农杆菌介导的枳壳转化系统建立研究初报

以枳壳为对象 ,进行了农杆菌介导的上胚轴转化系统建立研究 ,得到了携带柑桔衰退病病毒外壳蛋白基因的枳壳转化植株。研究表明 :枳壳各类外植体中 ,上胚轴是较好的转化材料 ;上胚轴出芽部位主要在形态学上端。以卡那霉素为选择剂 ,外植体水平面放置时 ,选择剂的剂量是 50～ 70mg/ L。转化研究中发现感染液中乙酰丁香酮 10 0μmol/ L的加入促进转化外植体 Gus表达阳性率的提高。延迟 1a的选择培养 ,有助于 Gus阳性芽的增多。所得 Gus阳性无性系经 Southern blot杂交检测 ,证明外源基因已稳定整合到了植物基因组中 ,所得 Gus阳性植株确为携带柑桔衰退病病毒外壳蛋白基因的转化植株     

玉米Lc基因植物表达载体构建及菊花转化

Lc基因是从玉米中分离得到的与花青素合成相关的调节基因,它在多种植物中的异源表达可以增加花青素的含量.本研究对pBI121载体Gus基因后的终止子进行2次PCR扩增,在原Sac Ⅰ酶切位点后添加了新的Sac Ⅰ酶切位点,利用组织化学染色法检测,结果表明改造后的载体上的Gus基因能正常表达,终止子功能正常,载体改造成功.用改造的pBI121N构建了含有Lc基因的植物表达载体pBl121N-Lc,利用农杆菌介导法转化菊花叶盘,获得了19株生根抗性苗.通过提取抗性苗基因组总DNA,PCR扩增Lc基因和CaMv35S启动子获得了11个阳性株系,PCR结果表明Lc基因已经转入菊花中.同时,在已获得的转基因植株中发现7个株系的根系有变红的现象.     

根癌农杆菌介导的芦荟遗传转化条件的研究

以美国库拉索芦荟(Aloe.arborescens)的横切薄层切片(transverse thin cell layer, tTCL)作为转化受体, 通过受体材料对抗生素的敏感性实验和Gus 基因瞬时表达率的研究, 找出了较适合的外植体转化条件。研究表明:芦荟对头雹霉素(cefotaxime) 和羧苄霉素(carbenicillin)不敏感, 而对卡那霉素(kanamycin)和潮霉素(hygromycin)敏感;用靠近顶芽的材料得到的横切薄层切片芽再生率高, 有较高的Gus 基因瞬时表达率;乙酰丁香酮(acetosyringone)在芦荟转化是不可缺少的, 对其转化有明显的促进作用。     

ACA基因启动子的克隆及功能初探

根据已知的ACA基因的5’端序列设计三个基因特异的反向引物(GSP-1,GSP-2,GSP-3)分别与11个简并引物(AD1-AD11)配对,进行热不对称嵌套PCR(Thermal asymmetric interlaced PCR,TAIL-PCR)扩增,获得了ACA基因起始密码子上游约700bp的片段。为检测其表达特性,构建了该片段与Gus嵌合基因的表达载体pBpAG,在真空条件下通过农杆菌介导,转化了植物的叶、果实、种子三种不同组织,Gus瞬时表达染色结果显示,该DNA片段具有种子特异的启动子活性?对该启动子的一些顺式元件进行了讨论。     

甘草不同功能基因拷贝数多态性组合类型与叶片形态及甘草酸含量的相关性分析

目的:探讨甘草功能基因拷贝数多态性(CNV)与叶片形态特征及甘草酸含量的相关性,为筛选高品质甘草奠定基础。方法:以来自7个产地的60株甘草为实验材料,采用实时PCR对甘草中的3-羟基-3-甲基戊二酰Co A还原酶(HMGR)基因、鲨稀合成酶1(SQS1)基因和β-香树脂醇合成酶(β-AS)基因进行拷贝数测定,采用HPLC测定甘草酸含量,并考察不同功能基因CNV组合类型甘草叶片的形态特征。结果:根据实时PCR测定结果,按照3个功能基因的拷贝数,将甘草分为6种类型,即A型(β-AS+HMGR+SQS1=1+1+1)、B型(1+2+1)、C型(2+1+1)、D型(2+1+2)、E型(2+2+1)和F型(2+2+2);不同类型甘草在叶片形态特征方面存在显著性差异,A型与D型叶片的整体面积较小,而B型与E型叶片的整体面积则较大;HPLC测定结果显示B型甘草的甘草酸平均含量最高,E型甘草的甘草酸含量较高,而A型和D型的甘草酸含量则较低。结论:甘草中功能基因CNV组合类型与甘草叶片的形态特征及甘草酸含量之间存在明确的相关性,β-AS基因与SQS1基因单拷贝、HMGR基因双拷贝的甘草植株,其叶片具有最大的面积,同时其甘草酸含量最高。本研究结果可为优质甘草筛选奠定基础。     

ACA基因启动子的克隆及功能初探

根据已知的ACA基因的 5 '端序列设计三个基因特异的反向引物 (GSP-1,GSP-2 ,GSP-3)分别与 11个简并引物 (AD1-AD11)配对 ,进行热不对称嵌套PCR(ThermalasymmetricinterlacedPCR ,TAIL-PCR)扩增 ,获得了ACA基因起始密码子上游约700bp的片段。为检测其表达特性 ,构建了该片段与Gus嵌合基因的表达载体pBpAG ,在真空条件下通过农杆菌介导 ,转化了植物的叶、果实、种子三种不同组织 ,Gus瞬时表达染色结果显示 ,该DNA片段具有种子特异的启动子活性。对该启动子的一些顺式元件进行了讨论。     

胸腺肽基因多元植物表达载体的构建

Gus基因是最为常用的报告基因之一,为了便于对胸腺肽目的基因进行检测,在该研究中,胸腺肽目的基因、PRIB分泌基因和GUS报告基因DNA片段用DNA纯化回收试剂盒回收,然后用T4 DNA连接酶连接,构建成几个基因相融合的多元植物表达载体,并通过酶切进行鉴定。用基因枪转化法对胡萝卜愈伤组织进行了遗传转化,转化的胡萝卜愈伤组织经Southern杂交检测和X-Gluc染色,结果表明,多元表达载体成功地被导入胡萝卜愈伤组织。     

分子系统发育分析揭示中国云南省间座壳属两个新种

间座壳属真菌在多种经济上重要的农作物、观赏植物和森林树木上常作为植物病原菌、内生菌或腐生菌被人熟知。本研究调查我国云南省患病植物叶片上的间座壳属真菌,通过核糖体内部转录间隔区、β微管蛋白基因、翻译延伸因子基因、钙调蛋白基因和组蛋白基因的DNA序列数据比较和形态学特征描述了2个新种。它们是分别采集自密花胡颓子和荔枝患病叶片上的密花胡颓子间座壳和荔枝间座壳。     

茶树实时荧光定量PCR分析中内参基因的选择

选择合适的内参基因是提高实时荧光定量PCR分析（qRT-PCR）准确性的先决条件。该文以茶树（Camellia sinensis）芽、叶、幼根、嫩茎、花瓣、种子和愈伤组织为材料,应用实时荧光定量PCR技术,分析了18S rRNA、GAPDH、β-actin和α-tubulin4个常用内参基因在茶树不同器官组织中的表达情况。经GeNorm和NormFinder软件分析发现,当利用荧光定量PCR分析比较茶树不同器官组织中的基因表达差异时,可选择β-actin作为校正内参基因;而比较不同成熟度的叶片和愈伤组织时,可以选择GAPDH作为校正内参基因。     

蝴蝶兰PhRCAβ基因的克隆及表达特性分析

为研究蝴蝶兰Rubisco活化酶(RCA)基因对低温胁迫的响应机制,以蝴蝶兰"满天红"为材料,分析了蝴蝶兰Rubisco活化酶基因PhRCAβ的全长序列(Gen Bank登录号为KU954548)及其表达特性。结果表明,PhRCAβ基因全长1 695 bp,编码440个氨基酸;其编码的蛋白具有RCA的特异位点,含叶绿体转运肽,定位于叶绿体中,其成熟蛋白的分子量为42.52 k D,等电点为蛋白5.54,属于RCA小亚基基因;该基因在绿色组织中转录表达;13℃/8℃(昼/夜)的低温胁迫抑制Ph RCAβ基因的转录表达,并随着低温胁迫时间的延长,PhRCAβ基因的表达水平逐渐降低,在温度恢复正常时其表达水平升高;4℃低温条件下,Ph RCAβ基因的表达水平随着低温处理时间的延长逐渐降低,并一直维持在较低水平。由此推测,PhRCAβ直接参与了蝴蝶兰叶片光合作用的调控。     

Construction of the cybrid transplastomic Brassica napus plants containing Lesquerella fendleri chloroplasts

Transferring of Lesquerella fendleri genetically transformed plastids to Brassica napus plants has been performed with the somatic hybridization method. The plastome of the previously engineered transplastomic L. fendleri plants contained the aadA16gfp selective marker gene conferring spectinomycin/streptomycin resistance and green fluorescence under UV light. The protoplasts of B. napus chlorophyll-deficient plants were fused with gamma-irradiated protoplasts of L. fendleri transplastomic plants. A total of 59 green hybrid colonies have been isolated followed by spectinomycin/streptomycin selection. Shoot regeneration has been observed for two cell lines. Morphologically normal plants have been regenerated for one of them. PCR and isozyme analyses showed that the plants were transplastomic cybrids containing B. napus nuclei and L. fendleri transformed chloroplasts.     

Phenological responses to temperature of an annual and a perennial Lesquerella species

BACKGROUND AND AIMS: The annual Lesquerella fendleri, native to the south-western desert of United States and Mexico, and the perennial L. mendocina, native to Argentina, may have potential as new crops for cold-arid environments. The introduction of a new crop requires an understanding of environmental influences on growth and development, particularly temperature, which has been recognized as the main factor affecting the rate of development in crops. The objective of this study was to examine differences in the phenology of L. fendleri and L. mendocina and in the response to temperature in both vegetative and reproductive phases. METHODS: Plants of each species were grown at a range of constant temperatures under controlled conditions and developmental responses were analysed and quantified. KEY RESULTS: The rate of development of L. fendleri increased linearly with temperature in the phase from emergence (EM) to floral bud appearance (FBA) over the range 9-20 degrees C, and for the phase from FBA to first flower open (FL) over the range 9-24 degrees C. In contrast, the rate of development of L. mendocina was insensitive to temperature during the phase EM to FBA. In the phase FBA to FL, L. mendocina had a lower sensitivity to temperature than L. fendleri. In addition, L. fendleri exhibited a quantitative response to supra-optimal temperatures (reducing rate of development with further increases in temperature) whereas L. mendocina showed a qualitative response, with development ceasing to progress at temperatures above the optimum. CONCLUSIONS: This differential behaviour at high temperatures could explain the biennial habit found for L. mendocina sown during late spring under field conditions, whereas it behaves as an annual when sown in autumn-winter. The possibility is discussed of using this information for establishing the coincidence of critical stages with environmental conditions that can limit potential and actual yield through agronomic practices.     

Reproductive allocation of biomass and nitrogen in annual and perennial Lesquerella crops

* BACKGROUND AND AIMS: The use of perennial crops could contribute to increase agricultural sustainability. However, almost all of the major grain crops are herbaceous annuals and opportunities to replace them with more long-lived perennials have been poorly explored. This follows the presumption that the perennial life cycle is associated with a lower potential yield, due to a reduced allocation of biomass to grains. The hypothesis was tested that allocation to perpetuation organs in the perennial L. mendocina would not be directly related to a lower allocation to seeds. * METHODS: Two field experiments were carried on with the annual Lesquerella fendleri and the iteroparous perennial L. mendocina, two promising oil-seed crops for low-productivity environments, subjected to different water and nitrogen availability. * KEY RESULTS: Seed biomass allocation was similar for both species, and unresponsive to water and nitrogen availability. Greater root and vegetative shoot allocation in the perennial was counterbalanced by a lower allocation to other reproductive structures compared with the annual Lesquerella. Allometric relationships revealed that allocation differences between the annual and the perennial increased linearly with plant size. The general allocation patterns for nitrogen did not differ from those of biomass. However, nitrogen concentrations were higher in the vegetative shoot and root of L. mendocina than of L. fendleri but remained stable in seeds of both species. * CONCLUSIONS: It is concluded that vegetative organs are more hierarchically important sinks in L. mendocina than in the annual L. fendleri, but without disadvantages in seed hierarchy.     

Microsatellite development for an endangered riparian inhabitant, Lilaeopsis schaffneriana subsp. recurva (Apiaceae)

? Premise of the study: Microsatellite markers were developed and characterized to evaluate genetic diversity and population structure in Lilaeopsis schaffneriana subsp. recurva, an endangered species endemic to wetlands dispersed throughout southeastern Arizona, USA, and northern Sonora, Mexico. ? Methods and Results: Eight loci (one of which was monomorphic) were developed and characterized in 48 individuals from two populations. The total number of alleles was 35, ranging from one to 10 per locus. Many of the primers amplified in L. carolinensis, L. chinensis, L. masonii, L. occidentalis, L. schaffneriana subsp. schaffneriana, Oxypolis fendleri, and Eryngium lemmonii. ? Conclusions: Development of these novel microsatellite loci will facilitate a deeper understanding of genetic diversity, mode of reproduction, and population structure not only in L. schaffneriana subsp. recurva, but also in apiaceous relatives.     

Metabolism of Hydroxy Fatty Acids in Developing Seeds in the Genera Lesquerella (Brassicaceae) and Linum (Linaceae)

Members of the genus Lesquerella produce seed oil that contains a high proportion of hydroxy fatty acids (HFAs). There are three groups of Lesquerella species that are distinguished by their most abundant seed oil fatty acid: lesquerolic acid (20:1OH; e.g. Lesquerella fendleri), densipolic acid (18:2OH; e.g. Lesquerella kathryn), and auricolic acid (20:2OH; e.g. Lesquerella auriculata). To investigate the biochemistry of HFA production in Lesquerella species, the conversion of putative radiolabeled intermediates of HFA biosynthesis, including 18:1, 20:1,18:1OH, 18:2OH, and 20:1OH, was examined in developing embryos of L. fendleri, L.kathryn, and L. auriculata. The results are consistent with (a) 18:1OH formation by hydroxylation of 18:1, (b) elongation and desaturation of 18:1OH to produce 20:1OH and 18:2OH, respectively, and (c) desaturation of 20:1OH to produce 20:2OH. The desaturation of 20:1OH was also found to occur in developing embryos of high, but not low, linolenic acid flax. This suggests that the desaturation is catalyzed by the extraplastidial linoleate desaturase. Confirming this suggestion, it was notable that 18:1OH and 18:2OH were found in low and high linolenic flax (Linum usitatissimum) seeds, respectively, at levels of 0.2 to 1%.     

Resistant potato selections contain leptine and inhibit development of the Colorado potato beetle (Coleoptera: Chrysomelidae).

We recently described a new source of host-plant resistance to the Colorado potato beetle, Leptinotarsa decemlineata (Say), in a tetraploid potato (Solanum tuberosum L.) selection, ND2858-1. This genotype, and selected backcross progeny, had little damage while check cultivars were defoliated in open-choice field assays. To further characterize the observed deterrence, we determined foliar glycoalkaloids and conducted no-choice assays with ND2858-1 backcross progeny genotypes (ND4382-n). Development of neonate L. decemlineata in detached leaf assays on resistant progeny genotypes was delayed and larval weight gain after 4 d was inhibited by 75% relative to larval development and weight gain on susceptible genotypes. Inhibition of larval development in detached leaf assays with the selected progeny genotypes was equivalent to that of high-leptine genotypes of S. chacoense Bitter. Foliar glycoalkaloids of resistant genotypes included low levels of leptines I and II. The unlikely nature of this cross and the presence of leptine in this and resistant progeny selections cast doubt on the recorded pedigree. Molecular analyses were conducted by restriction fragment-length polymorphism and amplified fragment-length polymorphisms. Both methods established a high degree of relatedness to S. tuberososum and S. chacoense but not to S. fendleri. We conclude that ND2858-1 did not originate from a cross with S. fendleri, but is likely derived from S. chacoense. Oviposition and larval survival were reduced when adult L. decemlineata were placed in cages with resistant genotypes; an effect that was enhanced by inclusion of Perillus bioculatus F. Therefore, the nonpreference previously observed in open-choice field defoliation assays is also associated with antibiotic effects on L. decemlineata. The resistance may be caused by leptines, but is greater than would be expected by the leptine content. This source of host plant resistance could be a cost-effective management strategy, especially if combined with other resistance mechanisms or compatible control measures to delay development of resistance in the target insects.