大白菜基因组DNA快速提取方法的研究

快速高效的DNA提取是作物大规模分子育种的关键一步。旨为构建一种快速提取大白菜基因组DNA的方法,以大白菜叶片为试验材料,比较了CTAB法、二步CTAB法以及4种碱裂解法提取DNA的质量,分析了不同方法提取的DNA为模板的PCR扩增效果,还对不同方法提取的基因组DNA的保存时间和保存条件进行了比较,选择最优的方法在抗根肿病基因分子标记辅助选择中进行了应用。结果表明,CTAB法和3种碱裂解法提取的基因组DNA作为模板的PCR扩增产物,都可以通过8%的非变性聚丙烯酰胺凝胶电泳检测到清晰的条带。其中碱裂解法Ⅲ,不仅提取质量好,而且提取过程简单、快速,能够满足大白菜高通量DNA提取的需要,提取的DNA在4℃和-20℃的条件下保存,将保存至30 d的基因组DNA作为模板进行PCR扩增,产物仍然可以通过聚丙烯酰胺凝胶电泳检测到清晰的条带,说明这种方法保存时间较长,经验证,该方法在抗根肿病基因分子标记辅助选择中的应用效果也较好。碱裂解法Ⅲ显著提高了大白菜分子标记辅助筛选的效率,可广泛应用于大白菜分子标记辅助选择育种。     

一种玉米叶片基因组DNA快速提取新方法的初步研究

在玉米分子标记辅助育种工作中,需要对大量群体的个体样本进行分子标记分析,其中DNA的高效快速提取是关键的技术环节。以异硫氰酸胍为主要提取试剂,结合使用组织研磨器(Qiagen Tissuelyser),建立了一种玉米叶片基因组DNA的快速提取新方法。利用此方法提取玉米叶片基因组DNA,具有使用药品少、操作步骤简单、快速、成本低、DNA质量稳定等优点,通常情况下每人每个工作日可以提取300个DNA样品。此DNA提取方法可有效地用于玉米分子标记分析。     

一种快速提取棉花干种子基因组DNA的新方法

利用SSR(Simple sequence repeats)分子标记技术,能够快速有效地进行杂交棉纯度鉴定。为了攻克SSR分子标记技术中存在的技术难关,本文专门对棉花干种子胚的基因组DNA提取方法进行了研究。利用该方法,只需要2次加液、1次离心,从基因组DNA提取到SSR-PCR扩增,最后电泳,进行谱带分析,整个过程仅需4.5~5 h。本方法成本低、速度快、操作简单,适用于批量化DNA的提取,且整个提取过程无毒、无污染,是一种资源节约型和环境友好型的高效提取方法,适用于大批量样本的SSR分子标记鉴定工作。     

三种水稻基因组DNA快速提取方法的比较

用分子标记辅助选择进行精细定位和遗传改良时,首先就需要大批量提取植物DNA,作为PCR反应的模板。水稻基因组DNA常用的简易提取方法有碱处理法和高温水煮法等,本文改进了常规的CTAB方法,提出另一种简便的高质量DNA提取方法,称“简化法”,并比较了这三种方法提取的DNA在数量、质量上的差异,以及作为PCR模板时扩增效果,确定了在精细定位和遗传改良时“简化法”是一种较优的方法。     

种快速提取棉花干种子基因组 DNA 的新方 法简

 利用SSR(Simple sequence repeats)分子标记技术,能够快速有效地进行杂交棉纯度鉴定。为了攻克SSR分子标记技术中存在的技术难关,本文专门对棉花干种子胚的基因组DNA提取方法进行了研究。利用该方法,只需要2次加液、1次离心,从基因组DNA提取到SSR-PCR扩增,最后电泳,进行谱带分析,整个过程仅需4.5～5 h。本方法成本低、速度快、操作简单,适用于批量化DNA的提取,且整个提取过程无毒、无污染,是一种资源节约型和环境友好型的高效提取方法,适用于大批量样本的SSR分子标记鉴定工作。     

一种简单、极快的植物叶片DNA提取方法

为了满足分子标记辅助育种中基因型鉴定、基因克隆、测序的需要,本研究借用棉签等具吸附功能的材料代替移液器,依次直接在2种DNA提取液中操作,建立了一种简单、快速、环保的水稻叶片DNA提取方法。结果表明,提取溶液通过PCR扩增,满足基因扩增对模板的要求;同时,对其进行了灵敏度测试,稀释107倍仍然能够扩增出目标片段;最后通过PCR、克隆载体连接等分子生物学手段,验证此方法也满足测序的需要。本方法能够在90s内完成植物叶片DNA的提取,解决了目前植物基因组DNA提取方法需要离心机等昂贵特殊设备、提取步骤繁锁、提取过程中刺激味大、提取时间长等问题,有利于大规模开展分子标记辅助育种中材料基因型的鉴定,具有简单、快速、廉价、环保等优点。     

小麦基因组的一种简易提取方法

摘要：【研究目的】在综合分析多种提取小麦DNA方法的基础上,改良发展了一种快速简便高效的小麦基因组DNA提取方法。【方法】以春小麦Thatcher和以Thatcher为背景的近等基因系TcLr19,TcLr20, TcLr28为材料,改进后的方法提取的基因组DNA用抗叶锈基因Lr20的STS标记、Lr19和Lr28的SCAR标记、Lr28的SSR 标记进行PCR扩增,【结果】各分子标记都扩增出条带清晰正确的目的片段【结论】这表明该方法能够获得适用于STS、SCAR、SSR标记PCR扩增的高质量的DNA,而且该DNA简易提取方法加快了DNA提取速度、降低了污染源和成本,为大批量提取DNA提供了技术支撑。     

基于HRM体系开发抗稻瘟病基因Pi2特异性分子标记

Pi2是一个具有广谱抗性的稻瘟病抗性基因,利用分子标记辅助选择技术选育含有Pi2抗性基因的水稻品种已得到越来越广泛的应用。本研究根据稻瘟病抗性基因Pi2及其复等位基因的序列,利用HRM技术开发与Pi2完全共分离的分子标记,并结合水稻DNA快速提取方法,建立中高通量辅助选择体系。该方法体系准确高效、简便快速、成本低廉,能够大大提高抗病育种选育的效率,为分子标记辅助选择育种或者水稻种质资源Pi2基因型鉴定提供技术选择。     

棉花耐盐性的SSR鉴定研究

土壤盐碱化现已成为危害农业发展和生态环境的全球性问题,培育和鉴选棉花耐盐品种是合理开发利用盐碱地的有效途径。本研究选用25份耐盐(包括耐和抗)和23份盐敏感棉花种质为实验材料,采用SSR技术,展开了棉花耐盐性鉴定技术的有关研究。从DNA快速提取到PCR扩增和产物检测以及多标记组合鉴定等环节进行分析探讨,初步制定了一套适于棉花耐盐性分子鉴定的方法,即多标记组合鉴定法。并用11份材料对该方法进行了验证,结果表明和0.4%盐量胁迫法的鉴定结果的相符率达90.91%。初步研究结果表明多标记组合鉴定法可用于棉花耐盐分子标记辅助鉴定。     

棉花分子标记图谱的构建和一些重要性状的定位

在棉花上,虽然已有若干张不同的分子标记连锁图发表,但在育种中它们仍较难被应用,主要是因为在现有的分子标记连锁图谱中能用于育种的重要农艺性状的DNA分子标记尚不多.为此,本研究选用具有13个质量性状标记的两个陆地棉(Gossypium hirsutum L.)多标记基因系T582和T586作为亲本,用SSR和AFLP的DNA分子标记技术,对F2的作图群体进行DNA分子标记连锁图谱的构建,对棉花重要质量性状基因和数量性状的QTLs进行定位和标记,并建立分子标记连锁群与染色体的对应关系,为棉花分子标记辅助选择育种提供依据.     

Determination of Phytic Acid in Cottonseed Kernels by Ion Chromatography

Cottonseed meal is an important plant protein resource of feed industry and breeding industry, although it contains many antinutritional factors. Phytic acid, as one of the mainly antinutritional factor, cannot be digested easily by monogastric animals, such as fowls and pigs, consequently results in the decreased bio-availability of material elements and leads to the environment pollution by the phytate in excreta. Thus far, study of breeding cottons containing low phytic acid gradually becomes the hotspot, and the method of fully extracting phytic acid from cotton seeds and precise measurement is becoming more and more important and necessary. A new method for the rapid extraction and determination of phytic acid in cottonseed kernels by ion chromatography was developed in this study. After the diethyl ether treatment of dry cottonseed kernel powder, the samples were suspended in 10ml 0.66 mol·L^－1 HCl and boiled for 1 hour. Then the samples were shaken at 4 ℃ for 12 hours and centrifuged. The supernatant was diluted and purified for loading. To analyze the sample, DIONEX ICS-2000 ion chromatography, AG11-HC Guard (4 mm×50 mm) protection column and AG11-HC Guard (4 mm×250 mm) separation column were used in controlled conditions (washing buffer: 30.0 mmol·L^－1 KOH; ream speed: 1.0 mL·min^－1; collecting time: 25 min; 75 mA current; temperature: 30 ℃; loading amount: 20 μL). The results showed that R2 of linear regression is 0.999, which means the regression relation is great. In addition, the standard deviations of all the indexes are within 5% and the average recovery rate is 98.64%-102.31%. The detection limit, showing the minimum detectable concentration of analyte in the sample, is 0.059 μg·mL^－1, and the quantitation limit, showing the minimum amount of analyte in a sample can be quantitatively determined, is 0.196 μg·mL^－1. This method is easy and accurate with great reproducibility, showing a strong application value.     

Effects of Exogenous 24-epibrassinolide on Photosynthetic Physiology of CottonSeedlings under Low Temperature

[Objective] The objective of this study was to investigate the effects of exogenous 24-epibrassinolide (EBR) on the photosynthetic physiology of cotton seedlings under low temperature and to provide basis for improving the cold tolerance of cotton by using EBR as growth regulator. [Method] Taking CCRI 60, Lumianyan 28 and Simian 3 as materials, a field experiment was carried out in Institute of Cotton Research of CAAS(Anyang county, Henan province). Before the first low temperature treatment, the cotton seedlings were sprayed with distilled water (Control) and different concentrations of EBR (0.1 mg·L^－1 and 0.2 mg·L^－1), respectively. After 3 days, the relative electrical conductivity, chlorophyll content, rapid chlorophyll fluorescence induction kinetic curve (OJIP) and fluorescence parameters were measured. [Result] Under low temperature, the relative conductivity of CCRI 60, Lumianyan 28 and Simian 3 spraying with EBR decreased by 17.7%～32.8% compared with control, and there was no significant difference between CCRI 60 and Lumianyan 28 in different concentrations of EBR treatments, butthe relative conductivity of Simian 3 treating with 0.2 mg·L^－1 EBR was significantly lower than those treatments with 0.1 mg·L^－1 EBR . The chl a and chl b contents increased by 9.7%～32.6% and 15.0%～18.9%, respectively. The maximum photochemical efficiency of photosystemⅡ (F_v/F_M) and photosynthetic performance index on absorption basis(PI_ABS) increased significantly. PI_ABS of CCRI 60 increase the maximum by 75.6% using 0.1 mg·L^－1 EBR. Lumianyan 28 and Simian 3 increased the maximum by 101.1% and 265.6% using 0.2 mg·L^－1 EBR, respectively; Absorbed photon flux per cross section (ABS/CSm), electron transport flux (further than QA) per active reactive center (ETo/RC) and probability for electron transport (φEo) are significantly increased. [Conclusion] Exogenous EBR can enhance the ability of low temperature tolerance of cotton seedlings and alleviate the inhibition of photosynthesis in cotton at low temperature. The study showed that 0.1 mg·L^－1 EBR performs well in CCRI 60 and 0.2 mg·L^－1 in Lumianyan 28 and Simian 3.     

Effects of Melatonin on the Antioxidant Enzyme Activities and Seed Germination of Cotton (Gossypium hirsutum L.) under Salt-Stress Conditions

[Objective] Cotton (Gossypium hirsutum L.) seed germination, in which melatonin plays an important regulatory role, is seriously affected by soil salinization. Cotton seed germination, antioxidant enzyme activity levels and other physiological indicators were analyzed to clarify the regulatory effects of exogenous melatonin on cotton seed germination under salt-stress conditions. [Method] The experiment was conducted in a greenhouse of Hebei Agricultural University in Baoding City, Hebei Province from 2018 to 2019 using Guoxin Cotton 9 as the material. The germination rate, germination potential, seed biomass after germination, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, as well as the malondialdehyde (MDA) content, of cotton seeds were measured after exposure to different melatonin concentrations (0, 10, 20, 50 and 100 μmol·L^－1) and 150 mmol·L^－1 NaCl treatments. [Result] After germination, the germination rate, germination potential, radical length and seed biomass significantly decreased, as did the SOD, POD and CAT activities. However, the MDA content significantly increased under salt-stress conditions. Low exogenous melatonin concentrations (10 and 20 μmol·L^－1) increased the germination potential, germination rate and biomass of cotton seeds, and promoted the elongation of radicals; however high melatonin concentrations (50 and 100 μmol·L^－1) inhibited cotton seed germination and decreased seed biomass after germination. Low melatonin concentrations (10 and 20 μmol·L^－1) increased the SOD, POD and CAT activities and decreased the MDA content in cotton seeds. [Conclusion] Low melatonin concentrations could promote germination and improve the salt tolerance of cotton seeds, while high melatonin concentrations could inhibit their germination. A 20 μmol·L^－1 melatonin concentration is appropriate for regulating cotton seed germination under salt-stress conditions.     

Effects of Low Phosphorus Stress on Root Morphology and Physiological Characteristics of Different Cotton Genotypes at the Seedling Stage

[Objective] Phosphorus is one of three major required nutrients for cotton growth and development. A phosphorus deficiency will lead to retarded cotton growth, undeveloped roots, and the abscission of buds and bolls, which results in serious reductions in cotton yield and quality. While applied phosphorus is easily fixed in soils, it is mostly transferred to occluded P, which is not available to plants. Therefore, this study aimed to provide theoretical support for the study of response mechanisms to low phosphorus stress and the efficient phosphorus uptake and utilization of different cotton genotypes. [Method] The effects of phosphorus accumulation and utilization, leaf photosynthetic characteristics and root morphology of two genotypes of cotton (CCRI 79 and SCRC 28) were studied in hydroponic cultures having different phosphorus concentrations. [Result] CCRI 79 had higher phosphorus utilization rates of 90.92 mg·mg^－1 at the low phosphorus level (KH2PO4 1.0×10^－5 mol·L^－1) and 23.09 mg·mg^－1 at the high phosphorus level (KH2PO4 0.5 mol·L－1) compared with SCRC 28. When phosphorus was deficient, the total root length, total root surface area and total root volume of SCRC 28 increased by 13.05%, 18.78% and 10.50%, respectively, while the total root length, total root surface area and total root volume of CCRI 79 decreased significantly. [Conclusion] Here, we found that SCRC 28 had a root morphology and physiological characteristics that allowed it to adapt to low phosphorusstress, while CCRI 79 utilized phosphorus more efficiently.     

不同咸水利用方式对棉花叶绿素荧光参数及土壤盐分的影响

【目的】本研究旨在科学合理地利用浅层咸水资源。【方法】依托短期定位试验开展了在第3年和第4年不同咸水利用方式下(CK:造墒和蕾期灌淡水;T1:造墒和蕾期灌咸淡混配矿化度3 g·L^-1的微咸水;T2:淡水造墒蕾期灌矿化度5 g·L^-1咸水;T3:造墒和蕾期灌矿化度5 g·L^-1咸水;T4:淡水造墒蕾期不灌水)棉花长势、叶绿素荧光参数、土壤盐分累积及其运移的变化。【结果】T1和T2处理的齐苗率、株高、干物质质量、叶面积指数、叶绿素荧光参数、产量及霜前花率与CK无显著差异,土壤盐分含量较CK有所增加,但未对棉花生长产生明显抑制。T3处理的棉花长势指标、叶绿素荧光参数较CK显著降低,0~100 cm土壤盐分含量较CK明显增加。【结论】从土壤质量安全和咸水高效利用的角度考虑,连续4年用3 g·L^-1的咸淡混合水灌溉或用淡水与5 g·L^-1的咸水轮灌不仅能节约淡水,且不影响棉花产量。本研究结果为当地在棉花生产中安全利用咸水提供技术参考。     

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。     

A Regeneration System for Cotton Variety Xinluzao 45 via Somatic Embryogenesis

[Objective] The purpose of this study was to expand upland cotton genotypes suitable for regeneration via somatic embryogenesis, so as to provide excellent receptor materials for the genetic transformation of cotton. [Method] Six different hormone combinations were studied to establish a somatic embryo regeneration system for hypocotyl explants of Xinluzao 45, a widely planted variety in Xinjiang Province. [Result] Faint yellow callus was induced on a medium consisting of hormone combinations C1 (0.1 mg·L^－1 acetic acid dichlorobenzene oxide [2, 4-D] + 0.1 mg·L^－1 kinetin [KT]) or C5 (0.1 mg·L^－1 2, 4-D + 0.5 mg·L^－1 indo-3-butyric acid + 0.1 mg·L^－1 KT). The generated callus was able to successfully induce embryogenic callus on hormone-free medium containing doubled KNO3. The embryogenic callus then developed into somatic embryos and regenerated plants. Maximum rates of callus induction and regeneration were 92.2% and 40.3%, respectively. Although no significant difference was observed in the rate of callus induction of Xinluzao 45 hypocotyls between hormone combinations C1 and C5, the incidence of induced embryonic callus was higher on C1 than on C5. [Conclusion] The somatic embryo regeneration system developed in this study provides technical support that will facilitate Agrobacterium-mediated transformation of upland cotton Xinluzao 45.     

棕色棉茎尖基因枪遗传转化

为了建立高效的棉花茎尖遗传转化体系,以棕色棉品种新彩棉5号为材料,采用茎尖培养和基因枪遗传转化方法,通过对凝固剂种类、活性炭含量、卡那霉素浓度、渗透处理时间、轰击压力、轰击距离等因素的优化,建立了棕色棉新彩棉5号茎尖基因枪遗传转化技术体系。结果表明,不同凝固剂种类、活性炭含量、卡那霉素浓度、渗透处理时间、轰击压力和轰击距离对棕色棉茎尖转化率有明显影响。最佳的遗传转化体系为:前渗处理4 h,轰击压力7.586 MPa(1100 psi),轰击距离9 cm,轰击后恢复培养4 d,然后在MSB+8 g·L~(-1)琼脂+125mg·L~(-1)卡那霉素筛选培养基中筛选25 d。获得抗性芽后,在添加1 g·L~(-1)活性炭的生根培养基中诱导抗性芽生根,并获得抗性再生植株。通过PCR和q PCR检测,5200个茎尖共获得16株阳性转基因植株,转化率为0.31%。该技术体系的建立为棕色棉的遗传转化奠定了基础,为彩色棉育种提供了新的材料。     

陆地棉耐盐相关EST-SSR以及EST-InDel分子标记的开发

随着棉花基因组学、转录组学等重要学科的快速发展,针对候选基因进行序列多样性分析,开发基于候选基因的分子标记,将能够极大推进棉花分子育种研究。本研究利用高通量的测序技术对耐盐陆地棉品种Miscott 7913-83和盐敏感品种苏棉12盐胁迫前后根、叶总RNA的混样进行了转录组测序,对不同盐处理时期Miscott 7913-83和苏12根和叶分别进行表达谱分析。获得3232条盐胁迫下差异表达基因;基于差异表达基因利用生物信息学手段大规模开发了SSR以及In Del等分子标记;随机选择了部分SSR及In Del引物进行验证,进一步证实了分子标记的准确性。本研究为快速开发陆地棉品种间多态性分子标记提供了高效可行的分析方法,通过开发陆地棉耐盐相关功能标记,以期用于分子标记辅助育种改良陆地棉耐盐性。     

Applying Plant Growth Regulators at Early Stages Affects Maturation of Cotton

[Objective] Early initiation and early maturity are the foundation of high yield and good quality of cotton. The purpose of this study is to determine the effects of plant growth regulators applied at the seedling and squaring stage on the early initiation of flower bud and the rate of the opened cotton boll (ROCB) during later development period, and to provide practical measures for hastening the maturity of cotton. [Method] Several plant growth regulators were applied from cotyledonary to squaring stage under greenhouse and field conditions, water was used as the control. The first fruiting branch node (indicating the initiation of flower bud), the number of bud prior to blooming and the ROCB at mid-term of boll maturation period (23 September, 2017) were compared among treatments. [Result] Under greenhouse conditions, gibberellic acid (GA₃) applied at the cotyledonary stage with 140 μmol·L^－1 as well as the three consecutive applications of sodium nitrophenolate (CSN, 2.23 μmol·L^－1) at the cotyledonary, two-leaf and four-leaf stage made the first fruiting branch node move down by about 0.9 nodes. In field experiments, the application of gibberellin₄₊₇(GA₄₊₇, 288 and 576 μmol·L^－1) at the cotyledonary stage significantly decreased the first fruiting branch node by about 0.4 nodes. Also, the application of 6-benzylaminopurine (6-BA, 44.4 μmol·L^－1) at the three-leaf stage significantly decreased the first fruiting branch node by 0.2 nodes. However, there was no significant correlation between the first fruiting branch node and the ROCB in late September. Moreover, the application of Brassinolide (BR, 0.10 μmol·L^－1) at the bud stage increased the ROCB in late September, which was mainly associated with the increased boll set in the lower and middle fruiting branches. [Conclusion] The reasonable distribution of bolls (concentrated in the lower and middle fruiting branches as well as inner fruiting sites) is more important for the earliness of cotton than lowering the first fruiting branch node.