根癌农杆菌对甘蓝型油菜的转化及转基因植株的再生

用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ）共培养法把外源基因导入甘蓝型油菜（Ｂｒａｓｓｉ－ｃａ ｎａｐｕｓＬ．）主要栽培品种“云北２ 号”,获得转基因植株。所用外植体为带有１—２ ｍ ｍ 子叶柄的完整子叶,根癌农杆菌为Ａ２０８ＳＥ（ｐＴｉＴ３７－ＳＥ, ｐＲＯＡ９３）。Ｔｉ质粒ｐＲＯＡ９３ 带有ＮＰＴⅡ及ＧＵＳ嵌合基因。共培养２ 天后转到附加２５ ｍ ｇ／Ｌ卡那霉素的分化培养基（ＭＳ＋ ４．５ ｍ ｇ／ＬＢＡＰ）上。ＡｇＮＯ３ 和羧苄青霉素促进芽的分化,头孢霉素则有抑制作用。最高转化频率为２７％ 。把分化出的茎芽切下,插入含有２５ ｍ ｇ／Ｌ卡那霉素的生根培养基中。羧苄青霉素不利于根的形成。把完整抗性植株移入盛土壤的盆中,生长状况良好。测定β－葡糖苷酸酶活性,８４％ 明显高于对照。以ＮＰＴⅡ基因作探针进行Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分析,证实外源基因已插入到植物细胞基因组中     

芥菜型多室油菜与甘蓝型油菜的种间远缘杂交

通过对芥菜型多室油菜与甘蓝型油菜种间杂交 以下简写为芥×甘或甘×芥 的结实性、交配性以及不同甘蓝型油菜对交配性的影响等研究发现:芥、甘正反交形成的饱满种子数较少,其形成种子的能力弱,但是芥×甘与甘×芥杂交相比,芥×甘形成饱满种子的能力较强,受精能力以及杂种胚胎的发育能力也强,在授粉后的子房发育上二者无显著差异.所以,芥菜型多室油菜与甘蓝型油菜种间杂交创建新资源时宜采用芥×甘杂交方式;不同甘蓝型油菜品种与芥菜型多室油菜正反交的结角率、受精指数、结籽指数和可交配指数均不相同,但可交配指数的变异系数最大.因此,筛选可交配性强的甘蓝型基因型应着眼于可交配指数高的甘蓝型油菜亲本材料,根据本试验结果,芥菜型多室油菜与甘蓝型油菜93-221-1杂交形成的杂种胚具有较强的可发育性.     

子房注射法与农杆菌介导法转化甘蓝型油菜的比较研究

以甘蓝型油菜湘油１３号为试验材料,运用根癌农杆菌介导法和子房注射法成功地将Ｂｔ杀虫蛋白基因导入油菜,在特定的条件下,以子叶柄作为转化受体进行农杆菌介导转化,其转化效率受侵染时间和共培养时间的影响,最佳侵染时间为３－５ｍｉｎ,最佳共培养时间为６０－７２ｈ;子房注射法的最佳注射时间为授粉后第２０－３０ｈ,最适注射部位为子房中部,ＤＮＡ注射量为０．５－１．５μｇ,在最佳转化条件下它们分别获得抗卡那霉素植     

青藏高原早熟甘蓝型春油菜遗传资源研究

利用SSR和SRAP 2种分子标记研究了69份试验材料的遗传差异及其亲缘关系.29对SSR标记共扩增出118条多态性带,多态性位点占总扩增位点的97.5%,27对SRAP引物扩增出123条多态性带,多态性比率为70.3%.两种标记聚类结果表明.在相似系数0.566处所有材料可以分为A、B 2个大类群;B类在相似系数0.620处又可分为7个亚类,10个天然双低早熟甘蓝型品系、2个甘蓝型亲本和4个新型品系聚在第1亚类中,其余的51个新型甘蓝型油菜品系分别聚在其他6个亚类中.对55份新型品系进行遗传成分分析,结果表明,每个品系都合有4种带型,各品系所舍不同带所占比率不同.对各品系中含有白菜型亲本带所占比率分别与其对应的两亲本之间的遗传距离进行相关分析,结果表明新型甘蓝型油菜品系中白菜型亲本带所占比率与白菜型素本间的遗传距离为负相关(-0.52),且达到极显著水平;与甘蓝型亲本间的遗传距离为正相关(0.31),且达到显著水平.对试验材料之间的遗传距离及其来源进行分析(除与2个白菜型亲本间),遗传距离排名前20位的都来自新型品系之间或天然品系与新型之间,最大为0.544.     

甘蓝型油菜花瓣缺失基因的图谱定位

在无花瓣品系APT02和正常有花瓣品种中双4号构建的的F2分离群体中,运用AFLP和SRAP两种标记技术对甘蓝型油菜花瓣缺失基因进行分子标记和图谱定位。在两亲本间筛选20对AFLP引物和170对SRAP 引物,进一步通过BSA法筛选,获得了与甘蓝型油菜花瓣缺失基因WHB连锁的1个SRAP标记e8m3_4（600bp）和1个AFLP标记E3247_15（150bp）,标记与基因WHB之间的遗传距离分别为5 cM和13.5cM;构建了一个甘蓝型油菜(Brassica napus.L )的分子标记遗传连锁图谱,该图谱共包含213个AFLP标记、56个SRAP标记和1个形态标记,分布于17个主要连锁群、两个三联体和4个连锁对中,遗传图距总长2487.1cM,标记间平均距离为10.09 cM。通过图谱定位,控制花瓣缺失性状的基因WHB被定位到第4连锁群(LG4)上。     

根癌农杆菌对甘蓝型油菜的转化及转基因植的再生

用根癌农杆菌共培养法把外源基因导入甘蓝型油菜主要栽培品种“云北２号”,获得转基因植株。所用外植体为带有１－２ｍｍ子叶柄的完整子叶,根癌农杆菌为Ａ２０８ＳＥ（ｐＴｉＴ３７－ＳＥ,ｐＲＯＡ９３）。Ｔｉ质粒ｐＲＯＡ９３带有ＮＰＴⅡ及ＧＵＳ嵌合基因。共培养２天后转到附加２５ｍｇ／Ｌ卡那霉素的分化培养基（ＭＳ＋４．５ｍｇ／ＬＢＡＰ）上。ＡｇＮＯ３和羧苄青霉素促进芽的分化,头孢霉素则有抑制作用。最高转化频率为     

中国甘蓝型油菜遗传多样性的RAPD分子标记

本文利用ＲＡＰＤ方法和统计学分析,对我国７省市和国外引进的总计４０份甘蓝型油菜品种的遗传多样性进行了研究。结果表明,４０个品种的甘蓝型油菜存在着广泛的遗传变异,根据ＲＡＰＤ指纹图谱,通过在ＤＮＡ分子水平上的聚类分析可以将它们分为３大类群,反映出这些品种之间的亲缘关系,并对如何引进甘蓝型油菜资源进行了初浅的讨论。     

玉米遗传转化的研究进展

自从１９８８年首次获得玉米转基因完整植株以来,玉米遗传转化技术的研究取得了许多重要进展,抗虫、抗除草剂的转基因业已率先应用于玉米的商品化生产。作者对近年来玉米遗传转化技术所取得的重要进展及其在玉米品种改良中的应用前景进行了论述。     

苹果遗传转化的研究进展

苹果的遗传转化技术通过分子手段改良苹果,有助于缩短其育种周期。最近十年,在该领域的研究取得很大进展,涉及到一些重要的苹果基因型及有用的外源基因。迄今,苹果的遗传转化主要采用农杆菌介导法,侵染与转化材料的再生是影响其转化效率的关键过程,了解其影响因素,寻找有利因素以提高转化效率是目前苹果遗传转化研究的重点。     

甘蓝型油菜千粒重性状的QTL初步定位研究

千粒重是油菜重要的产量相关性状之一,构建油菜遗传连锁图谱是研究其产量性状基因的前提。本研究利用小孢子培养技术,选育出了甘蓝型油菜大粒品系(G-42)和小粒品系(7-9)的纯合DH系DH-G-42和DH-7-9,其千粒重分别为6.24 g和2.42 g,二者比值达2.58。以DH-G-42为母本、DH-7-9为父本,构建了含190个单株的F2遗传作图群体,利用SSR和SRAP标记技术绘制遗传连锁图谱,该图谱共包含20个连锁群,涉及128个SSR标记和100个SRAP标记,图谱总长1546.6cM,标记间平均图距为6.78cM。本研究共检测到3个与千粒重性状相关的QTL,分别位于A9和C1连锁群,其中qSW-A9-1和qSW-A9-2贡献率分别达到10.98%和27.45%,均可视为控制粒重的主效QTL。本研究为后续进行油菜千粒重性状QTL的精细定位分析、分子标记辅助选择育种及新基因的克隆等奠定了基础。     

根癌农杆菌介导的高粱遗传转化体系的研究

采用根癌农杆菌介导法将杀虫晶体蛋白基因cryIA(b)转入高粱胚性愈伤组织中,建立农杆菌转化高粱的遗传转化体系,获得70棵再生植株。经GUS及PCR检测,结果表明,cryIA(b)基因确实转移到高粱恢复系0-30中,报告基因GUS在再生植株中也得到表达。转化植株的抗病性鉴定正在进行中。     

Effect of Pyrolysis Temperature on Chemical and Surface Properties of Biochar of Rapeseed (Brassica napus L.)

The biochar is an important carbon-rich product that is generated from biomass sources through pyrolysis. Biochar (charcoal) can be both used directly as a potential source of solid biofuels and as soil amendments for barren lands. The aim of this study was investigate influence of pyrolysis temperature on the physicochemical properties and structure of biochar. The biochars were produced by pyrolysis of rapeseed (Brassica napus L.) using a fixed-bed reactor at different pyrolysis temperatures (400–700°C). The produced biochars were characterized by proximate and elemental analysis, Brunauer–Emmett–Teller (BET) surface area, particle size distributions, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy. The results showed that both chemical and surface properties of the biochars were significantly affected by the pyrolysis temperature. Aromatic hydrocarbons, hydroxyl and carbonyl compounds were the majority components of the biochar. The biochar obtained at 700°C had a high fixed carbon content (66.16%) as well as a high heating value, and therefore it could be used as solid fuel, precursor in the activated carbons manufacture (specific surface area until 25.38 m² g^?1), or to obtain category-A briquettes.     

农杆菌介导的小麦遗传转化几个影响因素的研究

采用携带gus和（或）bar基因双元表达载体（p3301,pBTAaB)的3个根癌农杆菌（Agrobacterium tumefaciens)菌株（AGL-1,EHA105和LBA4404）对普通小麦（Triticum aestivumL.)冬性栽培品种农大170和农大146的幼胚及幼胚愈伤组织进行了遗传转化,结果表明,菌液浓度OD6001.0和侵染时间1h对外植体的生存和转化最为有利;侵染前对外植体进行高渗处理较明显地提高了抗性愈伤获得率;乙酰丁香酮（AS）对小麦转化的作用随菌株和外植体的不同而异;菌株/质粒组合,受体基因型及外植体的类型,年龄和生理状态对转化效率有很大的影响,条件优化后,得到大量具有PPT抗性的愈伤和一些抗性植株,抗性愈伤的GUS染色阳性率在50%-60%之间,所检测的抗性苗呈GUS阳性,对6株抗性苗的PCR和Southern检测初步证明,外源基因已经整合到其中3株的基因组中。     

Tissue-Specific Distribution of Secondary Metabolites in Rapeseed (Brassica napus L.)

Four different parts, hypocotyl and radicle (HR), inner cotyledon (IC), outer cotyledon (OC), seed coat and endosperm (SE), were sampled from mature rapeseed (Brassica napus L.) by laser microdissection. Subsequently, major secondary metabolites, glucosinolates and sinapine, as well as three minor ones, a cyclic spermidine conjugate and two flavonoids, representing different compound categories, were qualified and quantified in dissected samples by high-performance liquid chromatography with diode array detection and mass spectrometry. No qualitative and quantitative difference of glucosinolates and sinapine was detected in embryo tissues (HR, IC and OC). On the other hand, the three minor compounds were observed to be distributed unevenly in different rapeseed tissues. The hypothetic biological functions of the distribution patterns of different secondary metabolites in rapeseed are discussed.     

Resistance to Water Absorption in Germinating Rapeseed (Brassica napus L.)

Dynamics of the imbibition phase of rapeseed (Brassica napusL.) was studied by evaluating water content-water potentialrelations and the permeability to water. It was found that resistanceto water absorption was very high (diffusivity was very low)during the early stages of water absorption. As seed water contentincreased, resistance decreased by more than six orders of magnitude. The concept of hydraulic conductivity of a seed was introduced.The change in hydraulic conductivity with change in seed watercontent was similar to that observed for diffusivity. It wasshown that at late stages of water absorption hydraulic conductivityapproached values found in soils at the dry end of the availablewater range. For this reason it was suggested that hydraulicconductivity in soil may have an influence on imbibition ratein the late stages of water absorption.     

Unraveling the Genetic Basis of Seed Tocopherol Content and Composition in Rapeseed (Brassica napus L.)

Background

Tocopherols are important antioxidants in vegetable oils; when present as vitamin E, tocopherols are an essential nutrient for humans and livestock. Rapeseed (Brassica napus L, AACC, 2 n = 38) is one of the most important oil crops and a major source of tocopherols. Although the tocopherol biosynthetic pathway has been well elucidated in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803, knowledge about the genetic basis of tocopherol biosynthesis in seeds of rapeseed is scant. This project was carried out to dissect the genetic basis of seed tocopherol content and composition in rapeseed through quantitative trait loci (QTL) detection, genome-wide association analysis, and homologous gene mapping.

Methodology/Principal Findings

We used a segregating Tapidor × Ningyou7 doubled haploid (TNDH) population, its reconstructed F₂ (RC-F₂) population, and a panel of 142 rapeseed accessions (association panel). Genetic effects mainly contributed to phenotypic variations in tocopherol content and composition; environmental effects were also identified. Thirty-three unique QTL were detected for tocopherol content and composition in TNDH and RC-F₂ populations. Of these, seven QTL co-localized with candidate sequences associated with tocopherol biosynthesis through in silico and linkage mapping. Several near-isogenic lines carrying introgressions from the parent with higher tocopherol content showed highly increased tocopherol content compared with the recurrent parent. Genome-wide association analysis was performed with 142 B. napus accessions. Sixty-one loci were significantly associated with tocopherol content and composition, 11 of which were localized within the confidence intervals of tocopherol QTL.

Conclusions/Significance

This joint QTL, candidate gene, and association mapping study sheds light on the genetic basis of seed tocopherol biosynthesis in rapeseed. The sequences presented here may be used for marker-assisted selection of oilseed rape lines with superior tocopherol content and composition.     

农杆菌介导的玉米遗传转化研究进展

本文就农杆菌介导的玉米遗传转化的技术要点及原理等进行了综述,并对各种影响农杆菌转化玉米效率的关键因子包括农杆菌的菌株与载体、标记基因、受体材料的基因型、来源和发育状态以及组织培养的条件等进行了讨论。     

根癌农杆菌介导的大豆遗传转化

农杆菌介导法是大豆遗传转化的重要方法之一 ,许多实验室应用该方法得到了转基因大豆 ,但目前使用该方法进行转化的效率还比较低 ,尚需深入研究。农杆菌菌株、大豆基因型、组织培养条件、T-DNA的转移效率和转化后的筛选模式都会影响大豆转化的效率。概述了近年来根癌农杆菌介导的大豆遗传转化的一些重要成果 ,以及转化过程中大豆的易感性与农杆菌的转化能力、乙酰丁香酮促进vir基因活化、转化的受体系统和巯基混合物减轻受体材料的褐化、提高T DNA的转移效率等几个重要因素的研究进展 ,并介绍了转化中常用的几个筛选标记基因 (nptⅡ、hpt、bar基因和突变的ahas基因 )及通过共转化法去除标记基因的方法 ,同时对今后研究的重点进行了讨论.     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.