栽培草莓叶绿体分离与cpDNA的提取方法

以栽培草莓‘红颊’(Fragaria×ananassa Duch.‘Benihoppe’)新鲜幼嫩叶片为材料,利用高盐-低pH的缓冲液结合Percoll密度梯度离心分离纯化叶绿体,再用SDS-蛋白酶K法提取叶绿体DNA(cpDNA)。经荧光显微镜、琼脂糖凝胶电泳和PCR扩增检测,结果表明:该方法分离的草莓叶绿体完整性高,叶绿体DNA质量好,纯度高,能够满足后续基因组测序等实验的基本要求,为草莓属及其他草本植物cpDNA的提取提供参考。     

一种高效提取普通小球藻叶绿体DNA的新方法

本文采用尿素-月桂酰肌氨酸钠（urea-sarkosyl）法, 用于分离带有坚硬细胞壁小球藻的高纯度叶绿体DNA （cpDNA）。将对数生长期的小球藻收集后置于冰上研磨, percoll密度梯度离心收集叶绿体层, 显微观察表明叶绿体经梯度离心后形态完整。采用尿素-月桂酰肌氨酸钠法、蛋白酶K消化及酚/氯仿/异戊醇抽提, 获得了高纯度的cpDNA。检测结果显示, cpDNA分子长度为22 kb, A260：A280值为1.87±0.01, 产率达（2.52±0.01） μg？g-1 （DW）; cpDNA编码的16S rDNA扩增呈阳性, 而由细胞核编码的18S rDNA扩增呈阴性。表明cpDNA纯度高, 没有受到核基因组DNA的污染, 符合小球藻cpDNA高通量测序的要求。同时, 该方法也适合提取具有相似细胞壁成分的其他微藻的基因组DNA和cpDNA。     

适合于胞质基因组扩增的红麻成熟叶片DNA提取改良方法

为了从成熟红麻叶片中提取高质量、高产量的基因组DNA,针对红麻成熟叶片中多糖、多酚含量较高的特性,利用改良CTAB法及改良SDS法分别提取红麻品种福红952成熟叶基因组DNA,并通过琼脂糖凝胶电泳和紫外分光光度计测定进行DNA质量检测。结果表明:改良CTAB法提取的基因组DNA电泳时点样孔干净,条带整齐无拖带,OD260/OD280为1.9左右,产率可达1.84μg/g,其质量、产量都高于改良SDS法,所提取的DNA可用于红麻RAPD分子标记、线粒体DNA、叶绿体DNA通用引物PCR扩增。改良CTAB法是提取成熟红麻叶片DNA的有效方法,并且可用于红麻分子标记及胞质基因组学研究。     

一种纯化高等植物叶绿体DNA的有效方法

１９８４年,Ｂｏｏｋｊａｎｓ ｅｔ ａｌ．建立了高盐介质纯叶绿体ＤＮＡ的方法。在我们进行的高粱叶绿体光系统基因研究中发现,用Ｂｏｏｋｊａｎｓ的方法提取的ｃｔＤＮＡ酶切效果欠佳,产率太低。应用我们反进的高盐低ＰＨ方法,纯化了高粱、小麦、水稻和速效豌豆等植物的ｃｔＤＮＡ。结果表明,这种改进的方法具有有得率高,酶切效果好和操作简便等优点。     

基于叶绿体基因组SNP的天台鹅耳枥谱系结构与分化分析

天台鹅耳枥为中国特有的濒危植物,仅间断分布于浙江省境内,种群数量稀少,已处于极危状态。该文通过对6个自然居群(包含所有居群的母株)叶绿体基因组(cpDNA)单核苷酸多态性(SNP)研究,探讨天台鹅耳枥谱系结构与系统分化,以评估濒危状况,并提出相应的保护策略。使用TIANGEN试剂盒法提取基因组DNA,用Illumina NovaSeq 6000进行高通量测序,对获得叶绿体全基因组序列,使用在线程序OGDRAW制作cpDNA图谱,用DnaSP分析核苷酸多样性,用PopART软件进行单倍型网络构建,使用RAxML软件构建极大似然树(ML tree),用MrBayes构建Bayes tree。结果表明：(1)通过天台鹅耳枥叶绿体全基因组序列分析,发现大多数蛋白质编码基因和氨基酸序列显示出明显的密码子偏好,检测到cpLTR正向重复32个、回文重复25个、反转重复22个;SSR重复序列不同类型87个,其中大多数富含A/T,单核苷酸的数量最多。(2)在cpDNA中鉴定了314条SNPs,单核苷酸取代显示天台鹅耳枥群体属单系,分为天台县居群(THS)和景宁县居群(JST),居群单倍型之间演化关系呈现...     

胡椒叶片基因组DNA提取方法比较

目的：研究建立胡椒叶片中提取高质量DNA的方法。方法：采用各种CTAB法和SDS法的改良方法,提取胡椒叶片中的总DNA,并对DNA进行紫外和电泳检测。结果：改良CTAB法4和5提取的DNA经电泳检测,有一条明亮主带,且无拖尾现象,样品槽无荧光出现,说明抽出的DNA纯度较高,一致性好;测定其OD260和OD280值,并计算其比值,OD260/OD280值在1.8-2.0之间,提取率在51.667-60.000μg/g之间,获得的基因组DNA质量高。结论：改良CTAB法4和法5可从胡椒幼叶中提取高质量DNA。     

两种方法制备模板DNA在PCR-SSCP中的应用研究

目的:比较两种方法(DNA试剂盒提取法和FTA卡法)提取的DNA在PCR-SSCP反中的可靠性.方法:用DNA试剂盒从全血中提取DNA和用NaOH方法从FTA卡中提取DNA后,用分光光度计检测两种方法提取DNA的浓度及纯度,进行PCR反应之后,用2％的琼脂糖凝胶电泳检测其质量,接着进行SSCP检测,观测其效果.两种方法提取的样品相同,进行PCR反应和SSCP检测的条件完全一致.结果:用DNA试剂盒提取法和FTA卡法提取的DNA纯度分别为OD260/280=1.817,OD260/280=1.806.48份贵州荷斯坦奶牛DNA后续PCR反应和SSCP的检测结果表明,两种方法提取的DNA用于PCR反应和SSCP检测其效果没有明显差别,成功率为100％.结论:FTA卡结合DNA较稳定,用NaOH法提取DNA效果可靠且比试剂盒方法简便、快捷、经济,值得推广.     

猕猴桃干叶片DNA的提取及叶绿体基因PCR-RFLP反应

采用CTAB法猕猴桃干叶片提取总DNA,运用PCR技术扩增出rbcL和psbA两个叶绿体基因片段,分别用两种限制性内切酶对它们进行酶切分析,实验结果表明,当CTAB提取液体积数为750ul,猕猴桃叶片质量为10mg时,所得到的猕猴桃DNA的质量较为理想：DNA模板量为250ng时,扩增到的特异性条带明亮,无拖尾,2个基因的酶切结果共得到25个限制性位点,其中24个具有多态性,为从cpDNA水平探讨猕猴桃属植物的系统发育打下了坚实的基础。     

牛肉干中牛基因组DNA提取方法的比较研究

为得到一种快速、稳定、准确、优质的牛肉干DNA的提取方法,本研究比较了SDS法、改良CTAB法、酚-氯仿抽提法以及4种试剂盒提取法的提取效果。通过比较DNA的提取质量、提取效率,并对提取的DNA进行PCR扩增分析。DNA提取结果表明,7种提取方法均能从牛肉干中提取出DNA,其中采用SDS法、酚-氯仿法、试剂盒1法和试剂盒4法所提取的DNA质量较高,A260/A280比值在1.7~1.9之间。试剂盒3法提取所花的时间最少,DNA得率最高,但DNA的纯度最低。PCR扩增结果表明,7种方法所提取的DNA均能满足后续PCR扩增实验的要求。实验室可根据具体的实际情况选择使用DNA提取方法。     

野生山梨基因组DNA提取方法和部位比较

以野生山梨(Pyrus ussuriensis Maxim)嫩叶、一年生枝韧皮部和成熟叶片为材料,分别用CTAB,改良CTAB+高盐+5%PVP和试剂盒3种方法提取基因组DNA,通过测OD值、DNA琼脂糖凝胶电泳和SSR引物扩增检验.结果表明:韧皮部和嫩叶是最理想的提取DNA材料,改良CTAB法是比较理想的方法,试剂盒是提取DNA最简便快捷的有效方法.     

衣藻叶绿体遗传转化的研究

叶绿体遗传转人是近几年发展起来的新领域。本文主要介绍了叶绿体遗传转化的特点、基本原理和衣藻叶绿体遗传转化的方法与技术;     

叶绿体基因工程简介

叶绿体是植物细胞中一种特殊的细胞器。自1988年开始,人们认识到叶绿体在植物基因工程中的特殊地位。叶绿体基因工程的特点,特别是其高效表达和安全性,使其受到越来越多的重视,本文对叶绿体转化作了较为全面的介绍,包括其优势、方法、用途及不足等内容。 Abstract:Chloroplast is a kind of special cell organin plant cells.Since 1988,Scientists have realized its advantages in plant gene enginearing.It′s high efficient expression and safety made it been attached more and more importance to.This paper introduces the chloroplast transfer mation,including its advantages,methods,uses and defects.     

植物基因工程新途径：叶绿体转化

在植物基因工程研究中,叶绿体是继核转化之后又一新的遗传转化和表达受体,叶绿体转化体系具有可同时进行多基因转化,表达原核性,超量表达,后代遗传稳定,定点整合,不会产生基因沉默及母性遗传和安全性好等特点,本文着重介绍叶绿体转化体系的特点,国内外研究动态,存在的问题及未来发展方向。     

Chloroplast phylogenomics resolves key relationships in ferns

Studies on chloroplast genomes of ferns and lycophytes are relatively few in comparison with those on seed plants. Although a basic phylogenetic framework of extant ferns is available, relationships among a few key nodes remain unresolved or poorly supported. The primary objective of this study is to explore the phylogenetic utility of large chloroplast gene data in resolving difficult deep nodes in ferns. We sequenced the chloroplast genomes from Cyrtomium devexiscapulae(Koidz.) Ching (eupolypod I) and Woodwardia unigemmata (Makino) Nakai (eupolypod II), and constructed the phylogeny of ferns based on both 48 genes and 64 genes. The trees based on 48 genes and 64 genes are identical in topology, differing only in support values for four nodes, three of which showed higher support values for the 48-gene dataset. Equisetum L. was resolved as the sister to the Psilotales–Ophioglossales clade, and Equisetales–Psilotales–Ophioglossales clade was sister to the clade of the leptosporangiate and marattioid ferns. The sister relationship between the tree fern clade and polypods was supported by 82% and 100% bootstrap values in the 64-gene and 48-gene trees, respectively. Within polypod ferns, Pteridaceae was sister to the clade of Dennstaedtiaceae and eupolypods with a high support value, and the relationship of Dennstaedtiaceae–eupolypods was strongly supported. With recent parallel advances in the phylogenetics of ferns using nuclear data, chloroplast phylogenomics shows great potential in providing a framework for testing the impact of reticulate evolution in the early evolution of ferns.     

甘薯叶绿体表达载体构建及其融合基因在叶绿体中的瞬间表达

利用叶绿体基因组进化中高度保守的特点,根据烟草叶绿体基因组全序列设计引物,从甘薯(Ipomoea batatas)叶绿体基因组中克隆了2个相邻的功能基因rbcL(GenBank登录号为AY942199)和accD(GenBank登录号为AY942200),并以此作为定点整合外源基因的同源重组片段.以来自叶绿体基因组的强启动子Prrn和RpsbA-pro分别驱动选择标记基因aadA及phaC-gfp融合基因,构建成表达盒prrn-aadA-TpsbA-ter与RpsbA-pro-phaC-gfp-RpsbA-ter,然后将这2个表达盒串联在一起克隆进甘薯叶绿体同源片段中,获得甘薯叶绿体定点整合表达载体pSC-GFP.酶切分析证明,所构建的载体符合预期设计;采用该载体对甘薯叶片进行基因枪转化,结果显示,phaC-gfp融合基因可在叶绿体特异启动子和终止子的调控下在甘薯幼嫩叶片中瞬间表达,证明构建的载体pSC-GFP可用于甘薯叶绿体转化.     

Chloroplast Transformation in Oilseed Rape

The chloroplast transformation vector pNRAB carries two expression cassettes for the spectinomycin resistance gene aadA and the insect resistance gene cry1Aa10. The two cassettes are sited between the rps7 and ndhB targeting fragments. Biolistic delivery of the vector DNA, followed by spectinomycin selection, yielded chloroplast transformants at a frequency of four in 1000 bombarded cotyledon petioles. PCR analysis and Southern blot of PCR products confirmed the site-specific integration of aadA and cry1Aa10 into the chloroplast genomes of transgenic oilseed rape. When transgenic oilseed rape leaves were fed to second instar Plutella xylostera larvae, 47% mortality was observed against this insect and the surviving larvae had significantly lower weight than the control. This is the first report of chloroplast transformation in oilseed rape and the introduction of novel genes between the rps7 and ndhB genes in the chloroplast genome. This offers an opportunity for improvement of oilseed rape by chloroplast genetic engineering.     

Chloroplast Proteases

The chloroplast within the plant cell has a dynamic environment where proteases play an important role in processing of precursor proteins, degradation of incomplete proteins lacking cofactors, stress-induced degradation and removal of damaged proteins. A number of proteases in the chloroplast are well characterized and found to be localized within different compartments such as stroma, thylakoids and lumen. In recent years, an increasing number of proteases in chloroplasts have been discovered and identified as bacterial protease homologues. These include the stromal Clp, thylakoidal FtsH and lumenal DegP. The current focus is to understand their role in chloroplast regulation both at the enzyme-substrate and genetic levels.     

叶绿体——疫苗生产新工厂

以叶绿体为受体的叶绿体基因工程技术已经在工农业及医药生物技术领域发挥了重要作用。利用叶绿体为基因转移受体的叶绿体基因工程技术来生产疫苗是目前人们关注的焦点。现着重在叶绿体作为遗传工程受体的特点、叶绿体基因工程的研究进展,它在疫苗生产中的应用及发展前景等方面进行综述。     

Chloroplast DNA from three archegoniates

1. DNA from female and male Sphaerocarpos donnellii (liverwort) plants exhibits at least two species with buoyant densities of 1.703 (main band) and 1.691 (satellite) g cm^-3 in CsCl equilibrium gradients. At least part, if not all, of the satellite DNA is localized in plastids. It consists of up to 90% of uniformly sized circular molecules of an average circumference of 38.5 m. Compared to other Chlorophyta, the liverwort's cpDNA is unusually low both in diensity and contour length. — 2. On the hand, cpDNA from the ferns Asplenium nidus and Pteris vittata resembles those of higher plants in buoyant density (1.697 g cm^-3) and circumference (about 44.8 m). — 3. Analysis of DNA from the archegoniate chloroplasts with restriction endonucleases indicates chat the cyclic molecules are monomers. — 4. The results show that the circular molecules found in cpDNA of higher plants do not represent the functionally required minimum size of DNA in plastids.Abbreviations cpDNA chloroplast - DNA nucDNA=nuclear - DNA Sal I=restriction endonuclease from Streptomyces albus S - Eco RI restriction endonuclease from Escherichia coli, carrying resistance factor 1 - DTT dithiothreitol (Cleland's reagent) - Saline-EDTA 0.15 M NaCl, 0.1 M ethylene diamine tetraacetic acid, pH 8.0 - SSC 0.15 M NaCl, 0.015 M Na citrate, pH 7.2 - DNAase deoxyribonuclease - Md Megadalton Dedicated to the memory of Prof. Dr. Edgar Knapp     

外源基因在叶绿体中表达的研究

综述了叶绿体表达系统的特点,及国内外此方面的研究动态,提出了外源基因在叶绿体中表达存在的问题和解决策略.