Deciphering codon usage patterns and evolutionary forces in chloroplast genes of Camellia sinensis var. assamica and Camellia sinensis var. sinensis in comparison to Camellia pubicosta

Codon usage bias(CUB) is a unique property of genome which refers to non-random usage of synonymous codons in coding sequences. The present study makes an attempt to find out the pattern of CUB in chloroplast(cp) genes among three tea species, i.e., Camellia sinensis var. assamica(Assam tea), Camellia sinensis var. sinensis(Chinese tea) and Camellia pubicosta(wild tea species) as no work on CUB was reported earlier. To understand the patterns of codon usage among the cp genes of three tea groups, we used bioinformatic tools to investigate the protein coding sequences of cp genes. In our present study, the mean nucleobase T was the highest whereas C was the lowest in all the three tea groups. The overall AT content was more than GC content, i.e., genes were AT rich. The scaled chi-square(SCS) value indicated that the CUB of cp genes was low. The codon CGT(Arg) was over-represented in C. sinensis var. sinensis whereas GGA(Pro) was over-represented in C. pubicosta species. Heatmap study revealed that most of the GC ending codons showed positive correlations between codon usage and GC3 while AT ending codons exhibited negative correlations. From neutrality plot analysis, it was evident that natural selection had played a major role, while mutation pressure exerted a minor effect in the CUB of cp genes in three tea groups. Highly significant(P<0.01) positive correlation was found between SCS and synonymous codon usage order(SCUO) of cp genes which suggested that high expression of cp genes was associated with high degree of CUB.

Expression of Six Chloroplast Genes in <i>Jatropha curcas</i>Callus under Light and Dark Conditions

The induction of genes encoded in the open reading frames (ORFs) of chloroplast genomes have been posited to be influenced by ambient light condition. The current study focused on determining which of the six ORFs, encoding the genes ycf 1, ycf 2, psbD (photosystem II), rbcl (Rubisco), matK (Maturase K) and rpoC1 (RNA polymerase) were influenced by light. Characterization of gene expression at the whole plant level and callus stage facilitates the identification of transcripts which are differentially regulated under these environmental conditions. Specificity of these primers was tested against genomic DNA and total RNA. Transcripts of six targeted genes were detected in all three replicates of the green and white callus under light and dark conditions, except for ycf 2 gene in green callus under light. The result showed that a partial transcript of the gene ycf 2 located on the J. curcas chloroplast genome was not detectable using reverse transcription PCR. This finding was then validated using quantitative real-time PCR. The gene was suspected to be post-transcriptionally modified. The transcripts of the remaining five ORFs could be detected using quantitative real-time PCR. Specific transcripts can be identified for application as biomarkers for selection of callus for plantlet regeneration.

Chloroplast gene expression:Recent advances and perspectives

Chloroplasts evolved from an ancient cyanobacterial endosymbiont more than 1.5 billion years ago.During subsequent coevolution with the nuclear genome,the chloroplast genome has remained independent,albeit strongly reduced,with its own transcriptional machinery and distinct features,such as chloroplast-specific innovations in gene expression and complicated post-transcriptional processing.Light activates the expression of chloroplast genes via mechanisms that optimize photosynthesis,minimize photodamage,and prioritize energy investments.Over the past few years,studies have moved from describing phases of chloroplast gene expression to exploring the underlying mechanisms.In this review,we focus on recent advances and emerging principles that govern chloroplast gene expression in land plants.We discuss engineering of pentatricopeptide repeat proteins and its biotechnological effects on chloroplast RNA research;new techniques for characterizing the molecular mechanisms of chloroplast gene expression;and important aspects of chloroplast gene expression for improving crop yield and stress tolerance.We also discuss biological and mechanistic questions that remain to be answered in the future.

Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants

In contrast to the situation of random integration of foreign genes in nuclear transformation, the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination. To establish an expression system for alien genes in rice chloroplast, the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study. Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique, and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator ofpsbA gene 3 sequence. Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct. Subsequently, the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained. Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome. Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome. Thus, the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome, but PPT-resistant trait for rice plants as well. It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.

The Research of Bt and OC Gene Cotransformation in Tobacco Chloroplast

The Bt Cry IA (C) chloroplast expression cassette and OC chloroplast expression cassette were constructed. The Bt expression cassette contained the 3.5 kb wild type Bt Cry I4(C) gene under the control of the strong light-induced psbA promoter and terminator from rice (Oryza saliva . L) chloroplast, the gene: trnH-psbA-trnk from tobacco (Nicotiana tabacum. L) as the homologous fragment. The OC chloroplast expression cassette contained the OC gene under the control of 16S promoter and terminator from tobacco, the tobacco gene: psbA-ORF512 as homologous fragment. The two cassettes both had the aadA gene expression cassette as the selectable marker. Leaves of tobacco were cotransformed with the particle bombardment method. After selection by spectinomycin, the transformants were obtained. The integration of Bt and OC gene were confirmed by Southern-blotting analysis, and Western-blotting analysis. Proteinase inhibitor assays showed that the Bt and OC gene had expressed. Bioassays showed that the transgenic tobacco had a significant resistance to the larvae of cotton bollworm (helicoverpa zea).

Expressing PHB synthetic genes through chloroplast genetic engineering

Chloroplast integration and expression vector containing expression cassettes for phbB, phbA, phbC and aadA genes was constructed and bombarded into the tobacco chloroplast genome. Transplastomic plants were analyzed with PCR and Southern blot. Their homoplastomy was also judged. Northern dot and RT-PCR analysis were employed to investigate transgene expression at transcriptional level. The results indicate that the chloroplast transformation system is compatible for poly-3-hydroxybutyrate (PHB) production.

桑树基因组学研究进展

栽桑养蚕的历史对整个人类文明产生了重要的影响。桑树是多年生木本植物,具有重要的经济价值、药用价值和生态价值。自2013年桑树基因组测序项目实施以来,桑树的研究进入了基因组学时代,为桑树的分子育种奠定了基础。本文从桑树基因组测序的实施开始,系统总结了桑树基因组学研究的最新进展,包括了测序技术、拼接策略、组装质量以及基于全基因组重测序应用等方面。未来,随着技术的不断创新,桑树基因组学研究将迎来更广阔的发展前景。本文旨在推动桑树产业的发展并为林木基因组学的研究提供理论与技术参考。

茶树叶绿体基因组的研究与应用进展

多数植物叶绿体基因组为双链环状DNA,具有保守的四分体结构、非重组、单倍体、单亲遗传、进化速率适中、序列和结构高度保守等特征,可为植物进化等提供有用信息。茶树种质资源丰富,其复杂的起源、进化、分类等方面缺乏有效鉴评,而有碍于对其高效保护与创新利用。目前,叶绿体基因组测序技术快速发展并向三代过渡,33份茶树资源的叶绿体基因组已在NCBI公布,茶树叶绿体基因组基因类型、基因序列特征已被部分揭示,且已应用于茶树资源的分类鉴定、起源进化、白化机制等方面研究,但仍有不少茶树资源的叶绿体基因组未被破译。本文简述了植物叶绿体基因组的起源、遗传方式、基本特征;重点述评了茶树叶绿体基因组测序技术、基因组特征、基因类型、基因序列等最新研究成果;概述了茶树叶绿体基因组应用现状;最后探讨了茶树叶绿体基因组未来的应用与发展方向。

Molecular phylogeny of tribe Atraphaxideae(Polygonaceae)evidenced from five cpDNA genes

Traditionally, Atraphaxis, Calligonum, Pteropyrum and Parapteropyrum are included in the tribe Atraphxideae. Recently, sequence data has revealed that this tribe is not monophyletic. The structure of the tribe was examined by adding more taxa and sequences to clarify the congruence between morphology and molecular phylogeny, the systematic placements of four genera in Polygonaceae, as well as the infra-generic relationships of Atraphaxis and Calligonum within Atraphaxideae. Five chloroplast genes, atpB-rbcL, psbA-trnH, trnL-tmF, psbK-psbl, and rbcL of Atraphaxis, Calligonum, Pteropyrum, and Parapteropyrum were sequenced. The non-monophyly of Atraphaxideae was confirmed. Atraphaxis and Calligonum, respectively, formed a monophyletic group that was well supported. Calligonum is closely related to Pteropyrum; Atraphaxis is sister to Polygonum s. str. and Parapteropyrum is allied with Fagopyrum. Although the morphology suggested the four genera should form a tribe, the molecular data indicated Atraphaxideae was not one monophyletic group. The clades identified within Atraphaxis corresponded well with the current sectional classification based on morphological features. As for Cal- ligonum, Medusa was identified as a non-monophyletic section.

植物间水平基因转移——基因交流新途径及其农业利用潜力

水平基因转移(horizontal gene transfer, HGT)指通过受精以外的方式进行的跨物种遗传物质传递,是原核和真核生物基因组构成的重要来源,对生物进化具有重要推动作用。近年来,基因组测序技术的发展进一步证明了植物之间存在着大量的水平基因转移事件。文章主要对国内外植物间水平基因转移的途径、转移的基因分类,以及水平基因转移在农业中的应用潜力等方面进行了综述和讨论,分析了当前植物水平基因转移研究存在的问题,并就未来基础理论研究及利用的方向作了展望。

甜瓜CmEPF基因家族的鉴定及表达分析

【目的】表皮模式因子(epidermal patterning factor,EPF)是一类植物特有的分泌蛋白,在植物生长发育特别是气孔形态建成过程中发挥着重要作用,旨为揭示甜瓜EPF基因的特性和功能。【方法】对甜瓜CmEPF基因家族进行基因组鉴定,利用生物信息学手段对其基因结构、系统进化以及组织表达特性进行分析。【结果】甜瓜基因组存在11个可分为3个亚家族的CmEPF成员,它们的基因结构相对保守,含有1-3个内含子,且大都分布于甜瓜的叶绿体上;基于系统进化分析将其分为3个亚家族。不同器官RT-qPCR分析发现,CmEPFs基因在甜瓜器官中广泛表达,但不同家族成员在不同器官中的表达模式存在差异,其在第二片叶表达量较高,且气孔密度、光合速率和蒸腾速率最高,其表达量与光合参数和气孔密度显著相关。【结论】CmEPFs基因通过影响甜瓜的气孔密度,影响其光合作用,从而调控甜瓜叶片气孔对外界环境的响应。

Analysis and characterization of the Salix suchowensis chloroplast genome

By screening sequence reads from the Salix suchowensis chloroplast （cp） genome that were generated by next-generation sequencing platforms, we assembled a complete circular pseudomolecule for the cp genome. This pseudomolecule is 155,508 bp long and has a typical quadripartite structure that contains two single copy regions, a large single copy region （LSC, 84,385 bp）, and a small single copy region （SSC, 16,209 bp） separated by inverted repeat regions （IRs, 27,457 bp）. Gene annotation revealed that the S. suchowensis cp genome encoded 119 unique genes, including four ribosome RNA genes, 30 transfer RNA genes, 82 protein-coding genes, and three pseudogenes. Analysis of the repetitive sequences revealed 31 tandem repeats, 16 forward repeats, and five palindromic repeats. In addition, a total of 148 perfect microsatellites, which were characterized as A/T dominant in nucleotide composition, were detected. Significant shifting of the IR/SSC boundaries was revealed by comparing this cp genome with those of other rosid plants. We also constructed phylogenetic trees to demonstrate the phylogenetic position of S. suchowensis in Rosidae based on 66 orthologous protein-coding genes present in the cp genomes of 32 species. Sequencing 30 amplicons based on the pseudomolecule for experimental verification revealed 99.88% accuracy for the S. suchowensis cp genome assembly. Therefore, we assembled a high-quality pseudomolecule of the S. suchowensis cp genome, which is a useful resource for facilitating development of this shrub willow into a more productive bioenergy crop.

洪泽湖周边水域浮萍生物多样性研究

本文通过对洪泽湖周边水域的20个浮萍样品的叶绿体保守基因atp H-atpF和psbK-psbI间隔区序列的克隆、测序、序列比对分析,研究该区域的浮萍生物多样性。研究表明20个浮萍样品分属5个不同物种,分别为Spirodela polyrhiza、Landoltia punctata、Lemna aequinoctialis、Lemna turionifera和Wolffia globosa,其中以Spirodela polyrhiza最多,占样品总数的60%。通过浮萍叶绿体的保守基因不仅可以确定物种的生态型,这些数据也为未来区域浮萍种质资源保护和应用研究提供了理论依据。

水稻致死突变体基因克隆与分子机制研究进展

水稻致死突变体主要有叶片白化致死突变体和叶片早衰致死突变体2大类,这2类突变体相关基因的克隆和功能分析对于解析水稻白化致死和早衰致死具有重要意义,主要涉及光合色素的合成与降解、叶绿体发育与调控、蛋白质的合成与降解、激素合成与调控途径、细胞程序性死亡与转录因子调控等相关基因。本文综述了水稻致死突变基因的克隆与分子机制,并分析了它们的育种价值;并提出在阐明水稻衰老调控机制的基础上,从分子水平和栽培技术上设计调控水稻衰老的策略,发掘其育种和生产价值。

兰科植物叶绿体基因组研究进展

叶绿体基因组(cpDNA)中含有大量功能基因,在系统发育研究及物种识别鉴定中的应用价值已经受到研究者们的重视和认可。兰科物种数量庞大,基因多样性极高,是叶绿体基因组研究的一个热点,本文就兰科主要植物的叶绿体基因组特征、基因类型分布和序列特征、密码子偏好性以及兰科主要植物叶绿体基因组在系统发育中的应用等方面进行了综合分析,为下一步将叶绿体基因组的研究应用于兰科植物的系统发育关系、野生植物资源保护、遗传育种等方面的研究奠定基础。

尾叶桉叶绿体ROS代谢相关基因表达与不定芽分化的关系

为研究不同类型愈伤组织的叶绿体活性氧(ROS)代谢相关基因表达,探讨尾叶桉再生型愈伤组织转绿和不定芽分化之间的关系,将9 d苗龄的尾叶桉(Eucalyptus urophylla S. T. Blake)幼苗下胚轴接种在添加N-苯基-N-噻唑基脲、6-苄氨基腺嘌呤、吲哚-3-乙酸的标准平板计数琼脂培养基(SPCA)中,外植体先膨大形成白色愈伤组织,8周后分化形成不同类型的愈伤组织(绿色带芽点愈伤组织、绿色愈伤组织、红色愈伤组织、白色愈伤组织)。4种愈伤组织中叶绿素质量分数分别为0.24、0.18、0.07、0.03 mg/g。实时荧光定量多聚核苷酸链式反应(qPCR)检测叶绿体rbcL基因和6个ROS代谢相关基因(TDP1、SOD2、DAR1、TRM4、TRF2、WTR1)的表达。qPCR检测到8周前的白色愈伤组织中4个叶绿体基因rbcL、TDP1、SOD2和DAR1的转录。其中,rbcL、TDP1、SOD2的转录水平随愈伤组织块的膨大不断升高,DAR1基因转录水平则较低。8周后,叶绿体rbcL基因和6个活性氧代谢相关基因均在绿色带芽点愈伤组织、绿色愈伤组织、红色愈伤组织中转录。设定红色愈伤组织中对应基因的表达量为1,绿色愈伤组织中,上述7个叶绿体基因(rbcL、TDP1、SOD2、DAR1、TRM4、TRF2、WTR1)相对表达量分别为8.84、3.36、2.89、2.65、2.53、1.28和7.49,绿色带芽点愈伤组织中7个基因的相对表达量分别为19.57、3.72、4.59、3.53、4.73、3.08和4.12。结果表明,愈伤组织转绿和不定芽分化过程中,叶绿体发育开始启动、叶绿素大量合成、rbcL转录显著增强,叶绿体中与活性氧代谢相关基因的表达水平升高。

西南地区野生刺梨的遗传多样性和遗传结构研究

为了探究西南地区野生刺梨(Rosa roxburghii)的遗传多样性和起源演化,该研究基于2段单拷贝核基因(GAPDH和ncpGS)和3段叶绿体基因(atp F-trn H、trn L-trn F和trn G-trn S)的拼接序列,对刺梨27个野生居群共320个个体进行PCR扩增和测序,并用相关软件对测序结果进行分析。结果表明:(1)在单拷贝核基因和叶绿体基因水平上刺梨均表现出较低的遗传多样性(scnDNA:H d=0.4692,π=0.00049;cpDNA:H d=0.6534,π=0.00065),并且不同居群间存在较大差异。(2)分子方差分析(AMOVA)结果均显示,遗传变异主要发生在居群内,表明居群内的变异是野生刺梨遗传变异的主要来源,居群间存在明显的遗传分化(cpDNA:F_(ST)=0.33647,G_(ST)=0.273,N_(ST)=0.308;scnDNA:F_(ST)=0.09487,N_(ST)=0.076,G_(ST)=0.056),刺梨的分布不具有明显的谱系地理结构(P>0.05)。(3)中性检验Tajima’s D值均为不显著负值,符合中性进化模型。Fu’s Fs值均为显著负值,结合失配分析曲线,推测刺梨种群在小范围内经历过扩张,但总体上维持稳定状态。(4)根据单倍型多样性得出,毕节地区的居群遗传多样性水平较高并且拥有丰富的单倍型,推测可能为冰期避难所,因此应对其实施就地保护,对于拥有特殊性状和特有单倍型的居群也应采取优先保护措施。该文为野生刺梨资源保护和遗传育种提供了一定的参考价值。

BnABCI8影响甘蓝型油菜叶绿体发育

成熟叶绿体是高等植物光合作用的重要场所,是影响作物产量的重要器官。BnABCI8是ABC转运蛋白I亚族的成员,在甘蓝型油菜中有2个功能拷贝,分别是BnA09.ABCI8和BnC09.ABCI8,其氨基酸序列在不同物种中是非常保守的。表达模式分析发现,BnABCI8在油菜植株各个组织中均有表达,且在叶和花中表达量较高;亚细胞定位证明,BnABCI8能够定位在叶绿体中;表型鉴定发现,BnA09.ABCI8和BnC09.ABCI8的同时突变及BnA09.ABCI8的单突变均会导致黄色的子叶和褪绿的真叶,且双突变体褪绿更为严重;透射电镜结果显示,双突变体中叶绿体不能够形成正常的类囊体膜;BnABCI8的敲除导致叶绿素合成途径相关基因的表达下调,且叶片中积累了大量的Fe离子。这些结果表明,BnABCI8的突变造成叶绿体结构异常,叶绿素合成受阻,叶片中Fe离子大量积累,而Fe离子的积累又可能会引发一系列的反应如活性氧积累,细胞死亡和叶绿素降解等,最终导致了叶色突变。

7种作物叶绿体基因的密码子偏好性及聚类分析

研究作物叶绿体蛋白编码基因的编码特点,对指导农作物的叶绿体基因工程研究设计,促进外源基因在受体作物中的高效、稳定表达具有重要作用。为此,综合运用了多种分析软件,对7种大田作物的叶绿体蛋白编码基因进行分析。结果表明:叶绿体蛋白编码基因的总碱基构成在7种作物中差异不大,都是A含量最高,而G,C的含量较低;但在密码子第3位的碱基构成上却有明显差别,3种双子叶作物偏爱以C,T结尾的密码子,而禾本科的4种作物则偏爱以T,C结尾的密码子;RSCU(同义密码子的相对使用度)值显示出禾本科作物有26个密码子具有偏好性,而双子叶作物有25个密码子具有偏好性,但二者之间有22个相同的偏好性密码子。基于RSCU值的聚类结果显示,植物的密码子偏好性具有种族特征,表明基于叶绿体蛋白编码基因的密码子使用特性的聚类结果可以作为系统发育分析的重要补充。

水稻条斑花叶突变体st(t)的鉴定与遗传定位

利用EMS诱变育成优良籼型恢复系缙恢10号,从其后代中鉴定出一个白色条斑花叶突变体st(t),在三叶期开始表现白斑,拔节期白斑变为不规则线状,一直保持到成熟。突变体叶绿素含量明显下降,类胡萝卜素含量显著升高。透射电镜观察表明,突变体的绿色叶片部位与野生型相比,在细胞结构上无明显差异,叶绿体发育正常;突变体的白化部位细胞结构异常,质体内多含有积聚在一起的嗜锇小球,不能发育出正常叶绿体所具有的类囊体和基质片层结构。遗传分析表明该性状受一对隐性核基因调控,利用1500株西农1A/st(t)的F2隐性定位群体,最终把St(t)基因定位在第6染色体SSR标记RM19745和RM19762之间,遗传距离分别为0.07cM和0.27cM,根据9311基因组序列推测,两标记之间的物理距离约为345kb。这为St(t)基因的图位克隆和分子标记辅助育种奠定了基础。