Genome-Wide Analysis of the Sus Gene Family in Cotton

Sucrose synthase (Sus) is a key enzyme in plant sucrose metabolism. In cotton, Sus (EC 2.4.1.13) is the main enzyme that degrades sucrose imported into cotton fibers from the phloem of the seed coat. This study demonstrated that the genomes of Gossypium arboreum L., G. raimondii Ulbr., and G. hirsutum L., contained 8, 8, and 15 Sus genes, respectively. Their structural organizations, phylogenetic relationships, and expression profiles were characterized. Comparisons of genomic and coding sequences identified multiple introns, the number and positions of which were highly conserved between diploid and allotetraploid cotton species. Most of the phylogenetic clades contained sequences from all three species, suggesting that the Sus genes of tetraploid G. hirsutum derived from those of its diploid ancestors. One Sus group (Sus I) underwent expansion during cotton evolution. Expression analyses indicated that most Sus genes were differentially expressed in various tissues and had development-dependent expression profiles in cotton fiber cells. Members of the same orthologous group had very similar expression patterns in all three species. These results provide new insights into the evolution of the cotton Sus gene family, and insight into its members' physiological functions during fiber growth and development.     

Copy number lability and evolutionary dynamics of the Adh gene family in diploid and tetraploid cotton (Gossypium)

Nuclear-encoded genes exist in families of various sizes. To further our understanding of the evolutionary dynamics of nuclear gene families we present a characterization of the structure and evolution of the alcohol dehydrogenase (Adh) gene family in diploid and tetraploid members of the cotton genus (Gossypium, Malvaceae). A PCR-based approach was employed to isolate and sequence multiple Adh gene family members, and Southern hybridization analyses were used to document variation in gene copy number. Adh gene copy number varies among Gossypium species, with diploids containing at least seven Adh loci in two primary gene lineages. Allotetraploid Gossypium species are inferred to contain at least 14 loci. Intron lengths vary markedly between loci, and one locus has lost two introns usually found in other plant Adh genes. Multiple examples of apparent gene duplication events were observed and at least one case of pseudogenization and one case of gene elimination were also found. Thus, Adh gene family structure is dynamic within this single plant genus. Evolutionary rate estimates differ between loci and in some cases between organismal lineages at the same locus. We suggest that dynamic fluctuation in copy number will prove common for nuclear genes, and we discuss the implications of this perspective for inferences of orthology and functional evolution.     

雷蒙德氏棉HSP70基因家族的进化分析及其同源基因在陆地棉中的表达分析

热激蛋白70家族(HSP70)是一类在植物中高度保守的分子伴侣蛋白,在细胞中协助蛋白质正确折叠。文章利用隐马可链夫模型(HMM)在雷蒙德氏棉(Gossypium raimondii L.)全基因组范围内进行HSP70基因家族成员进化分析,共得到30个HSP70家族成员。利用生物信息学对雷蒙德氏棉HSP70基因的结构、染色体分布、基因倍增模式以及系统进化进行分析,结果表明,HSP70基因家族根据亚细胞定位结果可分为不同的基因亚家族,各亚家族中HSP70基因具有相对保守的基因结构;染色体片段重复和串联重复是雷蒙德氏棉HSP70基因家族扩增的主要方式。通过对不同物种的HSP70基因家族进行系统进化分析可知,HSP70亚组的分化发生在单细胞植物形成前,且细胞质型HSP70成员大量扩增。比较陆地棉棉纤维发育不同时期的深度测序表达谱,发现HSP70基因可能参与棉纤维的生长发育。本研究结果有助于了解棉属植物HSP70基因家族的功能,以期为深入研究棉纤维发育过程中的分子调控机理提供基础。     

Molecular analysis of an alcohol dehydrogenase (Adh) gene from chromosome 1 of wheat

We have cloned and determined the nucleotide sequence of a gene encoding alcohol dehydrogenase (Adh) from Triticum aestivum cv. Millewa. Southern analysis using cv. Chinese Spring nullisomic-tetrasomic and ditelosomic lines established that the cloned gene mapped to the long arm of chromosome 1A and does not correspond to any previously identified wheat Adh locus. Southern analysis also provided evidence for triplicate copies of this Adh gene on the homoeologous group 1 chromosomes, while Northern blots indicated that the homoeologous group 1 Adh genes, like several other plant Adh genes, are transcribed under anaerobic conditions. Sequence analysis indicates that the cloned gene has a structure similar to both monocot and dicot Adh genes with an open reading frame encoding a polypeptide of 379 amino acids. Sequences important for eucaryotic gene expression such as the TATA box, polyadenylation signal, and intron splice sites were found in the expected positions. The open reading frame is interrupted by 8 introns which are in identical positions with 8 of the 9 introns in maize and pea Adh genes, suggesting that during evolution there are processes occurring that result in the loss of introns. Sequence analysis also revealed that the cloned wheat Adh gene shared extensive homology with the barley Adh3 gene not only in the coding region but also in the noncoding regions. However, this homology is discontinuous as a result of a 1.8-kbp insertion (TLM), which is present in the cloned wheat Adh gene and absent in the barley Adh3 gene. Sequence analysis of this insertion reveals features characteristic of the short terminal inverted repeat class of eucaryotic transposable elements. We have no evidence for the transposition of the TLM element. However, Southern blots reveal multiple copies of sequences related to TLM in the wheat genome and in other closely related species, suggesting that transposition may once have played an important role in the evolution of the Gramineae family.     

Cloning and Expression Studies of Novel Small RNAs in Tetraploid Cotton

Small RNAs are a group of non-coding RNAs that downregulate gene expression in a sequence-specific manner to control plant growth and development. The objective of the present study was to clone and characterize several small RNAs in cotton. To identify small RNAs that are involved in the development of cotton bolls and fibers, we generated cDNA libraries from cotton bolls at 13?days post-anthesis from two cotton cultivars, Pima Phy 76 (Gossypium bardadense) and Acala 1517?C99 (Gossypium hirsutum). Screening of these libraries identified eight small RNAs, seven of which have not been reported in other plant species and appear to be absent in the known sequences of other plant species. Their predicted target genes are known to be involved in cotton fiber development. The cloned small RNAs displayed lower and differential expression in the examined boll developmental stages using RT-PCR and quantitative RT-PCR. The genetic polymorphism of the small RNAs at the DNA level was evaluated by miRNA-amplified fragment length polymorphism (AFLP) analysis using primers designed from the small miRNA genes in combination with AFLP primers. Homologous small RNA gene sequences were further isolated using this homology-based genotyping approach, and potential hairpin structures were identified. The results represent a novel method to isolate small including miRNA genes at the RNA and DNA levels in many plant species where genome sequences are not available or expressed sequence tags are limited.     

Species-Specific Expansion and Molecular Evolution of the 3-hydroxy-3-methylglutaryl Coenzyme A Reductase (HMGR) Gene Family in Plants

The terpene compounds represent the largest and most diverse class of plant secondary metabolites which are important in plant growth and development. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) is one of the key enzymes contributed to terpene biosynthesis. To better understand the basic characteristics and evolutionary history of the HMGR gene family in plants, a genome-wide analysis of HMGR genes from 20 representative species was carried out. A total of 56 HMGR genes in the 14 land plant genomes were identified, but no genes were found in all 6 algal genomes. The gene structure and protein architecture of all plant HMGR genes were highly conserved. The phylogenetic analysis revealed that the plant HMGRs were derived from one ancestor gene and finally developed into four distinct groups, two in the monocot plants and two in dicot plants. Species-specific gene duplications, caused mainly by segmental duplication, led to the limited expansion of HMGR genes in Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max after the species diverged. The analysis of Ka/Ks ratios and expression profiles indicated that functional divergence after the gene duplications was restricted. The results suggested that the function and evolution of HMGR gene family were dramatically conserved throughout the plant kingdom.     

Complete nucleotide sequence of the cotton (Gossypium barbadense L.) chloroplast genome with a comparative analysis of sequences among 9 dicot plants

Recently, the complete chloroplast genome sequences of many important crop plants were determined, and this can be considered a major step forward toward exploiting the usefulness of chloroplast genetic engineering technology. Economically, cotton is one of the most important crop plants for many countries. To further our understanding of this important crop, we determined the complete nucleotide sequence of the chloroplast genome from cotton (Gossypium barbadense L.). The chloroplast genome of cotton is 160,317 base pairs (bp) in length, and is composed of a large single copy (LSC) of 88,841 bp, a small single copy (SSC) of 20,294 bp, and two identical inverted repeat (IR) regions of 25,591 bp each. The genome contains 114 unique genes, of which 17 genes are duplicated in the IRs. In addition, many open reading frames (ORFs) and hypothetical chloroplast reading frames (ycfs) with unknown functions were deduced. Compared to the chloroplast genomes from 8 other dicot plants, the cotton chloroplast genome showed a high degree of similarity of the overall structure, gene organization, and gene content. Furthermore, the sequences of the genes showed high degrees of identity at the DNA and amino acid levels. The cotton chloroplast genome was somewhat longer than the chloroplast genomes of most of the other dicot plants compared here. However, this elongation of the cotton chloroplast genome was found to be due mainly to expansions of the intergenic regions and introns (non-coding DNA). Moreover, these expansions occurred predominantly in the LSC and SSC regions.     

Identification and analyses of miRNA genes in allotetraploid Gossypium hirsutum fiber cells based on the sequenced diploid G. raimondii genome

The plant genome possesses a large number of microRNAs（miRNAs）mainly 21-24 nucleotides in length.They play a vital role in regulation of target gene expression at various stages throughout the whole plant life cycle.Here we sequenced and analyzed～10 million non-coding RNAs（ncRNAs）derived from fiber tissue of the allotetraploid cotton（Gossypium hirsutum）1 days post-anthesis using ncRNA-seq technology.In terms of distinct reads,24 nt ncRNA is by far the dominant species,followed by 21 nt and 23 nt ncRNAs. Using ab initio prediction,we identified and characterized a total of 562 candidate miRNA gene loci on the recently assembled D₅ genome of the diploid cotton G.raimondii.Of all the 562 predicted miRNAs,22 were previously discovered in cotton species and 187 had sequence conservation and homology to homologous miRNAs of other plant species.Nucleotide bias analysis showed that the 9th and 1 st positions were significantly conserved among different types of miRNA genes.Among the 463 putative miRNA target genes,most significant up/down-regulation occurred in 10-20 days post-anthesis,indicating that miRNAs played an important role during the elongation and secondary cell wall synthesis stages of cotton fiber development.The discovery of new miRNA genes will help understand the mechanisms of miRNA generation and regulation in cotton.     

The 28.5-kDa iron-sulfur protein of mitochondrial complex I is encoded in the nucleus in plants

 The intrinsic 28.5-kDa iron-sulfur protein of complex I in the mitochondrial respiratory chain is encoded in the nucleus in animals and fungi, but specified by a mitochondrial gene in trypanosomes. In plants, the homologous protein is now found to be encoded by a single-copy nuclear gene in Arabidopsis thaliana and by two nuclear genes in potato. The cysteine motifs involved in binding two iron-sulfur clusters are conserved in the plant protein sequences. The locations of the seven introns, with sizes between 60 and 1700 nucleotides, are identical in the A. thaliana and the two potato genes, while their primary sequences diverge considerably. The A+T contents of the intron sequences range between 61% and 73%, as is characteristic for dicot plants, but are in some instances not higher than in the adjacent exons. Here, differences in T content may instead serve to discriminate exons and introns. In potato, both genes are expressed, with the highest levels found in flowers. Sequence similarities between the homologous nuclear and mitochondrial genes indicate that the nuclear forms in animals and plants originate from the endosymbiont genome. Received: 28 May 1996 / Accepted: 22 August 1996     

Characterization and expression of plasma and tonoplast membrane aquaporins in elongating cotton fibers

Cotton fiber (Gossypium hirsutum L. and G. barbadense L.) is a good model for studies of plant cell elongation and cell wall biogenesis. Aquaporins are ancient membrane channel proteins that facilitate the permeation of water across biological membranes. We studied GhPIP1-2, encoding plasma membrane intrinsic protein, and GhgammaTIP1, encoding tonoplast intrinsic protein, during cotton fiber development. The full-length cDNAs of GhPIP1-2 and GhgammaTIP1 were obtained through 5' RACE. The deduced amino acid sequences of GhPIP1-2 and GhgammaTIP1 share high sequence identity with aquaporins from diverse plant species. Phylogenetic analysis of GhPIP1-2 and GhgammaTIP1 with other plant aquaporins showed that GhPIP1-2 belongs to the PIP1 group of the PIP subfamily and GhgammaTIP1 belongs to the gammaTIP group of the TIP subfamily. GhPIP1-2 and GhgammaTIP1 contain three and two introns, respectively. Genomic Southern blot analysis indicated that GhPIP1-2 and GhgammaTIP1 have several copies and multiple homologous genes in allotetraploid cotton. Northern blot analysis with gene-specific probes and real-time PCR demonstrated that GhPIP1-2 and GhgammaTIP1 are predominantly expressed during cotton fiber elongation, with the highest expression levels at 5 days post-anthesis. Moreover, expression patterns of the two genes in G. hirsutum and G. barbadense are similar, whereas the expression levels in G. barbadense are much lower than that in G. hirsutum. The high and preferential expression of GhPIP1-2 and GhgammaTIP1 during fiber cell elongation suggests that they may play important roles in supporting the rapid influx of water into vacuoles during cotton fiber cell expansion.     

陆地棉GST基因家族全基因组分析

谷胱甘肽转移酶(glutathione-S-transferase, GST)是一种普遍存在的具有多功能的超家族蛋白,在植物初次生代谢、逆境胁迫、胞间信号传递等方面具有重要作用;同时,作为配体其在植物激素代谢以及物质转运方面也发挥作用。为了解析陆地棉(Gossypium hirsutum L.) GST基因家族的信息,本研究对该基因家族成员的种类、进化关系、物理定位、基因结构和保守基序以及表达模式进行了分析。结果显示,在陆地棉全基因组中共含有70个GST基因,进化树和基因结构分析将该家族分为U族、F族、T族、Z族、EF1Bγ族和TCHQD族。基因定位分析发现,除了AD/At2、AD/At4、AD/At5、AD/Dt5、AD/Dt10号染色体上没有GST基因外,其他染色体上都有GST基因,并且在AD/At9、AD/Dt7、AD/Dt12、AD/Dt13这4条染色体上出现基因簇。对F族(Phi类) 9个GST基因进行荧光定量分析,结果表明,除GhGSTF1可能为假基因外,GhGSTF2~9等8个基因在陆地棉根、茎、叶以及各个发育时期的纤维中均有表达;结合生物信息学分析,推测GhGSTF8可能参与原花青素/花青素的转运和积累;GhGSTF4、GhGSTF6和GhGSTF9可能在调节陆地棉的生长和胁迫反应中起作用,而GhGSTF2、GhGSTF3、GhGSTF5和GhGSTF7的功能还有待进一步研究。本研究为陆地棉GST基因家族的分子进化及功能研究提供了理论依据。     

Chloroplast-expressed glutamine synthetase (ncpGS): potential utility for phylogenetic studies with an example from Oxalis (Oxalidaceae).

Chloroplast-expressed glutamine synthetase (ncpGS), a nuclear-encoded gene containing several introns, is introduced as a tool for phylogenetic studies at lower taxonomic levels. This gene is a member of a multigene family, but it diverged long ago from the cytosolic-expressed members of the family and appears to be single copy in the majority of taxa examined to date. The conservation of both coding sequence and position of introns has allowed the design of primers for use in a broad range of dicot taxa to amplify and sequence a region of ncpGS that contains four introns. The utility of this region in phylogenetic studies of congeneric species is illustrated by an example using eight Oxalis species. The four introns in these taxa are typical in size (76 to 136 bp), base composition (high T content), and structure (e.g., sequence of splice sites and putative branch points) for plant internal introns. Levels of variation among these ncpGS sequences compare favorably with those of the internal transcribed spacer of nuclear ribosomal DNA (ITS) from the same taxa, and results of phylogenetic analysis of ncpGS data are generally congruent with previous results using ITS.