转基因棉花的Bt基因流

通过两年实验检测转基因陆地棉品种间和海岛棉与陆地棉种间的Bt基因流.将转基因陆地棉国抗12号种植在12m×12m的样方内,周围分别种植非转基因陆地棉品种中棉所12号和海岛棉品种新海13号,在离转基因棉花不同距离选取样点,采集非转基因棉花种子,利用标记选择基因、Dot朎LISA和PCR扩增检测Bt基因流.结果表明在0～6m内陆地棉品种间显示较高频率的基因流,随着距离的增加Bt基因流降低,最大可达36m;提高样方内转基因棉花纯度,仅增加0～3m内基因流,对较远距离基因流无影响.海陆种间Bt基因流在0～6m内比陆地棉品种间低,但Bt基因流随距离增加下降幅度小,最远达72m.因此,在小规模转基因棉花环境释放实验,可选用同种不同品种棉花作为缓冲隔离带,并讨论了棉花转基因逃离至我国棉属种类的可能性.     

茎尖法转基因棉花植株真实性鉴定方法探究

棉花茎尖转化法具备不受基因型限制、转化周期短的优点,是理想的棉花转化体系,但据报道其所获得的转基因植株普遍存在遗传不稳定、高代植株基因丢失的现象。以陆地棉TM?1品种为受体材料,利用茎尖转化法将DsRed2载体转入棉花,经卡那霉素筛选获得16株抗性植株,进一步PCR扩增靶基因,获得6株DsRed2基因片段PCR检测为阳性的植株,初步判断该6株为茎尖转化法获得的转基因植株,但经紫外照射,6个转基因植株均未检测到红色荧光。对其进行靶基因RT?PCR,发现DsRed2基因在6个转基因植株中仅有极低量的表达或无表达。进一步对DsRed2载体的非T?DNA片段,即载体骨架部分进行PCR以及植株内生菌培养检测,结果表明,6个转基因植株均含有完整的DsRed2载体,植株可培养出含有完整载体的内生菌,且内生菌经农杆菌16S核糖体RNA（16S rRNA）片段PCR检测结果为阳性,推测由于茎尖侵染形成农杆菌与植株共生关系,造成假阳性株的现象,进而导致高代转基因植株基因丢失、遗传不稳定的现象。旨在建立一套完整的茎尖法转基因棉花植株真实性的鉴定方法,为进一步深入研究提供参考依据。     

影响陆地棉遗传转化的外部因子研究

以我国培育的陆地棉载品种为主,研究了农杆菌介导遗传转化过程中外植体材料、菌液浓度、感染时间、抗菌素等对论效率的影响以及愈伤组织的诱导分化过程,明确了影响睦棉遗传转化的外界条件。     

棉花转基因技术和转基因棉花

概述了棉花转基因育种技术和转基因抗虫棉、转抗除草剂基因棉花、转抗病基因棉花、转品质改良基因棉花及天然彩色棉花等的研究概况。     

小麦转基因方法及其评述

小麦是遗传转化比较困难的作物之一。为了克服小麦基因工程育种和功能基因组学研究的障碍,人们分别尝试利用基因枪、花粉管通道、超声波、离子束注入、激光微束穿刺、PEG(Polyethylene glycol)、电击和农杆菌等方法转化小麦,涉及的受体材料包括幼胚、成熟胚、花药愈伤组织、幼穗、芽尖和花器官。文章对小麦主要遗传转化方法及其应用进行了介绍、回顾和评述,分析、比较了获得安全型转基因小麦的几种策略,以期增强读者对小麦转基因技术和进展的了解,促进小麦转化技术的持续改进和提高。     

棉花转基因恢复系杂种F1小孢子发生的细胞学观察

对转gst基因棉花恢复系浙大强恢\"配制的杂种F1(三系杂交棉)的成熟花药和花粉育性进行了研究.结果表明,在花药的长、宽、鲜重和成熟花粉粒的育性方面,浙大强恢\"所配的F1和保持系DES-HAB277\"接近,无显著性差异,但比受体恢复系DES-HAF277\"所配的F1分别提高47.7%、61.8%、28.5%和39.6%.以不育系DES-HAMS277\"和保持系DES-HAB277\"的花药为对照,对浙大强恢\"和受体恢复系所配的F1的小孢子发生进行了细胞学观察发现,不育系小孢子败育主要发生在造孢细胞增殖期和小孢子母细胞形成期,且在减数分裂期彻底败育,不能形成四分体;受体恢复系所配的F1在小孢子发生和雄配子形成的各个发育时期都有部分败育,平均败育率约为20%,且主要发生在小孢子母细胞减数分裂期和小孢子单核期;而浙大强恢\"所配的F1与保持系一样,花药的发育、小孢子的发生以及雄配子的形成均正常.研究结果从细胞形态学方面证明gst基因对三系杂交棉具有防止部分小孢子败育和提高花粉育性的功能.     

农杆菌介导棉花大规模高效转化体系的研究

对农杆菌介导法转化棉花技术体系进行了综合改进,突破基因型的限制,使多品种或基因型(10个以上)的我国主栽棉花品种(系)转化成功,其中中棉所24号发展成为快速模式化转化品种(转化周期5～7个月,转化率8．1％),CCRI27、CCRI36等多个品种转化体系基本成熟。不同转化体系的愈伤组织诱导率为10％～40％,愈伤组织胚性愈伤诱导率达到10％～40％,转化率总体平均达到1．8％,并对大批量再生苗的鉴定予以程序化。实现棉花转基因规模化,达到年产转基因棉花植株2000～4000株以上的水平。     

转基因棉花的遗传研究

选用不同来源的转Ｂｔ基因抗虫棉中棉所３０号和新棉３３Ｂ、转ｔｆｄＡ基因抗除草剂棉花材料ＴＦＤ,通过与不同主栽推广品种正反交和回交研究外源Ｂｔ基因和ｔｆｄＡ基因在棉花体内的遗传和分离行为。结果表明,由外源基因提供给转基因的抗虫和抗除草剂性状均是由一对基因控制的显性性状,且该性状不受细胞质效应的影响,符合孟德尔遗传规律。     

Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex

 Transgenic cotton (Gossypium hirsutum L.) plants of a Texas cultivar CUBQHRPIS were obtained using Agrobacterium-mediated transformation coupled with the use of shoot-apex explants. After inoculation with A. tumefaciens strain LBA 4404 containing the pBI121 plasmid, regeneration of primary plants was carried out in a medium containing kanamycin (100  mg l^-1). Progeny obtained by selfing were germinated in the greenhouse and selected for expression of the T-DNA marker gene encoding neomycin phospho-transferase II (NPTII) by painting kanamycin (2%) on the leaves. Plants that survived the leaf painting were analyzed by DNA blots. Evidence for integration of the transgene (GUS) was observed in two successive generations from the regenerants (T₀). The transformed plants appeared to have more than one copy of the T-DNA. Received: 1 June 1998 / Accepted: 28 July 1998     

Increase of cotton cotyledon resistance to the herbicide endothall by abscisic acid

The herbicide endothall (7-oxabicyclo 2.2.1 heptane-2, 3-dicarboxylic acid) was applied to cotton ( Gossypium hirsutum L. cv. Acala SJ-1) cotyledon discs. Treatment with 10⁻⁴ M endothall for 24 h resulted in injury which was expressed by increased leakage of electrolytes, development of necrotic areas, increased level of polyphenols and tissue browning. We examined whether treatments which decrease chilling injury would also decrease injury caused by the herbicide. Tissue from seedlings grown at 28°C was more sensitive to endothall than that from seedlings grown at 15°C. Pretreatment with 10⁻⁵ M abscisic acid greatly decreased the leakage of electrolytes, necrotic areas, and tissue browning caused by endothall. Similar pretreatment did not prevent the increase of polyphenols caused by the herbicide. The treatment with abscisic acid was more effective in protection against the herbicide injury when applied several hours prior to the herbicidal treatment. This time requirement indicates that the mechanism by which abscisic acid induces resistance to the herbicide depends, at least partially, on active metabolism. We suggest that the increased resistance to herbicide stress by abscisic acid is another example of a common resistance mechanism to various stresses in which abscisic acid is involved.     

陆地棉脂肪酶基因克隆及氨基酸序列分析

根据陆地棉(Gossypium hirsutum L.)EST序列设计一对引物,采用RT-PCR方法从萌发的棉籽中获得了脂肪酶(triacylglycerol acylhydrolases,EC 3.1.1.3)基因,该基因编码483个氨基酸;比对结果显示,棉籽脂肪酶与拟南芥、水稻、蓖麻等脂肪酶相似性较低,具有由Ser-Asp-His组成的三联体催化活性中心,在亲核Ser残基周围有GXSXG保守序列.软件预测显示该脂肪酶为可溶性蛋白,分子量约为55.4 kD,等电点为9.07,不含N端信号肽,亚细胞定位可能是过氧化物酶体或胞质溶胶.     

陆地棉抗病基因同源序列的克隆与分析

根据已知抗病基因NBS保守区的P-loop和GLPL区设计一对简并引物F1/R1,以7个抗黄萎病陆地棉品种和2个感黄萎病品种的基因组DNA为模板进行PCR扩增.在9个品种中均扩增出500 bp左右的条带.对目的条带进行回收,连接、转化克隆得到350个阳性克隆,进行测序.在8个棉花品种中克隆到74条具有完整开放读码框的棉花RGAs序列.这74条序列共有64种不同的基因型,有10条与其他品种中的RGAs序列相同.用MEGA软件对8个棉花品种的74条RGA序列以及12个已知的抗病基因的NBS区域进行聚类分析,可分为4类;4类RGAs之间的相似性较低,各类之内的RGAs虽然来自不同品种,氨基酸序列的相似度却非常高.推测各大类中相似性较高的序列分别属于同一个基因家族,从位点上说可能处于同一个基因簇.     

A genetic approach to in vitro regeneration of non-regenerating cotton (Gossypium hirsutum L.) cultivars

Selections were made among individual plants of Gossypium hirsutum cv `Coker 310' for high-frequency in vitro regeneration by somatic embryogenesis. After three generations of selection, a pure line for high-frequency somatic embryogenesis was selected and named Coker 310 FR (FR, fully regenerating). Coker 310 FR could be regenerated by following previously published protocols (see Materials and methods) and a modified protocol developed in this study that reduced the time necessary for in vitro regeneration. Coker 310 FR was crossed with individual plants of major cotton cultivars grown in India, namely `MCU 5', `MCU 7', `Khandwa 2', `Bikaneri Nerma', `F 846' that have been shown to be recalcitrant to in vitro regeneration, to evaluate the regeneration potential of F₁s. All the F₁s showed regeneration by somatic embryogenesis. However, the F₁ of G. barbadense×G. hirsutum Coker 310 FR did not regenerate. Received: 16 September 1997 / Revision received: 1 April 1998 / Accepted: 15 May 1998     

Effects of drought on [1-14C]-oleic and [1-14C]-linoleic acid desaturation in cotton leaves

The effects of water stress on [1-¹⁴C]-oleic and [1-¹⁴C]-linoleic acid desaturations were studied in leaves of two varieties of cotton ( Gossypium hirsutum L.), one drought-sensitive (Reba) and the other more resistant (Mocosinho). After 24 h incorporation, [1-¹⁴C]-oleate led to the appearance of linoleate in phospholipids and, additionally, of linolenate in galactolipids. [1-¹⁴C]-Linoleate was desaturated to linolenate only in galactolipid fractions. Water stress markedly inhibited the incorporation of the precursors into the leaf lipids. The two desaturation steps were affected, particularly the transformation of linoleate to linolenate in monogalactosyldiacylglycerol in the drought-sensitive variety of cotton. The metabolic implications of the inhibition of the biosynthesis of C₁₈-polyunsaturated fatty acids are discussed.     

基因工程在克服移植免疫排斥反应及诱导免疫耐受中的应用

有效地控制慢性移植排斥反应和诱导移植免疫耐受是当今移植免疫学研究的热点。基因工程由于在克服移植免疫排斥反应和诱导免疫耐受中具有独特优势而倍受人们关注。然而,在临床应用基因治疗克服移植器官排斥之前,必须确定有效、安全的载体及适宜的靶基因。对该领域的新进展进行了简要综述。     

陆地棉品种和骨干品系黄萎病抗性鉴定

选育和推广抗病品种是防治陆地棉黄萎病的主要措施,为了早日实现多类型、多区域大面积抗病品种的应用,本研究选取107份遗传背景差异较大的种质,利用河北省农林科学院棉花研究所小安舍试验站黄萎病病圃进行了3年黄萎病抗性重复鉴定。鉴定得到抗病品系8个,占7.5%;耐病品种(系)20个,占18.7%。本研究表明,当前被作为育种亲本的抗病品系还太少,需要深入开展抗病遗传机制,以及与其他经济性状协同改良的关系,为陆地棉抗病育种提供理论指导;达到抗病或接近抗病水平的大部分品种(系)来自于海陆野远缘后代,具有外源基因血统,证明了远缘杂交是陆地棉黄萎病抗性改良的有效手段。     

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467. The text was submitted by the authors in English.