陆地棉4-香豆酸辅酶A连接酶基因Gh4CL30的功能分析

【目的】研究4-香豆酸辅酶A连接酶(4CL)家族基因Gh4CL30的生物学功能,为棉花株型育种提供理论依据和基因种质资源。【方法】利用病毒诱导的基因沉默技术和基因编辑技术获得Gh4CL30沉默和编辑植株,测定该基因抑制及敲除植株的黄酮和木质素含量,调查棉花田间表型性状、种子大小和纤维品质。【结果】Gh4CL30基因沉默和敲除植株中木质素合成相关基因表达量显著下降,4-香豆酸辅酶A连接酶含量显著减少,茎秆中木质素含量显著降低,其株高、种子大小和纤维长度显著降低。【结论】Gh4CL30通过调控棉花木质素生物合成功能影响其生长发育。

The ability of biostimulants and copper-containing fungicide to protect cotton against chilling stress

Background Cotton(Gossypium hirsutum L.),adapted to tropical and subtropical regions of the world,is highly sensitive to low temperatures throughout its life cycle.The objective of this study was to evaluate the mitigating effects of different doses of animal-derived(0.25%,0.50%,and 1.00% Isabion■),seaweed-based(0.165%,0.330%,and 0.660% Proton■)biostimulants,as well as a copper(Cu)-containing fungicide application,on cotton cultivar Lazer seedlings at the four true leaves(V4)stage.The plants were exposed to a low temperature of 5℃for 48 h,and the changes in morphological(seedling fresh and dry weight,plant height,and stem diameter)and physiological parameters(leaf temperature,chlorophyll content,relative water content,electrolyte leakage,and relative injury)were examined.Results The results revealed that chilling stress reduced plant growth,while biostimulants helped protect the plants and overcome the adverse effects of chilling.Under chilling stress,there was a considerable reduction in seedling fresh weight(SFW),seedling dry weight(SDW),plant height(PH),stem diameter(SD),leaf temperature(LT),and relative water content(RWC).Cotton seedlings treated with the animal-derived biostimulants showed significantly enhanced SFW,SDW,PH,SD,LT,chlorophyll content(Chl),electrolyte leakage(EL),and relative injury(RI),although there were no positive changes in RWC.No significant differences in the morphological traits were observed among the doses of seaweed biostimulants.For SDW,PH,EL,and RI,the best results were obtained with the application of a fungicide containing copper.Conclusion These results show the efficiency of the biostimulant and fungicide treatments in mitigating low-temperature stress in cotton seedlings.Applying a copper-containing fungicide to cotton seedlings helped to counteract the negative effects of low-temperature stress and to protect the plants from damage by maintaining electrolyte balance.Among the biostimulant applications,all levels of animal-derived biostimulant applications,as well as the 0.660% level of the seaweed-derived biostimulant,led to increased tolerance of cotton plants to chilling stress.

多环境下陆地棉(Gossypium hirsutum L.)重组自交系铃重与衣分性状的QTL分析

本研究利用以SGK9708为母本,0-153为父本构建的196个陆地棉重组自交系(F6:8)构建了包含186个标记,总长827.84cM,标记间平均距离4.45cM,覆盖棉花基因组18.6%的遗传连锁图谱,并对7个环境下的铃重和衣分性状进行QTL定位和上位性互作分析。利用两种分析软件(WinQTLcart2.5和QTLNetwork2.0)共同定位了多个环境下稳定表达的5个主效QTLs(qBW-1-1,qBW-1-2,qLP-2-1,qLP-2-2和qLP-4-2)。利用QTLNetwork2.0分别检测到4对铃重上位性互作QTLs和7对衣分上位性互作QTLs,以背景位点间的互作和加性效应位点与背景位点间的互作为主。除主效QTL外,上位性效应也是陆地棉铃重和衣分性状的重要遗传基础。本研究定位的5个主效QTLs为选择高铃重、高衣分品种的棉花分子标记辅助育种提供了重要依据。

陆地棉（G．hirsutumL．）茎尖分生组织培养及其在基因导入上的应用

陆地棉栽培品种的种子无菌发芽１～３天。将萌动种子的顶端分生组织培养在无激素或附加低浓度激素ＭＳ基本培养基上，直接再生完整小植株。从顶端分生组织到再生植株所需的最短培养时间约１０天。顶端分生组织供体种芽的萌芽天数及其所连接的胚轴长度对植株再生频率有明显影响，但与品种的关系较小，不同品种均能获得再生植株。应用基因枪轰击法将ＣＰＴⅠ基因和ＮＰＴⅡ基因的重组质粒导入到棉花茎尖分生组织，培养获得卡那霉素抗性株。

棉花(Gossypium hirsutum L.)原生质体培养的体细胞胚胎发生及植株再生

以陆地棉(G.hirsutum L.)3118、晋棉4号和柯字棉312等3个品种为材料,取种子无菌苗的下胚轴诱导愈伤组织,建立胚性细胞悬浮培养系。以2％纤维素酶Ono-zuka RS和0.1％离析软化酶Y-23组成的混合酶液,从细胞悬浮培养物游离出原生质体。用含低融点琼脂糖的K_3培养基包埋原生质体,采用园柱及薄层漂浮平板培养方式。3个品种的原生质体培养都得到了愈伤组织,其中晋棉4号和柯字棉312品种的愈伤组织转到低激素水平或无激素的MS-2培养基上诱导产生胚状体,胚状体在无激素的MS-1培养基上形成了植株。

陆地棉（G．hirsutumL．）×克劳茨基棉（G．klotzschianumAn─derss）的研究和利用

以陆地棉泗棉２号×克劳茨基棉，得到５粒种子，获Ｆ１１株，其他４粒种子都是空的。以陆地棉５２－１２８×克劳茨基棉，得到３粒种子，子叶平展后，抽不出顶芽，苗期夭折。泗棉２号×克劳茨基棉，其Ｆ１短日照性很强，叶枝多，果枝少，第一果枝着生节位高。花冠黄色，花瓣基部有淡红色斑点，花药不开裂，无花粉。以Ｆ１及其宿根为母本，直接与陆地棉回交，很容易得到种子，１铃最多结７粒，纤维棕色。ＢＣ１Ｆ１的株型基本上与陆地棉相似，经５２－１２８回交的，花瓣基部有淡红色斑点；泗棉２号回交的则无斑点，育性比Ｆ１显著提高，自交也能收到种子，絮色洁白。ＢＣ１Ｆ４已获得纤维高品质株系，２．５％跨距长度达３０．９～３１．６ｍｍ，比强度为２５．３～２６．８（ｇｆ／ｔｅｘ），麦克隆值为４．３～４．８。作者认为克劳茨基棉具有纤维高品质潜力基因，与陆地棉的亲缘关系比较密切。文中分析了Ｆ１苗期致死的原因。

陆地棉(Gossypium hirsutum L.)单体的诱发和鉴定

以隐性亲本(芽黄 v_1,v_6,v_8,v_(10),v_(11),T582,TM-1)作母本,授以辐射处理的显性亲本(TM-1,2256-61,陕1155,泗棉2号,Rd,T586)的花粉,进行单体的诱发研究。通过对 F_1表型为隐性或畸形的植株,在现蕾时观察其减数分裂时的染色体构型,发现有单倍体的存在。并将单体作母本,用相应的易位系测验,鉴定了诱发得到的单体株分别为单体1、6、7。辐射剂量为800伦琴和500伦琴,其诱致单体的频率分列为20.5%和9.8%。发现高剂量比低剂量更能诱致染色体的丢失,特别是小染色体的丢失,并且高剂量诱致的染色体结构变异也增加。

陆地棉(G.hirsutum L.)N-乙酰氨基葡萄糖转移酶基因(GhGnT)的克隆及耐盐性功能分析

在耐盐相关基因芯片基础上,我们筛选到盐诱导显著上调表达(Ratio>2)的N-乙酰氨基葡糖转移酶基因。选取耐盐材料中9806为材料,根据耐盐性抑制消减文库EST序列设计引物,利用RACE及RT-PCR技术获得该基因cDNA全长1970bp,命名为GhGnT。通过Blast比对,发现该基因与蓖麻乙酰氨基葡萄糖转移酶基因相似性最高,达到82%,认为GhGnT与该基因属于同类基因。利用TargetP1.1和toppred分析,GhGnT蛋白跨线粒体膜。由Real-time分析发现,该基因受盐胁迫诱导上调表达,在耐盐材料中9806的表达水平明显高于盐敏感材料中S9612,与芯片结果一致。有研究发现在小麦中发现糖基转移酶TaUGT1和TaUGT2受盐诱导上调表达,而且转化糖基转移酶亚基STT3a基因的杜氏盐藻耐盐性得到提高。因此,我们认为该基因与耐胁迫密切相关,并推测GhGnT的高表达对提高陆地棉耐盐性有一定作用。GhGnT基因的克隆及分析为以后的功能验证及耐盐种质的创新奠定基础。

施氮量对不同开花期棉(Gossypium hirsutum L．)铃纤维细度和成熟度形成的影响

为兼顾试验的重复性和生态区域性,选用高品质棉(科棉1号)和常规棉(美棉33B)品种为材料,于2005年分别在江苏南京(118°50'E,32°02'N,长江流域下游棉区)和江苏徐州(117°11'E,34°15'N,黄河流域黄淮棉区)设置施氮量(低氮N 0 kg/hm2;适氮N 240 kg/hm2;高氮N 480 kg/hm2)试验,研究施氮量对不同开花期棉铃纤维细度、成熟度和马克隆值形成的影响。结果表明,(1)施氮量显著影响棉纤维细度、成熟度和马克隆值的形成过程,但三者在不同开花期对氮素水平的响应不同,施氮量与开花期对棉纤维细度、成熟度和马克隆值的形成存在互作效应。8月10日前开花的棉铃,铃期[花后0～50 d(DPA)]日均温在23.3℃以上,纤维细度、马克隆值以N 0 kg/hm2施氮量下最大,棉纤维马克隆值与纤维细度的相关性较大;8月25日开花的棉铃(铃期日均温在20.8～23.3℃之间),纤维成熟度、马克隆值以N 240 kg/hm2施氮量下最大;9月10日开花棉铃(铃期日均温低于20.8℃),纤维细度、成熟度和马克隆值均以N 480 kg/hm2最大,棉纤维马克隆值与纤维成熟度的相关性增强。(2)影响不同开花期间纤维细度、成熟度和马克隆值的主要因素是铃期日均温;最终纤维细度、成熟度和马克隆值在不同施氮量之间的变异与不同开花期(铃期日均温不同)间的变异比较,前者显著小于后者。综上,因开花期不同而形成的铃期日均温是决定纤维细度、成熟度和马克隆值的最重要因素,施氮量可通过对位叶叶氮浓度NA影响棉纤维细度、成熟度和马克隆值的形成过程,增加施氮量可减小上述指标在不同开花期间的差异。

棉花(Gossypium hirsutum L.)花粉壁的发育

用光镜和透射电镜观察研究了棉花花粉壁的发育过程.棉花花粉壁的发生始于四分孢子时期,四分体形成后,在小孢子的质膜与胼胝质壁之间逐渐形成多糖性质的原外壁.游离小孢子时期,在一定间隔的原外壁中小孢子质膜表面积累外壁物质并形成基粒棒,之后在基粒棒的下部和上部分别横向延展并接合形成基足层和覆盖层.花粉内壁发生时期较晚,大约在大液泡形成之前.但内层和外层花粉壁均在小孢子有丝分裂之前同时建成.构建花粉壁的物质主要来自绒毡层的周原质团和小孢子原生质体的代谢分泌.小孢子胞质中在四分体时期形成的巨型内质网复合体是较为特殊的细胞结构,在游离小孢子时期,巨型内质网不断产生大量的内含微粒物的运输小泡,这种小囊泡表现出向质膜趋向运动并进一步与质膜融合.推测由内质网复合体产生的这些小囊泡是小孢子原生质体向质膜外运输花粉壁构建物质的主要形式.

利用重组自交系进行陆地棉(GossypiumhirsutumL.)棉籽油分含量和蛋白质含量的QTL定位

本研究利用以0—153为父本和SGK9708为母本构建的196个陆地棉重组自交系（F6：8）为材料对棉籽油分含量和蛋白质含量进行了遗传分析和QTL定位。通过四个环境下的群体材料的棉籽油分含量和蛋白质含量分析表明棉籽油分含量和蛋白质含量均为典型数量性状，其中棉籽油分含量存在超低亲本的超亲分离，而其蛋白质含量呈现超高亲本的超亲分离。相关性分析显示棉籽油分含量和蛋白质含量呈极显著负相关，同步提高两者在棉籽的的含量较为困难。基于包含186个标记，总长827．84cM，标记间平均距离4．45cM，覆盖棉花基因组18．6％的遗传连锁图谱，应用WinQTLcart2．5软件对四个环境下的棉籽油分含量和蛋白质含量进行了QTL定位，共检测到8个油分含量QTLs，解释表型变异5．42％～13．15％，其中稳定的QTL1个。4个蛋白质含量QTLs，解释表型变异4．35％～14．93％。本研究结果可为进行陆地棉种子营养品质性状的分子遗传改良奠定基础。

Screening of drought resistance indices and evaluation of drought resistance in cotton(Gossypium hirsutum L.)

Sixteen cotton cultivars widely planted in China were sowed under five different drought concentrations(0,2.5,5,7.5,and 10%)using PEG6000 to screen the indices of drought resistance identification and explore the drought resistance of different cotton cultivars.Eighteen physiological indices including root,stem,and leaf water contents(RWC,SWC,and LWC),net photosynthetic rate(Pn),the maximum photochemical quantum yield(Fv/Fm),the actual photochemical quantum yield(ΦPSII),non-photochemical quenching coefficient(NPQ),leaf water potential(LWP),osmotic potential(Ψs),leaf relative conductivity(REC),leaf proline content(Pro),leaf and root soluble protein contents(LSPC and RSPC),leaf and root malondialdehyde(MDA)contents(LMDA and RMDA),root superoxide dismutase,peroxidase,and catalase activities(RSOD,RPOD,and RCAT)were measured.Results indicated the 18 physiological indices can be converted into five or six independent comprehensive indices by principal component analysis,and nine typical indices(Fv/Fm,SWC,LWP,Pro,LMDA,RSPC,RMDA,RSOD,and RCAT)screened out by a stepwise regression method could be utilized to evaluate the drought resistance.Moreover,the 16 cotton cultivars were divided into four types:drought sensitive,drought weak sensitive,moderate drought resistant,and drought resistant types.The resistance ability of two selected cotton cultivars(drought resistant cultivar,Dexiamian 1;drought sensitive cultivar,Yuzaomian 9110)with contrasting drought sensitivities were further verified by pot experiment.Results showed that the responses of final cotton biomass,yield,and yield composition to drought were significantly different between the two cultivars.In conclusion,drought resistant cultivar Dexiamian 1 and drought sensitive cultivar Yuzaomian 9110 were screened through hydroponics experiment,which can be used as ideal experimental materials to study the mechanism of different cotton cultivars with contrasting drought sensitivities in response to drought stress.

Multi-Location Investigation of Optimum Planting Density and Boll Distribution of High-Yielding Cotton (G.hirsutum L.) in Hubei Province,China

Cotton yield per unit ground area has stagnated for a dozen years in Hubei Province, China, although a series of new high- yielding varieties have been commercialized. A multi-location investigation was carried out in 2008 and 2009 in 13 counties to determine if increased planting population density （PPD） would break the stagnant yield. The results showed that significant differences among the fields existed in theoretical yield, PPD, and bolls per square meter （BPM）. The lowest yield of 1 641.1 kg ha-I was resulted from the lowest PPD of 1.7 plants m-2 and the lowest BPM of 71.8 bolls m-2, while the highest yield of 2 779.7 kg ha-~ was resulted from the highest PPD of 2.5 plants m-2, and the highest BPM of 129.4 bolls m-z. Plant mapping revealed that boll retention rate （BRR） was maintained over 30 or 40% for the first 17-18 fruiting branches （FBs） and decreased dramatically thereafter, rotten boll rate （RBR） decreased, but open boll rate （OBR） rose first and dropped later with rising FB from the bottom to the top. But BRR, RBR, and OBR were all dropped with the fruiting positions （FPs） extending outwards. The optimum range of plant density would be 2-3 plants m-2 and the proper individual plant structure would be 16-19 FBs with 5-7 FPs for cotton production in Hubei Province.

Relationship between plant canopy characteristics and photosynthetic productivity in diverse cultivars of cotton(Gossypium hirsutum L.)

Genotype and plant type affect photosynthetic production by changing the canopy structure in crops.To analyze the mechanism of action of heterosis and plant type on canopy structure in cotton(Gossypium hirsutum L.),we had selected two cotton hybrids(Shiza 2,Xinluzao 43) and two conventional varieties(Xinluzao 13,Xinluzao 33) with different plant types in this experiment.We studied canopy characteristics and their correlation with photosynthesis in populations of different genotypes and plant types during yield formation in Xinjiang,China.Canopy characteristics including leaf area index(LAI),mean foliage tilt angle(MTA),canopy openness(DIFN),and chlorophyll relative content(SPAD).The results showed that LAI and SPAD peak values were higher and their peak values arrived later,and the adjustment capacity of MTA during the flowering and boll-forming stages was stronger in Xinluzao 43,with the normal-leaf,pagoda plant type,than these values in other varieties.DIFN of Xinluzao 43 remained between0.09 and 0.12 during the flowering and boll-forming stages,but was lower than that in the other varieties during the boll-opening stage.Thus,these characteristics of Xinluzao 43 were helpful for optimizing the light environment and maximizing light interception,thereby increasing photosynthetic capability.The photosynthetic rate and photosynthetic area were thus affected by cotton genotype as changes in the adjustment range of MTA,increases in peak values of LAI and SPAD,and extension of the functional stage of leaves.Available photosynthetic area and canopy light environment were affected by cotton plant type as changes in MTA and DIFN.Heterosis expression and plant type development were coordinated during different growth stages,the key to optimizing the canopy structure and further increasing yield.

Somatic Embryogenesis and Plant Regeneration from Two Recalcitrant Genotypes of Gossypium hirsutum L.

An improved protocol has been developed for somatic embryogenesis and plant regeneration of recalcitrant cotton cultivars. High callus frequencies and embryogenic tissue were developed in MSB medium supplemented with gradient concentrations of KT and 2,4-D, their concentration decreasing from 0.1 to 0.01 mg·L^-1. Somatic embryos were successfully incubated in 1/2 macronutrient MSB suspension supplemented with 0.5 g· L^-1 glutamine and 0.5 g·L^-1 asparagine. Decrease in macronutrient concentration of MSB significantly alleviated browning and was beneficial to suspension cells. Transformation of somatic embryos into plants was induced in MSB medium supplemented with 3% sucrose, 0.5 g·L^-1 glutamine, 0.5 g·L^-1 asparagine, and 6.0 g·L^-1 agar. The effect of sucrose as carbohydrate was better than that of glucose for plant germination. Using this protocol, regenerated plantlets from the CCRI521 and Zhongzhi86-6 reached to as much as 19.6 and 18.5% somatic embryos, respectively.

Comparative Analysis of Salinity-Induced Proteomic Changes in Cotton (Gossypium hirsutum L.)

Salt stress on cotton varieties of distinct salinity tolerance can induce expression of different proteins. Zhong 07, a salt-tolerant variety and Zhong s9612, a salt-sensitive variety, were utilized as experimental materials. The leaves of trefoil seedlings treated with or without 0.4% NaCl for 24 h were harvested for whole-protein extraction. Two-dimensional technology, combined with mass spectroscopy (MS) analysis and protein database searching, was employed to detect differentially expressed proteins and determine their identities and biological functions. Compared with the control, Zhong 07 showed 10 differentially expressed proteins under salt stress, of which 6 were upregulated and 4 were downregulated. Meanwhile, 12 differentially expressed proteins were detected in Zhong s9612 under salt stress, of which 10 were upregulated and 2 were downregulated. In the matrix-assisted laser desorption-ionization/time of flight-time of flight/MS analysis, 14 differentially expressed proteins were successfully identified, including the ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisco) large subunit-binding protein subunit alpha (RuBisco α), luminal binding protein (LBP), heat shock protein 70 (Hsp1, 2, 3), pathogenesis-related protein class 10 (PR-10), quinoneoxidoreductase-like protein (QOR), S-adenosylmethioninesyn-thetase (SAMS), enolase (EN), and RuBisco large subunit-binding protein subunit beta (RuBisco β). Cellular function is ultimately executed by proteins, and cotton varieties with different salt tolerance can be influenced by salt stress to various degrees, which can provide certain theoretical foundation for the identification of salt tolerance of cotton varieties. The findings also provide some proteins, such as the RuBisco large subunit binding proteins α and β subunits, OEE2 protein, HSP70, and S-adenosylmethionine synthetase, which can be used as protein markers of salt-to-lerance before- and post-treatment, making a big difference in salt-tolerance identification in cotton.

Molecular Cloning and Characterization of Three Novel Genes Related to Fatty Acid Degradation and Their Responses to Abiotic Stresses in Gossypium hirsutum L.

Fatty acid metabolism is responsible not only for oilseed metabolism but also for plant responses to abiotic stresses. In this study, three novel genes related to fatty acid degradation designated GhACX, Gh4CL, and GhMFP, respectively, were isolated from Gossypium hirsutum acc. TM-1. The phylogenetic analysis revealed that amino acid sequences of GhACXand GhMFP have the highest homology with those from Vitis vinifera, and Gh4CL has a closer genetic relationship with that from Camellia sinensis. Tissue- and organ-specific analysis showed that the three genes expressed widely in all the tested tissues, including ovules and fiber at different developing stages, with expressed preferentially in some organs. Among them, GhACX showed the most abundant transcripts in seeds at 25 d post anthesis （DPA）, however, GhMFP and Gh4CL have the strongest expression level in ovules on the day of anthesis. Based on real-time quantitative RT-PCR, the three genes were differentially regulated when induced under wounding, methyl jasmonate （MeJA）, cold, and abscisic acid （ABA） treatments. The characterization and expression pattern of three novel fatty acid degradation related genes will aid both to understand the roles of fatty acid degradation related genes as precursor in stress stimuli and to elucidate the physiological function in cotton oilseed metabolism.

Selection and Characterization of a Novel Glyphosate Tolerant Upland Cotton(Gossypium hirsutum L.) Mutant(R1098)

Stepwise selection approach was adopted to obtain glyphosate-tolerant upland cotton mutant(R1098) from the embryogenic calli of Coker 312(Gossypium hirsutum L.).The calli were transferred to selection medium and multi-step selection pressure process was carried out until the

Introduction of Bacillus thuringiensis(Bt)gene does not reduce potassium use efficiency of Bt transgenic cotton(Gossypium hirsutum L.)

Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.Results:The cotton variety,Jihe 321(wild type,WT)and its two Bt(Cry1Ac)-transgenic overexpression lines(OE-29317,OE-29312)were studied in field with low soil-test K+(47.8 mg·kg^(−1)).In the field with low soil-test K+,only OE-29317 had less biomass and K+accumulation than the WT at some growth stages.Both Bt lines produced similar or even greater seed cotton yield than WT in the field.When the Bt gene(~70%)in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing(VIGS),the VIGS-Bt plants did not produce more biomass than VIGSgreen fluorescent protein(control)plants.Conclusions:The introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.

An Integrated Genetic,Physical and Transcript Map of Homoeologous Chromosomes 12 and 26 in Upland Cotton(Gossypium hirsutum L.)

While Upland cotton(Gossypium hirsutum L.) represents 95% of the world production,its genetic improvement is hindered by the shortage of effective genomic tools and resources.The