棉花八氢番茄红素脱氢酶GhPDS1基因的克隆与表达谱分析

八氢番茄红素脱氢酶(PDS)在胡萝卜素生理代谢和病毒诱导外源基因沉默中起着非常重要的作用.本研究通过同源克隆方法,分析已报道植物八氢番茄红素脱氢酶氨基酸保守区,设计筒并引物,扩增出编码棉花PDS基因cDNA中间片段.通过RACE技术成功地克隆了棉花PDS基因的全长cDNA,命名为GhPDS1(GenBank No.HQ...     

Breeding high-yield upland cotton (Gossypium hirsutum) lines with high-quality fibre by mutation

The yield and fibre quality of upland cotton (Gossypium hirsutum) cultivars are very difficult to improve simultaneously. Attempts to breed upland cotton cultivars with high-quality fibre that can spin more than 40 yarn count (Ne) and present yields equal to those of commercial cultivars have not been successful. The seeds of the high-yield upland cotton line Tai 8033 were mutagenized in a Chinese Practice No. 8 recoverable satellite, and the mutated line was selected to produce high-quality and high-yield lines by pedigree selection. The eight mutated lines fit the Chinese standard of high-quality cotton class I, were suitable for spinning more than 40 Ne and had yields that were 12.7%, 7.1% and 3.5% higher than the current control cultivar yield in the cotton cultivation area tested in Jiangsu Province. The fibre of one line successfully spun 80 Ne that fit the high-quality yarn standards. After seeds were mutagenized by exposure to a space environment in a satellite, the fibre quality of upland cotton cultivars was improved through pedigree selection.     

新型陆地棉细胞质雄性不育系花器形态学和细胞学观察

 采用石蜡切片法,对一种来源于陆地棉的新型细胞质雄性不育材料的3份转育材料（G22A,H109A和1793A）的花药发育过程进行了花器形态及细胞学观察。花器形态和细胞学观察结果表明,G22A,H109A和1793A三个不育系均花瓣小,花丝短,柱头明显外露,败育彻底,无花粉。败育时期主要发生在造孢细胞增殖时期至小孢子母细胞减数分裂前,表明这3份不育材料不育性状不受细胞核基因的影响。对不育材料与其保持系细胞学观察发现,不育材料与正常花药发育细胞学形态上主要差别是绒毡层发育。不育系绒毡层的着色不明显,大小与中层细胞相似且在整个花药发育过程中不降解,而保持系绒毡层的着色深,在小孢子发育过程中逐渐降解。因此,根据细胞学研究结果可以说明花药中绒毡层发育异常是该来源于陆地棉的新型细胞质雄性不育材料败育的细胞学基础。     

Genetic analysis of earliness in upland cotton ( Gossypium hirsutum L.). II. Yield and lint percentage

Inheritance and interrelationships of seed cotton and lint yields were evaluated in a diallel analysis involving seven early maturing parents of different origin and a commercial variety. Lint yield showed relatively little additive variance and low heritability, whereas lint percentage showed the opposite. Highest yields were shown by the least determinate and slowest-maturing genotypes; yields generally decreased as determinacy increased and rate of maturity accelerated. Except for date for first open boll, components of earliness showed no associated with yield.     

Inheritance of resistance to Verticillium wilt (Verticillium dahliae) in cotton (Gossypium hirsutum L.)

Verticillium wilt, caused by Verticillium dahliae Kleb., is a major constraint to cotton production in almost all countries where cotton is cultivated. Developing new cotton cultivars resistant to Verticillium wilt is the most effective and feasible way to combat the problem. Little is known about the inheritance of resistance to Verticillium wilt of cotton, especially that caused by the defoliating (D) and nondefoliating (ND) pathotypes of the soil‐borne fungus V. dahliae. The objective of this study was to determine the inheritance of resistance in cotton against both pathotypes of V. dahliae. Crosses were made between the susceptible parent ‘Cukurova 1518’ and each of four resistant parents PAUM 401, PAUM 403, PAUM 405 and PAUM 406 to produce F₂ generations in 2002 and F_2:3 families in 2003. Disease responses of parent and progeny populations to the D and ND pathotypes were scored based on a scale of 0‐4 (0, resistant; 4, susceptible). F₂ populations inoculated with the D pathotype showed a 3 : 1 (resistant : susceptible) plant segregation ratio. Tests of F_2:3 families confirmed that resistance was controlled by a single dominant gene. In contrast, analysis of data from F₂‐ and F₂‐derived F₃ families suggested that resistance to the ND pathotype is controlled by dominant alleles at two loci.     

玉米光敏色素A1基因(ZmPHYA1)在棉花中的转化及分子鉴定

为评价玉米ZmPHYA1基因在棉花种质资源改良中的价值,本研究利用农杆菌介导法在陆地棉(Gossypium hirsutum)R15材料中进行了玉米ZmPHYA1基因的遗传转化。经过愈伤组织诱导、抗性愈伤筛选、体细胞分化诱导后获得棉花转基因再生植株。通过田间草铵膦除草剂筛选鉴定抗性植株,并利用PCR扩增其草铵膦抗性基因和目的基因ZmPHYA1进行分子鉴定,发现阳性植株对草铵膦除草剂具较好抗性,并可扩增到256 bp的草铵膦抗性基因和217 bp ZmPHYA1基因的特异条带。进一步通过免疫印迹检测表明,3个不同转基因株系中外源ZmPHYA1基因可正常表达约170 kD大小的蛋白,且在不同组织中该外源蛋白均可正常表达。此外,对转基因植株的不同农艺性状分析表明,转基因株系株高明显低于受体对照,而铃重和纤维长度等性状无明显差异。本研究成功获得具有草铵膦抗性和外源ZmPHYA1基因的棉花新种质材料,为进一步利用光敏色素基因创新种质资源提供了材料来源。     

Two-year study on combining ability and heterotic potential in functional traits under salt stress in upland cotton (Gossypium hirsutum)

Salt tolerance is a physiologically and genetically complex trait controlled by multiple genes. To analyze the genetic basis of salt tolerance we evaluated 18 F₁ along with their nine parents under three salt stress levels i.e. control, 10 dS m⁻¹ and 15 dS m⁻¹ in the summer of 2017 and 2018. Data were recorded for the number of bolls, seed cotton yield, boll weight, Na⁺, K⁺, K⁺/Na⁺ ratio, H₂O₂, SOD, POD, CAT and TSP. Line × tester analysis indicated that the contribution of lines was greater than testers. Except for Na⁺, most of the traits were controlled by non-additive genes. With the rise of salt stress, SCA variances increased and were higher than GCA variances for most of the traits indicating the traits were controlled by a few largely dominant genes. KEHKSHAN, FH-118 and FH-114 were found good general combiner whereas the cross KEHKSHAN × FH-114 was a good specific combiner and also indicated significant better parent heterosis for most traits during two years under salt stress and can be utilized in a breeding programme for salt tolerance.     

陆地棉开花相关基因GhFLP1的克隆与功能验证

为了明确陆地棉开花促进因子基因的作用,以中棉所36叶片基因组DNA和cDNA为材料克隆得到了开花相关基因Gh FLP1,分析了其特征及功能。该基因全长为537 bp,包含339 bp开放阅读框,编码112个氨基酸。预测分子量约为12.176 kDa,理论等电点为9.13。组织特异性表达分析表明,该基因在花蕾中优势表达,且在早熟品种(中棉所36、中棉所74)中表达量高于中熟品种(中棉所60、鲁棉研28)。启动子序列分析和外源激素处理实验表明该基因受水杨酸和赤霉素调控。农杆菌介导转化拟南芥发现,该基因能促使拟南芥莲座叶减少,开花期提前。荧光定量结果表明,在转基因拟南芥中,内源开花相关基因AtFT、AtLFY、AtAP1和AtSOC1表达量不同程度上调,AtFUL表达量基本不变,AtFLC表达量下调。研究结果显示该基因可能参与陆地棉开花时间的调控,为创制转基因早熟棉花新材料打下基础。     

半野生棉与亚洲棉对草甘膦抗性评价及比较分析

采用叶片棉球定位鉴定法,初步鉴定并评价了半野生棉和亚洲棉对草甘膦的自然抗性,结果表明:参试材料抗性水平从高抗至高感分6级,极端抗性水平相差33.3倍(0.2%对0.006%)。其中,半野生棉对草甘膦的自然抗性分级丰富,抗性水平分布自高抗至高感;但大部分半野生棉不耐草甘膦,近86%(346份)的材料抗性水平处于低耐级别以下。而亚洲棉抗性水平都处于高抗至低耐的4个级别,抗性多样性较差,但整体抗性水平较高,70.3%(71份)表现为耐或以上级别。从半野生棉和亚洲棉中分别筛选到2份和5份高抗材料,半野生棉中有1份高感材料,亚洲棉中无对草甘膦敏感材料。本研究报道的极端材料为棉花草甘膦抗性遗传机制研究、新品种的培育、内源抗性基因的挖掘和感抗机理研究,奠定了良好的材料基础。     

棉花转录因子GhWRKY4基因的克隆及特征分析

 本研究从陆地棉中克隆得到GhWRKY4基因,全长cDNA是1281 bp,包含1083 bp的开放阅读框,编码的360个氨基酸属于IIcWRKY转录因子家族。分析GhWRKY4基因结构表明其有三个外显子和两个内含子。GhWRKY4蛋白的亚细胞定位实验表明其定位在洋葱表皮细胞核。实时荧光定量分析结果显示GhWRKY4在棉花的根、茎、叶和花中均有表达。染色体步移得到了GhWRKY4的5’端侧翼序列,生物信息学软件预测表明GhWRKY4包含一系列和胁迫基因调控相关的反式作用元件,表明GhWRKY4参与植物对盐害和冷害胁迫的反应。     

陆地棉GhERF8基因的克隆与表达分析

 乙烯响应元件结合因子（Ethylene-responsive element-binding factor,ERF）是植物中最大的转录因子家族之一。本研究以棉花叶片cDNA文库为基础,从陆地棉中棉所10号中克隆得到一个新的ERF基因,命名为GhERF8（GenBank：JN656957）。该基因编码265个氨基酸,蛋白序列中包含一个AP2保守结构域。采用荧光定量PCR（RT-PCR）的方法,对GhERF8基因在棉株不同生育时期叶片和不同组织中的表达水平进行了定量分析。结果表明,GhERF8基因在各组织中均有表达,但在成熟后期的叶片中表达量最高。GhERF8表达量与叶片衰老过程中叶绿素含量出现相反的变化趋势,推测GhERF8可能与叶片衰老有一定关系。在乙烯利和茉莉酸处理下GhERF8基因在叶片中上调表达,而在脱落酸处理下GhERF8表达量无显著变化,推测GhERF8可能处于乙烯和茉莉酸信号转导网络中,且表达途径为非ABA依赖途径。     

脱落酸对棉花体细胞胚胎发生的影响

【目的】研究脱落酸(Abscisic acid, ABA)对棉花体细胞胚胎发生过程中下胚轴脱分化和再分化的影响,优化体细胞胚胎发生体系和初步解析脱落酸调控棉花体细胞胚胎发生分子机制。【方法】以棉花品种中棉所24(CCRI 24)下胚轴为外植体,设置5个ABA浓度0、0.02、0.04、0.06、0.08μmol·L^-1,分别以A0、A1、A2、A3、A4表示,添加至MSB(MS培养基+B5维生素)培养基诱导愈伤和胚性愈伤,研究ABA对棉花下胚轴初始细胞脱分化、愈伤组织诱导和胚性愈伤组织诱导的影响。【结果】ABA促进下胚轴初始细胞脱分化;显著提高愈伤组织的脱分化率和增殖率;0.02μmol·L^-1ABA显著提高胚性愈伤分化率,0.04~0.08μmol·L^-1ABA显著降低胚性愈伤分化率。ABA处理后胚性愈伤和非胚性愈伤的增殖率均显著提高且质地受到影响。0.02~0.08μmol ABA处理下,LBD和LBD在愈伤起始期上调表达。0.02μmol·L^-1ABA处理下,在愈伤增殖早期和中期BBM、LEC1和AGL15上调表达,愈伤增殖后期FUS3、LEA、ABI3基因上调表达。【结论】脱落酸调控的棉花体细胞胚胎发生与相关标记基因的时空性表达密切相关,这些基因表达水平的增加是ABA调控愈伤和胚性愈伤分化的分子基础。     

棉花微管结合蛋白基因GhMAP1-LC3的克隆与表达分析

应用RT-PCR从陆地棉(Gossypium hirsutum L.)石远321开花当天胚珠中分离到一个553 bp的片段,含有一个360 bp的开放读码框,推导的氨基酸序列（119个氨基酸）与水稻、拟南芥的MAP1-LC3 (Microtubule-associated Protein 1-Light Chain3)分别具有94%与90%的同源性,由此推测该基因为编码棉花微管结合蛋白基因家族的一     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

棉花GhFTL1基因的克隆及初步功能分析

通过RT-PCR结合RACE技术,从新疆陆地棉品种新陆早33中克隆到一个FT类似基因,命名为GhFTLl基因(登录号:HM631972).该基因ORF(Open Reading Frame)全长为525 bp,可编码174个氨基酸,与其它植物中克隆的FT同源蛋白高度相似,含有FT蛋白亚家族两个关键的氨基酸残基及保守氨基...     

陆地棉Dirigent-like基因(GhDIR)的克隆与分析

Dirigent-like基因(DIR)编码蛋白质参与木质素的形成,与植物体的病菌防御有关.本研究以抑制差减杂交( SSH)技术获得DIR的EST为信息探针,对棉花EST数据库进行同源搜索和序列拼接,获得了全长为768 bp的DIR基因cDNA全长,将该基因命名为GhDIR.该基因序列的开放阅读框(ORF)位于70 ～...     

陆地棉GAI基因原核表达与多克隆抗体制备

 DELLA蛋白是GA信号响应的关键负调节因子,本文采用基于EST的电子克隆方法,从棉花中克隆了DELLA蛋白家族的一个成员GhGAI。根据电子克隆序列,从陆地棉花胚珠cDNA中扩增得到GhGAI全长基因片段。将其在原核中表达,得到了分子量（Mr）为62000的蛋白质条带。表达蛋白经His-Tag亲和层析纯化后,对兔子进行免疫,制备的抗血清通过间接ELISA检测,具有较高的效价和特异性。     

Induction of Embryogenic Callus in Gossypium barbadense L. and Gossypium hirsutum L. by Hormone Treatments

The aim of this study was to induce embryogenic callus from various cultivars of cotton in tissue culture, so that a stable and efficient regeneration system could be developed to produce new cotton varieties for cultivation in Xinjiang. The explant materials were hypocotyls of the main cotton cultivars grown in Xinjiang, i. e. Xinhai 25, Xinhai 16, Xinluzao 39, and Xinluzao 42. We tested the effects of different combinations of two hormones (kinetin, KT; 2,4-dichlorophenoxyacetic acid, 2,4-D) on induction of callus from these explants. Calli were produced by the explants under four different combinations of hormones in the media. The optimal hormone combination to induce callus from Gossypium hirsutum explants was 0.1 mg·L^-1 KT + 0.05 mg·L^-1 2,4-D, while that to induce callus from Gossypium barbadense explants was 0.1 mg·L^-1 KT + 0.1 mg·L^-1 2,4-D. Hormone-free medium and medium containing double to the normal concentration of KNO₃ promoted the emergence of embryogenic callus. Filter paper placed under the medium promoted somatic embryo growth and regeneration of the root system. The differentiation and embryogenesis processes occurred more rapidly in G. hirsutum explants than those in G. barbadense explants. Using this protocol, normal plantlets of these cotton cultivars with strong roots were produced within 10 to 12 months. These methods could be used to increase the number of cotton genotypes that can be regenerated in tissue culture.     

陆地棉REM基因家族全基因组鉴定及表达分析

[目的]REM(Reproductive meristem)基因家族编码一类转录因子在植物生长发育过程中发挥重要作用,但在棉花中未见报道.[方法]基于陆地棉基因组数据和公共数据库中的转录组数据,运用生物信息学方法对陆地棉REM基因家族成员进行系统鉴定,并对该基因家族的理化性质、基因结构、组织表达特异性以及胁迫条件下的表...