陆地棉GhDAHP基因的克隆与表达分析

棉花黄萎病是制约棉花产业健康发展的一种重要病害,在生产上未发现有效的根治办法,使用现代基因工程技术,从基因库中挖掘抗黄萎病相关基因,为棉花抗黄萎病育种工作的提供理论基础。本研究根据在GenBank中检索到抗黄萎病基因SDAHP(GU479467,1,3-脱氧-阿拉伯庚酮糖-7-磷酸合成酶),从陆地棉基因组数据库中检索到3个同源基因(NM_016893351.1,XM_016849738.1和XM_0168151150.1),根据3个同源基因核苷酸序列设计引物,以陆地棉cDNA为模板进行克隆,获得3个目的基因,依次命名为GhDAHP-1、GhDAHP-2和GhDAHP-3。利用ProtParam和Prot Scale等在线软件对目的基因进行生物信息学分析;RT-PCR分析黄萎病菌诱导后目的基因在陆地棉叶片中的表达量。结果表明:GhDAHP-1序列全长1983 bp,氨基酸总数为539,分子质量约59.45378 kD,理论等电点pI为8.7;GhDAHP-2序列全长1832 bp,氨基酸总数516,分子质量约57.02998 kD,理论等电点pI为7.68;GhDAHP-3序列全长1652 bp,氨基酸总数517,分子质量约56.95491 kD,理论等电点pI为8.52。系统进化树分析,GhDAHP与雷蒙德氏棉(Gossypium raimindii)、木本棉(Gossypium arboreum)的亲缘关系最近。棉花叶片在黄萎病菌的处理下,GhDAHP基因表达量呈现先升高后降低的趋势,推测受到黄萎病诱导后,GhDAHP基因可能参与了黄萎病菌侵染的防卫过程。     

陆地棉抗黄萎病种质创新与抗病基因挖掘

抗病种质在抗病育种中的地位不可替代。远缘杂交材料创新是陆地棉黄萎病抗性种质创制的重要基础;基因工程提供了创造变异的新技术,但由于抗病机制复杂,导入一、两个主基因的效果还不明显;采用分子生物学技术揭示棉花对黄萎病的抗性机制,发掘棉花抗病基因和病程相关基因,有助于剖析棉花-黄萎病菌互作机制。本文概述了陆地棉抗黄萎病种质创制、抗病品种选育的成就,介绍了棉花黄萎病抗性机理与功能基因发掘等方面的最新进展,为抗病分子设计育种提供理论支持。     

Physiological Mechanism of Different Varieties and Potassium Application Amounts on Cotton Resistance to Verticillium Wilt

[Objective] It has theoretical guiding significance for the comprehensive control technology of Verticillium wilt of cotton to explore the effect of potassium nutrition on mechanism of cotton Verticillium wilt resistance under the condition of pathogen stress of Verticillium dahliae. [Method] Three cotton cultivars Jimian 11 (JM-11 as susceptible cultivar), Zhongzhimian 2 (ZZM-2 as control cultivar), Nongda 601 (ND-601 as resistant cultivar) were adopted, and each cultivar had 0, 75, 150, 225, 300 kg· hm－2 potassium levels represented by K0, K75, K150, K225, K300. Two factors split plot experiment was designed under artificial nursery inoculated Verticillium dahlia, to observe disease index of Verticillium wilt, and researched resistance associated substances content and enzyme activity. [Result] (1) The lignin, total phenol contents and POD, PAL activity in leaves or roots of JM-11 were more greatly influenced by Verticillium wilt compared with ZZM-2, ND-601; the crucial disease resistance substances and enzymes were lignin, total phenol and PAL for JM-11, total phenol for ZZM-2 and ND-601. (2) The lignin, total phenol contents and POD, PAL activities of JM-11 were more sensitive to root potassium than ND-601, but weaker than that of ZZM-2. The sensitivity of POD, PAL activities in functional leaves of JM-11 to potassium were more sensitive than ND-601 and ZZM-2. (3) The best effect of potash fertilizer for relieving disease index for JM-11, ZZM-2, ND-601 were 32.5%, 26.4%, 16.5%, and corresponding potassium application amounts were 290.5 kg·hm－2, 253.5 kg·hm－2 and 229.5 kg·hm－2, respectively. [Conclusion] Application of potassium could increase the resistance to Verticillium wilt and reduce the occurrence of cotton Verticillium wilt.     

棉花黄萎病菌与抗黄萎病遗传育种研究进展

简要综述了棉花黄萎病菌及抗黄萎病遗传育种的研究进展。研究表明 ,各地的棉花黄萎病菌均存在致病力的分化 ,其致病机理是病菌侵入棉花后菌丝及孢子在导管内大量繁殖 ,同时刺激邻近的薄壁细胞产生胶状物质及侵填体而堵塞导管 ,使水分和养分运输发生困难 ,更重要的是病菌在棉株体内产生的糖蛋白毒素作用的结果。棉花抗黄萎病的遗传方式争论较大 ,但一般在温室由单一菌系接种鉴定时棉花黄萎病抗性表现为单基因遗传 ,而在田间病圃或用多菌系混合鉴定时 ,棉花黄萎病抗性表现为多基因遗传。由于陆地棉内缺乏高抗黄萎病资源 ,给棉花抗黄萎病育种带来一定困难 ,但 90年代以来 ,已育成 86 - 6、川 73 7、川 2 80 2、豫棉 1 9号、豫棉 2 1号等一些抗黄萎病的新品种。上述抗黄萎病品种在棉花黄萎病综合防治中起了重要作用     

一种新的棉花黄、枯萎病快速接种方法的研究

 3种不伤根的棉花黄、枯萎病接种方法,即全生育期的田间病圃鉴定法、液体培养棉花苗期孢子悬浮液浸根法和土壤种植棉花苗期孢子悬浮液灌根法进行对比,结果表明,液体培养棉花苗期孢子悬浮液浸根法（接种浓度为10⁷个分生孢子·mL^-1）全周期只需要30 d。通过这种方法,不仅可以鉴定出棉花品种对黄、枯萎病菌的抗、感病性,还可以鉴定不同黄、枯萎病菌菌株的致病力。对比不同浸根接种时间（10 min、20 min、30 min、40 min、50 min、60 min）对结果的影响,发现浸根接种40 min可加快发病,鉴定结果和田间病圃鉴定的比较一致。     

Induction of Defense Enzymes and Control Effect of Endogenous and Rhizosphere Bacteria on Verticillium Wilt of Cotton

[Objective] The colonization in soil, promoting cotton growth effects, inducing resistance of cotton and controlling effects to cotton Verticillium wilt of Bacillus vanillea SMT-24, B. velezensis BHZ-29, B. subtilis SHT-15 and B. atrophaeus SHZ-24 were studied in this paper, which provides a scientific basis for better control of cotton Verticillium wilt. [Method] The antagonistic resistance of these bacteria to Verticillium dahliae was judged by observing the inhibition zone; the number of colonization in the rhizosphere soil was tested to represent the colonization ability of antagonistic bacteria; the growth promoting effect of strains on cotton was determined by analyzing cotton plant height, root length, root hair number and number of leaves; the effect of antagonistic strains on cotton induced resistance was explored by determining catalase (CAT), superoxide dismutase (SOD), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activities in cotton leaves, and investigating the disease index of cotton after inoculation with antagonistic bacteria solutions. [Result] SMT-24, BHZ-29, SHT-15 and SHZ-24 strains significantly inhibited the growth of V. dahliae and formed a clear zone of inhibition. After 2 to 22 days of the third inoculation, these strains of bacteria could survive in the root soil, increase the plant height, leaf number, and root hair number of cotton plants, as well as the activities of cotton CAT, SOD, PAL and POD enzymes, but reduced the disease index. [Conclusion] SMT-24, BHZ-29, SHT-15 and SHZ-24 strains can promote cotton growth, increase the activities of some related defense enzymes, and effectively control cotton Verticillium wilt.     

Proteomics Analysis of Gossypium barbadense Leaves with Resistance to Verticillium dahliae under Infection Stress

[Objective] The purpose of this study is to analyze the resistance mechanism of island cotton under Verticillium wilt stress and to find possible resistance genes. [Method] The cotton resistance mechanism under the stress of Verticillium dahliae was studied by protein two-dimensional electrophoresis and mass spectrometry at the protein level. [Result] In the leaves, eleven proteins were down-regulated and 15 proteins were up-regulated after 2 hours of inoculation with Verticillium dahliae. The down-regulated proteins were mainly related to photosynthesis and carbon assimilation, and then we deduce that Verticillium dahliae is mainly broken cotton photosynthetic system to cause cotton susceptibility. The up-regulated proteins were mainly related to photosynthesis and benzoquinone reductase, beta-D-galactosidase, 14-3-3f protein and other disease-resistant protein. [Conclusion] It is speculated that the defense mechanism of island cotton on Verticillium wilt may occur at two levels: one is passive defense, it is manifested that the isoproteins of the down-regulated proteins such as chloroplast II AB binding protein and ribulose diphosphate carboxylase are highly expressed to maintain the stability of the island cotton photosynthetic system, the histone and 14-3-3f protein are highly expressed to keep the cell stability; the second is active defense, it is manifested that the high expressed β-D-galactosidase and benzoquinone reductase may be involved in the resistance of island cotton to Verticillium wilt.     

棉花对黄萎病的抗病机制研究进展

本文概述了棉花黄萎病抗性的遗传规律和特点，从寄主与病原菌识别的相互作用、组织抗性、生理生化抗性、生态抗性4个方面综述了棉花对黄萎病抗病机制的研究进展。指出真菌诱导子是寄主与病原菌识别和互作的关键因素，不同棉株的固有组织结构抗性和诱导组织结构抗性对黄萎病的抗病表现也不相同；在生理生化抗性上重点介绍了植物抗毒素、酶、糖类物质、激素与棉花抗病性的关系；阐述了棉花对黄萎病的生态抗性，即棉花根系分泌物和根系微生物与黄萎病抗性的相互作用。在这4种抗性中，生理生化抗性起主导作用，但离不开多种抗性机制的相互作用和协调。此外，作者对棉花抗黄萎病分子育种的进展作了概述，简要介绍了RFLP、RAPD、AFLP、SSR等分子标记技术和转基因技术在分子育种中的应用与所取得的进展。最后对棉花黄萎病抗性机制及棉花抗黄萎病分子育种研究的未来发展方向和重点作了展望。     

棉花抗黄萎病鉴定与评价标准的商榷

【目的】通过系统研究黄萎病抗病鉴定的关键技术和评价标准,解决现有棉花品种黄萎病抗性评价标准中存在的问题,建立新的田间棉花黄萎病分级标准和评价标准。【方法】以14个2014年国家主推棉花品种为试验材料,从接种量、可靠样本量、病情分级和评价方法 4个方面系统研究黄萎病抗性鉴定的关键技术和评价标准。【结果】确定了小区接种和行沟接种的最佳接菌量分别为60 g·m-2和10 g·m-1,小区调查最低样本量为50株;制定了新的分级标准为0级(0%)、1级(1%~33%)、2级(34%~66%)、3级(67%~99%)和4级(100%)。连续2年利用相对病程发病面积和发病高峰期的相对病情指数2种评价方法判定品种抗病性,前者作为抗病性评价的依据更为科学可靠,一致率达到100%。【结论】本试验为完善抗病鉴定技术操作规程,规范抗性评价方法,促进棉花抗病品种的选育、审定和推广提供了科学依据。     

Genome-Wide Identification and Expression Analysis of TLP Genes in Gossypium hirsutum L.

[Objective] Thaumatin-like proteins (TLPs) are important pathogenesis-related proteins that function during disease defense-related responses, participating in the defense reactions triggered by several kinds of pathogen infections. A genome-wide analysis of TLP genes could help increase our understanding of their molecular mechanism in response to Verticillium dahliae infections in cotton (Gossypium hirsutum L.). [Method] The TLP family members in the genomes of the G. hirsutum L. were identified and expression analyses of TLP genes in response to Verticillium dahliae inoculation were conducted using bioinformatics and real-time fluorescent quantitative PCR methods, respectively. [Result] In total, 88 TLP genes were identified in G. hirsutum. These TLPs were classified into 10 groups based on their amino acid sequences and a phylogenetic analysis. A gene structure analysis revealed that the number of exons ranges from 1 to 5, and the number of introns ranges from 0 to 4. TLP genes in the same group shared similar structures. Additionally, most TLP proteins contain five motifs that are arranged in the following order: 5, 4, 2, 3 and 1. In total, 87 TLP genes are distributed on 20 of the 26 chromosomes, and 42 and 45 TLP genes are distributed in the A and D subgroups, respectively. The real-time fluorescent quantitative PCR analysis showed that the expression levels of six candidate TLP genes were induced in both tolerant cotton cultivar GZ-1 and susceptible cotton cultivar 86-1, and showed a higher expression in the former. [Conclusion] These results provide a foundation for future studies of the functions and regulatory mechanisms of TLP family genes.     

Creation of a New Resistant Germplasm to Verticillium Wilt by Distant Hybridization in Upland Cotton

To create new germplasm lines resistant to Verticillium wilt in upland cotton, 65 distant hybridization germplasm lines (DHGLs) in upland cotton genetic background were cultivated by interspecific hybridization between Gossypium hirsutum and wild species including G. anomalum, G. armourianum, G. aridum, G. raimondii, G. mustelinum, interspecific F₁ backcrossing with G. hirsutum for four generations, and selfing for four generations, followed by a conventional breeding program. The results of agronomic trait identification during 2011-2012 indicated that average plant heights of DHGLs were closely similar to commercial cultivars of upland cotton, while average fruit branches, fruit nodes, bolls of DHGLs individual plant were lower than those in commercial cultivars of upland cotton. Average single boll weight and lint percentage of DHGLs were lower than commercial cultivars of upland cotton. Fiber length, strength, fineness and maturity of DHGLs were reasonably collocated. Fiber of most lines was suitable for spinning extra high count yarn, but the main fiber quality indices of commercial cultivars of upland cotton were not well coordinated. Identification of resistance to Verticillium wilt in defoliation disease nursery during 2012-2013 indicated that five DHGLs resistant to Verticillium wilt . Suyuan 040, Suyuan 045 and Suyuan 061 were highly resistant to Verticillium wilt with a disease index of 8.33, 4.35 and 7.79, respectively. Suyuan 030 and Suyuan 034 were resistant to Verticillium wilt with a disease index of 12.35 and 13.70, respectively. The genetic relationship of new germplasm lines resistant to Verticillium wilt were traced and showed that Suyuan 040 and Suyuan 045 were DHGLs of G. raimondii, Suyuan 061 was DHGL of G.mustelinum, Suyuan 030 and Suyuan 034 were DHGLs of G. aridum.     

The Effect of Spraying Concentrated Biogas on Growth and Physiological Characteristics of Cotton Seedling

[Objective] The effect of concentrated biogas slurry on growth and physiological characteristics of cotton seedlings was studied in this experiment, and theoretical basis for the rational application of concentrated biogas slurry was provided. [Method] Potted cotton seedlings was sprayed by five different dosages of concentrated biogas slurry, the impact of different spraying quantity (163 mL·hm^－2, 326 mL·hm^－2, 489 mL·hm^－2, 668 mL·hm^－2, 815 mL·hm^－2) after spraying time (after spraying 2nd day, 4th day, 6th day) on cotton seedlings growth and physiological indexes were studied. [Results] The results showed that the suitable quantity of spraying of concentrated biogas slurry had promoting effect to the cotton growth and development, the excess concentrated biogas slurry could inhibit the growth of cotton seedlings. The plant height, dry weight per plant and leaf area of spraying 489 mL·hm^－2 processing are the highest, the average of two batches on the 6th day after spraying are 21.15%, 35.95%, 18.84% higher than CK, respectively. Spraying concentrated biogas slurry can improve cotton seedlings chlorophyll content, soluble sugar contents, soluble protein content. Spraying 489 mL·hm^－2 processing is the highest in both batches, the average of two batches is 18.84%, 24.03%, 3.16% higher than CK, respectively. The result of concentrated biogas slurry onseedlings leaves took effect quickly, especially in 0～4th days. [Conclusion] It is concluded that the suitable amount of concentrated biogas slurry has a positive effect on the growth and physiological characteristics of cotton seedlings, which is beneficial to the formation of strong seedlings of cotton and provides the basis for the later growth of cotton.     

半野生棉抗黄萎病筛选及根系分泌物对黄萎病菌的影响

 对陆地棉7个野生种系的220份材料进行黄萎病菌的抗性筛选,用8份不同抗性材料进行了根系分泌物分析。从220份材料中筛选出18份抗黄萎病材料。根系分泌物研究表明,抗病品种根系分泌物对黄萎病菌菌丝生长及孢子萌发均具有抑制作用,而感病品种根系分泌物对其则有促进作用。另外通过对根系分泌物中氨基酸和可溶性糖的分析发现,抗病品种根系分泌物中氨基酸种类少于感病品种,并且抗病品种根系分泌物中氨基酸总量和可溶性糖含量显著少于感病品种。同时发现,精氨酸为抗病品种特有氨基酸。     

棉花黄萎病研究进展

由大丽轮枝菌引起的棉花黄萎病已成为当前我国棉花可持续生产的主要障碍之一。该病害为土传种传维管束病害,具有病原菌寄主范围广,防治困难的特点。本文综述了近年来国内外有关其病原菌的致病力分化、全基因组测序与致病机制、微菌核形成与萌发机制,寄主的抗病分子机制以及病害防治措施等方面的最新研究进展。     

棉花黄萎病抗性遗传和分子生物学研究进展

介绍了棉花黄萎病菌的致病机制 ,棉花黄萎病抗病机制及其遗传以及棉花黄萎病的鉴定方法 ,并对分子生物学在棉花黄萎病研究中的应用状况进展进行了综述。     

棉花黄萎病相关基因GhAAT的克隆与功能鉴定

棉花黄萎病(Verticillium dahliae)是一种土传性真菌维管束病害,严重影响棉花的生产。本研究通过转录组数据分析筛选出一个与棉花黄萎病相关的氨基酸转运蛋白GhAAT基因,该基因在棉花根部响应黄萎病菌诱导表达。利用病毒诱导的基因沉默(virus-induced gene silencing, VIGS)技术研究其在棉花抗黄萎病中的功能。结果显示将该基因在棉花TM-1的叶片和根部沉默后,棉花对黄萎病抗性减弱,证明GhAAT基因参与调控了棉花抗黄萎病功能。发掘棉花抗黄萎病菌重要调控基因并揭示其分子机制对培育棉花抗病品种具有重要意义。     

基于人工病圃筛选和分子标记辅助的棉花抗黄萎病育种方法研究与应用

以抗病性较强的中植372为研究对象,和陆地棉感病品种军棉1号配制组合构建F2作图群体,筛选到和黄萎病抗性紧密连锁的SSR标记NAU1269。选育过程中,以中植372为父本或者母本,通过人工黄萎病病圃对种间杂交、回交、加代选育以及再杂交的材料进行了抗病性筛选,同时每代育种材料均对黄萎病抗性紧密连锁的SSR标记NAU1269进行跟踪检测,筛选出与黄萎病抗病性状紧密连锁的亲本及其后代材料,进而培育出抗黄萎病、产量高、品质优良的中植棉2号、新植5号、中植棉6号、中植棉8号等10余个国审及省审抗(耐)黄萎病棉花新品种。对育成的品种进行检测发现,中植2棉号、中植6棉号、中植8棉号和新植5号均能够检测到与黄萎病抗性紧密连锁的SSR标记NAU1269和已报道的抗黄萎病的分子标记NAU828和NAU1225,而且这3个标记在各个品种材料之间呈共显性分离。结果表明,基于人工病圃筛选和分子标记辅助育种相结合是选育棉花抗黄萎病材料可行、高效的育种方法。     

Identification of QTLs Related to Verticillium Wilt Resistance Based on RIL Population

[Objective] The objective of this study was to identify the stable quantitative trait loci (QTLs) related to Verticillium wilt resistance in cotton. [Method] In this study, a population of 111 recombinant inbred lines (RILs) strains was developed by crossing a highly resistant parental line "Changkangmian" to Verticillium wilt and the susceptible parent TM-1. The complete composite interval mapping method was adopted to detect QTLs by Verticillium wilt disease index in multiple environmental conditions and periods in Anyang and Verticillium wilt affected areas of Xinjiang. Simple sequence repeat (SSR) markers of polymorphism were screened for genetic mapping. [Result] The genetic map was constructed by 40 simple sequence repeat (SSR) markers, consisted of 12 linkage groups with total length of 212.5 centimorgan (cM). A total of six QTLs related to the resistance to Verticillium wilt were obtained. The likelihood of odd (LOD) values ranged from 2.51 to 5.55. The maximum phenotypic variation explained (PVE) 20.34%, and the minimum PVE 6.93% were recorded. Among detected QTLs, qVR-D05-1 was detected in both Verticillium wilt affected fields in Anyang and Xinjiang in July 2015 and July 2016 with PVE of 12.96% and 20.34%, respectively. [Conclusion] This study can provide a potential reference for mapping stable QTLs related to resistance to Verticillium wilt.