间存在明显差异。【结论】分离到8个未报道的普通烟草CDPK基因,结合已有的报道,目前在普通烟草中获得了15个CDPK基因,为在基因组范围内研究普通烟草CDPK基因家族的功能奠定了基础。     

Genome-wide analysis of the calcium-dependent protein kinase gene family in Gossypium raimondii

Plant calcium-dependent protein kinases(CDPKs) play important roles in diverse physiological processes by regulating the downstream components of calcium signaling. To date, only a few species of the plant CDPK gene family have been functionally identified. In addition, there has been no systematic analysis of the CDPK family in cotton. Here, 41 putative cotton CDPK(Gr CDPK) genes were identified via bioinformatics analysis of the entire genome of Gossypium raimondii and were classified into four groups based on evolutionary relatedness. Gene structure analysis indicated that most of these Gr CDPK genes share a similar intron-exon structure(7 or 8 exons), strongly supporting their close evolutionary relationships. Chromosomal distributions and phylogenetics analysis showed that 13 pairs of Gr CDPK genes arose via segmental duplication events. Furthermore, using microarray data of upland cotton(G. hirsutum L.), comparative profiles analysis of these Gh CDPKs indicated that some of the encoding genes might be involved in the responses to multiple abiotic stresses and play important regulatory roles during cotton fiber development. This study is the first genome-wide analysis of the CDPK family in cotton, and it will provide valuable information for the further functional characterization of cotton CDPK genes.     

木薯淀粉分支酶基因MeSBE2.2的克隆和表达分析

为了明确淀粉分支酶(SBE)在木薯淀粉合成中的功能,利用RT-PCR方法克隆得到了MeSBE2.2序列,并通过实时定量PCR(Q-PCR)分析其转录特点。结果表明,MeSBE2.2全长2 053bp,编码616个氨基酸,其编码蛋白质序列与蓖麻SBEII氨基酸序列的同源性最高,为91%。研究其转录特点发现:MeSBE2.2在块根和叶片中的表达模式有品种间差异性;器官特异性表达分析结果表明,MeSBE2.2主要在木薯块根中柱中表达;于3个不同生长时期的表达分析发现,MeSBE2.2在块根中表达平稳,在叶片中其表达量随植株生长呈显著下降趋势。本研究结果为进一步研究SBE家族在木薯块根淀粉合成中的功能提供了重要依据。     

梨树CDPK基因家族进化和表达分析

钙依赖蛋白激酶(CDPK或CPK)在植物生长和发育、逆境信号刺激及其对病原物的防御反应过程中发挥着非常重要的作用。本研究利用梨树全基因组数据,采用生物信息学的方法,在全基因组水平上对梨树CDPK基因家族进行系谱进化关系、基因结构、共线性关系及表达情况等分析。结果表明,梨树基因组中共存在31个CDPK基因,命名为PbCDPK1~PbCDPK31。系谱分析结果表明,这些CDPK基因归属于4个亚家族。共线性分析检测到12对CDKP基因间存在显著的共线性关系,其中10对由最近一次发生的全基因组复制事件形成。非同义/同义置换率的比率(dN/dS)说明CDPK基因在功能上进化得非常保守。荧光定量PCR结果发现,5个梨树CDPK基因对干旱逆境有响应。试验结果可为进一步开展梨树CDPK基因家族的功能鉴定和分子进化机制的研究提供参考。     

GhCDPK4基因的克隆和功能分析

为了研究棉花中GhCDPK4基因在响应非生物胁迫中所起的作用,通过PCR的方法克隆GhCDPK4基因,利用基因重组技术,构建植物过表达载体,采用农杆菌介导的叶盘法转化模式植物烟草,分析干旱和盐胁迫处理对转基因烟草表型和生理生化指标的影响。本研究成功克隆了属于棉花CDPK家族的基因GhCDPK4,构建了植物过表达载体pCAMBIA2300-GhCDPK4。实时荧光定量PCR(qRT-PCR)检测发现转基因烟草中GhCDPK4基因高水平表达,并且转基因烟草相比于野生型烟草表现出较强的耐旱和耐盐性,其中SOD、POD和CAT活性显著升高,而相对电导率和MDA含量降低。研究结果表明GhCDPK4基因可正向参与应答干旱和盐胁迫。     

Isolating the Mutator Transposable Element Insertional Mutant Gene mio16 of Maize Using Double Selected Amplification of Insertion Flanking Fragments (DSAIFF)

Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (mio16) was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments (DSAIFF), was employed to isolate the Mu flanking fragments (MFFs) of mio16. The target site duplications (TSDs) isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and mio16 was mapped in silico onto the physical position ranged from 229 965 021 to 229 965 409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of mio16 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of mio16 is alternated by Mu insertion.     

蕙兰Flowering locus T基因的克隆及其对开花的影响

【目的】 探讨CfFT基因在蕙兰成花中的作用。【方法】采用RT-PCR结合RACE技术从蕙兰(Cymbidium faberi)中克隆Flowering locus T（FT）同源基因CfFT。采用实时定量RT-PCR对不同组织及不同花发育时期CfFT进行表达分析。将该基因克隆到PBI121载体上导入烟草中,并对不同转基因烟草株系中的CfFT、NFL、NtFUL和NAP1基因进行实时定量RT-PCR分析。【结果】对蕙兰花芽分化不同时期的CfFT的表达分析表明,CfFT在花芽分化初期的表达量最高,之后随着花芽的成熟表达量逐渐降低。将CfFT导入烟草进行异源表达,转基因株系表现出明显的早花表型。对开花时间不一的转基因株系中的CfFT表达分析表明,其表达量与转基因烟草开花时间早晚成正比。进一步对这些株系内源的NFL、NAP1和NtFUL表达分析表明,NFL、NAP1和NtFUL基因的表达量与CfFT表达成正比,说明NFL、NAP1和NtFUL的表达受FT基因的上游调控。【结论】在烟草中异源表达蕙兰中的CfFT基因能促进烟草提前开花。     

Genome-wide identification of WOX gene family in apple and a functional analysis of MdWOX4b during adventitious root formation

The plant-specific WUSCHEL-related homeobox (WOX) genes are crucial for plant growth and development. Here, we systematically identified the MdWOX gene family in apple at the genome-wide level, and analyzed the phylogenetic relationships, conserved motifs, gene structure, and syntenic relationships of the MdWOX genes. A total of 18 MdWOX genes were identified and phylogenetic analysis placed them into three clades. The phylogenetic relationships among the WOXs were further supported by the analyses of gene structure and conserved motifs. Chromosomal distribution and synteny analysis revealed that whole-genome and segmental duplications have played key roles in MdWOX gene family expansion. Moreover, the MdWOX genes exhibit tissue-specific expression patterns and MdWOX4a, MdWOX4b, MdWOX5b, MdWOX11/12a, and MdWOX11/12b may play essential roles in adventitious root development. The adventitious rooting ability was enhanced in MdWOX4b transgenic tobacco lines. The results of this study provide useful information for future functional studies on MdWOXs in the development of apple rootstocks.     

玉米钙依赖蛋白激酶38(ZmCDPK38)的生物信息学及表达特性分析

[目的]研究ZmCDPK38与其它物种中同源基因之间的遗传进化关系,通过与亲缘关系较近且功能已知的同源基因比较,对ZmCDPK38的功能进行理论预判.确定ZmCDPK38基因在不同组织和不同胁迫条件下的表达特性.[方法]生物信息学分析使用在线分析工具及MEGAX软件;基因表达分析采用Real-time PCR.[结果]...