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.

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.

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.

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。

叶绿体基因编辑技术:进展与展望

叶绿体是植物进行光合作用,参与多个代谢途径和免疫反应的重要场所。叶绿体基因组非常小,只有100多个基因,但对植物生长发育具有重要意义。本文首先总结了叶绿体的起源、结构与功能及叶绿体基因组研究的最新进展。其次,梳理了同源重组、碱基编辑两种叶绿体基因编辑技术,重点介绍了碱基编辑技术中的胞嘧啶碱基编辑器和腺嘌呤碱基编辑器,认为相较于传统的同源重组技术,碱基编辑技术可以针对特定靶位点进行定向修饰,单次选择就能获得同质转化植株,且可以不用保留外源DNA片段,实现了非转基因的基因编辑。最后展望了叶绿体基因编辑技术的广阔发展前景,以期促进精准高效的叶绿体基因编辑工具开发,为研究叶绿体基因功能以及加速对叶绿体的合理利用开辟新思路。

一个新的水稻黄绿叶突变体ygl-9108的鉴定与基因定位

本研究旨在对水稻黄绿叶突变体ygl-9108进行表型分析和基因定位,为后续图位克隆该叶色相关基因奠定基础。以黄绿叶突变体ygl-9108为试材,利用突变体ygl-9108和‘五山丝苗’杂交的F2群体进行基因定位。结果表明,与野生型相比,突变体的株高、有效穗数、穗长、每穗粒数、结实率、粒宽、千粒重和单株谷重均显著下降,下降比例分别为21.5%、21.2%、14.6%、19.2%、11.0%、10.2%、12.9%和52.2%。透射电镜观察显示,突变体叶肉细胞的叶绿体数量大量减少,类囊体结构异常。遗传分析表明,突变体的表型由一个隐性核基因控制。利用图位克隆方法,ygl-9108被定位于水稻第11号染色体两InDel标记11Y39和11Y45之间,物理距离约为147 kb,该区间内未见有叶色相关基因的报道,表明ygl-9108是一个新的黄绿叶调控基因。本研究结果为ygl-9108的克隆和功能分析奠定了坚实的基础。

青藏高原蕨麻的分子谱系地理学研究

青藏高原的隆升以及第四纪冰期气候的循环波动,对于青藏高原及其周边地区动植物的分布和遗传结构具有很大的影响。蕨麻是青藏高原极富营养、药用和生态价值的特有植物资源,蕨麻和鹅绒委陵菜的分类关系及分布在学术界存在争议,分子谱系地理学研究将为蕨麻遗传多样性形成机制及推断该物种迁移演化历史提供依据。以蕨麻为研究对象,对采集的30个居群的810个个体进行了叶绿体trnL-trnF序列和核基因ITS测序,揭示遗传变异在居群内和居群间的分布格局,结合群体遗传学和系统发生学,分析该物种的遗传结构与历史事件之间的关联,揭示物种及物种内不同种群形成现有分布格局的历史原因和演化过程。主要结论有:1)蕨麻具有较高水平的遗传多样性。cpDNA trnL-trnF片段共检测到40种单倍型,16个为共享单倍型,占比40%,24个为居群特有单倍型,占比60%,遗传多样性h=0.7078,单倍型多样性Hd=0.8217,核苷酸多样性π=0.010641,总遗传多样性HT=0.849;nrDNA ITS片段共检测到128种单倍型,共享单倍型42种,占比32.8%,居群特有单倍型86种,占比67.2%,遗传多样性h=0.7633,单倍型多样性Hd=0.8168,核苷酸多样性π=0.003584,总遗传多样性HT=0.844。2)居群内的遗传多样性大于居群间的遗传多样性。序列分析结果为居群内和居群间的遗传多样性都很高(cpDNA trnL-trnF:HT=0.849,居群内平均遗传多样性HS=0.640;nrDNA ITS序列:HT=0.844,HS=0.763,HT均大于HS)。蕨麻居群分为3个组:青海高原组、横断山脉组和藏南谷地组。蕨麻的遗传变异主要来源于居群内部。3)蕨麻种群具有明显的谱系地理结构。cpDNA trnL-trnF序列和nrDNA ITS遗传多态性分析及地理分布模式检验,cpDNA trnL-trnF序列:遗传分化系数G_(ST)=0.246,N_(ST)=0.417,nrDNA ITS序列:G_(ST)=0.096,N_(ST)=0.522,N_(ST)均显著大于G_(ST)(P<0.001),表明蕨麻所有居群单倍型存在显著的谱系地理结构,两种方法的结果高度一致。分子变异分析(AMOVA)表明,大部分遗传变异(59.69%)存在于居群内部,居群间分化水平很高(F_(ST)=0.40313)。4)共享单倍型和特有单倍型均由古老单倍型衍生而来。cpDNA trnL-trnF和nrDNA序列中央连接网状图呈以共享单倍型M4和H9位于中心,M_(1)、M_(3)和H_(2)、H_(10)、H_(11)、H_(12)位于主干位置的星状结构,其余共享单倍型和特有单倍型均由这些古老单倍型衍生而来,两者结果一致。5)蕨麻种群大小和范围发生过大规模扩张。利用cpDNA trnL-trnF和nrDNA序列进行歧点分析,前者歧点分布呈单峰曲线,表明近期群体大小和范围有大规模扩张发生;后者的歧点分布呈双峰曲线,反映基因谱系的高度复杂性,但Tajima’s D,Fu and Li’s D和Fu and Li’s F均为负值,且结果显著,且离差平方和(SSD)和扩张评估指数(H_(Rag))的统计检验不显著,表明蕨麻居群近期有扩张的可能。6)蕨麻存在3个冰期避难所,即东喜马拉雅区域、青藏高原东南边缘及横断山脉区域。

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。

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。

高等植物DNA条形码最新研究进展及其应用

植物DNA条形码的筛选工作主要集中于叶绿体基因上,包括位于编码区的mat K、rbc L、UPA、rpo B、rpo C1、acc D、ndh J和YCF5等以及间隔区的trn H-psb A、trn L-trn F、atp F-atp H、trn D-trn Y和psb K-psb I等,其中mat K、rbc L和trn H-psb A研究最为成熟,可作为核心条形码序列。由于每个基因进化速率不同以及植物种间杂交较为频繁等诸多原因,不同类群植物的最适条形码序列有很大差别。本文综述了DNA条形码的最新研究进展以及条形码技术在物种鉴定、分类学问题修订、新种隐种的发现、植物药材真伪鉴定、食品质量控制和安全、出入境植物检验检疫等方面的应用,提出了cp DNA条形码在应用中的不足之处以及解决措施等,并对DNA条形码未来的发展进行了展望,以期为高等植物的应用提供强有力的分子依据。

水稻(Oryza sativa L.)苗期低温白化突变体al12的超微结构与基因定位(英文)

水稻苗期低温白化突变是水稻在发育早期对低温胁迫的一种适应性,是一种受发育和温度控制的条件表达,它与其他水稻白化突变有本质的不同。本研究利用便携式叶绿素测量仪测定了白化时期植株的叶绿素含量和用透射电镜观察了叶绿体的结构变化。结果发现叶绿素平均含量仅为1.2(SPAD),而叶绿体也不能正常发育仅有囊泡状结构。通过与9311的正反交实验及子代的分离表现证明该性状受一个隐性核基因的控制。另外利用SSR分子标记技术将该基因定位在第8染色体上,两侧最近的SSR标记RM5068和RM3702分别距基因0.5～1.1cM和4.9cM,基因被定位在约6个cM的区间内。我们将该基因暂时命名为al12。

基于4个叶绿体基因识别蓑藓属(Macromitrium)植物的可行性研究

我国的蓑藓属植物形态变异式样复杂,分类问题多。DNA条形码技术是一种新的物种鉴定技术。本研究以采自浙江、福建、云南、广西、四川等省的蓑藓属(Macromitrium)7个物种及其外类群直叶藓Macrocoma tenue sub-sp.sullivantii和火藓Schlotheimia grevilleana的38份标本为对象,获得了它们的叶绿体基因trnL、trnG、psbT和rps4序列,基于这些基因的不同组合构建了15棵贝叶斯系统发育树,获得了相应的蓑藓属植物的物种识别率、种内和种间的遗传距离。发现基于trnL-rps4、trnL-trnG-rps4、trnL-psbT-rps4、trnG-psbT-rps4和trnL-trnG-rps4-psbT等5个组合能够较好地识别本研究中蓑藓属植物,均得到了100%的物种识别率。考虑到扩增和测序的成功率和得到的7种蓑藓属植物的系统发育关系,建议将trnL-trnG-psbT组合用于蓑藓属植物的系统发育分析和物种识别的DNA条形码。