通过GbF3’H基因单独沉默及其与GbCHI和GbDFR基因共沉默研究其在海岛棉中抗枯萎病功能

【目的】通过沉默海岛棉GbF3’H基因及共沉默GbF3’H、GbCHI和Gb DFR基因,研究其在海岛棉抗枯萎病中的作用。【方法】以海岛棉抗病材料06-146为研究对象,GhCLA1基因为阳性对照,空载体为阴性对照,构建海岛棉TRV2-Gb F3’H沉默载体,协同课题组前期构建的TRV2-CHI和TRV2-DFR载体,利用病毒诱导的基因沉默技术(Virus-induced gene silencing,VIGS)分别进行Gb F3’H基因单独沉默以及GbF3’H、GbCHI和Gb DFR这3种基因共沉默试验。通过实时荧光定量聚合酶链式反应(Quantitative real time-polymerase chain reaction,q RT-PCR)分析各处理样品中基因沉默情况;设置室内接种枯萎病菌试验测定病情指数,分析各沉默材料对枯萎病的抗性差异。【结果】q RT-PCR结果显示,海岛棉GbF3’H基因沉默后其在海岛棉根、茎和叶中的表达量比空载体对照低,Gb F3’H、GbCHI和GbDFR这3种基因共沉默后其在海岛棉根、茎和叶中的表达量均比空载体对照低。病情指数调查结果显示,野生型<空载体对照相似文献   

Effect of Pathogen Synergistic Pathogenicity on Severity of Verticillium Wilt of Cotton

Verticillium dahliae is a soil-borne and vascular pathogen causing Verticillium wilt of cotton, which is one of the most severe diseases affecting cotton production in China. Recently, a soil-inhabiting opportunistic pathogen was isolated and identified as Scopulariopsis gossypii; it could infect the host through wounds and increase disease severity in combination with V. dahliae. S. gossypii was able to form conidia in a nutritionally poor substrate and had similar biological characteristics to other vascular pathogens, while ultrastructure observation also confirmed that it was present in vascular tissue of cotton plants and produced conidia. Nematode activity in soil could cause wounds on the surface of cotton roots resulting in infection by the pathogens. On the basis of our analysis of the biological characteristics and infection process of S. gossypii and its interactions with other pathogens in cotton plants, we propose establishing a research program for multipathogen-host interactions in the cotton rhizosphere, which will enable detailed investigation of the synergistic pathogenesis of S. gossypii and V. dahliae.     

P0 protein of cotton leafroll dwarf virus-atypical isolate is a weak RNA silencing suppressor and the avirulence determinant that breaks the cotton Cbd gene-based resistance

Cotton blue disease (CBD) is the most important disease present in cotton crops in South America and cotton leafroll dwarf virus (CLRDV) is the causal agent. The disease has been controlled by sowing cotton varieties resistant to CLRDV. However, in the 2009/10 growing season, an outbreak due to an atypical CLRDV isolate (CLRDV-at) occurred in northwest Argentina. Although CLRDV and CLRDV-at genomes are very closely related, the symptoms they produce in cotton plants are quite different. P0 is the most divergent protein between the isolates and in CLRDV is a silencing suppressor protein. This work characterized the silencing suppressor activity of the P0 protein encoded by CLRDV-at (P0^CL-at) and evaluated its role in Cbd-resistance break in cotton plants. It was demonstrated that P0^CL-at, despite having a mutation in the consensus of the F-box-like motif, was able to suppress local RNA silencing, but displayed lower activity than P0^CL. P0^CL and P0^CL-at showed no differences in the interaction with Gossypium hirsutum SKP1 orthologue (GSK1) and Nicotiana benthamiana SKP1 and both P0 proteins triggered destabilization of ARGONAUTE1. However, when the ability to enhance PVX symptoms was evaluated, P0^CL-at was shown to be a weaker pathogenicity factor than P0^CL in N. benthamiana. Interestingly, trans-expressed P0^CL-at enabled CLRDV to systemically infect CBD-resistant plants, and a chimeric CLRDV-P0^CL-at infectious clone succeeded in establishing infection in CBD-resistant cotton varieties with symptoms resembling those produced by CLRDV-at. These results strongly suggest that P0^CL-at is the avirulence (Avr) determinant involved in breaking cotton Cbd gene-based resistance.     

Agronomic traits at the seedling stage,yield, and fiber quality in two cotton (Gossypium hirsutum L.) cultivars in response to phosphorus deficiency

ABSTRACT

Cotton is critical for phosphorus demands and very sensitive for its deficiency. However, identifying the effect of low-phosphorus tolerance on cotton growth, yield, and fiber quality by reducing phosphorus consumption. This may help to develop phosphorus-tolerant high-yielding cotton cultivars. In a two-year repeated (2015 and 2016) hydroponic experiment (using 0.01 and 1 mM KH₂PO₄), two cotton cultivars with phosphorus sensitivity (Lu 54; a low-phosphorus sensitive and Yuzaomian 9110; a low-phosphorus tolerant) were screened on the base of agronomic traits and physiological indices through correlation analysis, cluster analysis and principal component analysis from 16 cotton cultivars. Low phosphorus nutrition reduced the plant height, leaf number, leaf area, phosphorus accumulation and biomass in various organs of seedlings. The deficiency negatively affected the morphogenesis of seedlings, as well as yield and fiber quality. Further, these screened cultivars were tested in a pot experiment with 0, 50, 100, 150, 200 kg P₂O₅ ha^?1 during 2016 and 2017. It was found to have a significant (P< 0.05) difference in boll number, lint yield, fiber strength, and micronaire at the harvest. Furthermore, after collectively analyzed the characteristics of Lu 54 and Yuzaomian 9110, there were six key indices that could improve the low phosphorus tolerance of cotton cultivars. These were root phosphorus accumulation, stem phosphorus accumulation percentage, leaf and total biomass of seedlings, seed cotton weight per boll and fiber length.     

Isolation and Characterization of GhLEA3 Gene from Upland Cotton and Its Expression in Response to Low Temperature Stress

[Objective] Based on the analysis of cotton GhLEA3 gene structure and its expression model in chilling stress, we investigated the resistance function of GhLEA3 in response to cold stress. [Method] We cloned GhLEA3 from upland cotton by homologous sequence cloning way. We analyzed the properties of the GhLEA3, and constructed phylogenetic tree by bioinformatics analysis. We constructed the transient expression vector 35S∷GhLEA3-GFP by In-Fusion connection technology and studied the subcellular localization of GhLEA3. The expression of GhLEA3 gene in leaves of TM-1 at three-leaf stage under low temperature stress treatments (4 ℃, 24 h) was performed. Agrobacterium tumefaciens mediated floral dipping method was used to transform the gene with expression vector 35S∷GhLEA3-GFP into Arabidopsis thaliana wild type. Low temperature germination experiment at 4 ℃ was conducted to verify germination ability of the transgenic Arabidopsis thaliana T₃ seed. The transgenic Arabidopsis seedlings were treated at 4 ℃(low temperature), and all the leaves were taken to measure their electrical conductivity. [Result] The coding sequence of GhLEA3 gene is 1 218 bp, which encodes 405 amino acids. GhLEA3 protein includes 4 functional domains of PF02987. Phylogenetic analysis showed that the similarity of amino acid sequences between upland cotton GhLEA3 and Arabidopsis thaliana AtLEA3 was 52.6%. GhLEA3 might be mainly localized in vacuoles and small vesicles. GhLEA3 was up-regulated in leaves after low temperature treatment. The germination rate of transgenic Arabidopsis thaliana of T₃ generation at low temperature was significantly higher than that of wild type, and its electrical conductivity of leaves after low temperature treatment was significantly lower than that of wild type. [Conclusion] GhLEA3 belonged to the member of LEA3 family. Phylogenetic analysis showed that GhLEA3 had the closest relationship with AtLEA3. GhLEA3 was induced by low temperature stress and may play an important role in improving cold resistance.     

棉花离体不定根系的发生对色素腺体密度和棉酚含量的影响

为探明根系对色素腺体密度和棉酚含量的影响，以离体条件下的无根幼苗、不同生根状态幼苗以及无菌种子苗作为研究对象，对其茎叶色素腺体密度、棉酚含量以及棉酚生物合成关键基因表达量进行分析。结果表明，不同生根状态幼苗中，色素腺体密度和棉酚含量与一定范围内生根数呈显著正相关，随后趋于平稳。荧光定量PCR显示，棉酚生物合成关键基因GhCDN1和GhWRKY1表达量在生根数多的无菌苗中显著增高，而对棉酚合成具有反馈调节作用的 GhCYP706B1表达量呈现相反趋势；无根幼苗茎叶腺体密度和棉酚含量均显著低于有根幼苗，棉酚生物合成关键基因GhCDN1、GhWRKY1和 GhCYP706B1表达量反而高于有根幼苗。有根幼苗中根的棉酚含量显著高于茎和叶，无菌种子苗根系中棉酚生物合成关键基因表达量显著高于茎和叶。说明棉花根系能对棉酚含量产生直接影响，同时影响腺体密度。本研究进一步验证了根系是棉酚主要合成部位，地上部分也存在少量的棉酚合成，只是不能引起棉酚发生显著变化。     

Electron Transport Through Photosystem II Is Not Limited By A Wide Range of Water Deficit Conditions In Field‐Grown Gossypium hirsutum

Despite exhaustive literature describing drought stress effects on photosynthesis in Gossypium hirsutum, the sensitivity of photosynthetic electron flow to water deficit is heavily debated. To address this, G. hirsutum plants were grown at a field site near Camilla, GA under contrasting irrigation regimes, and pre‐dawn water potential (Ψ_PD), stomatal conductance (g_s), net photosynthesis (P_N), actual quantum yield of photosystem II (Φ_PSII) and electron transport rate (ETR) were measured at multiple times during the 2012 growing season. Ψ_PD values ranged from ?0.3 to ?1.1 MPa. Stomatal conductance exhibited a strong (r² = 0.697), sigmoidal response to Ψ_PD, where g_s was ≤0.1 mol m^?2 s^?1 at Ψ_PD values ≤ ?0.86 MPa. Neither Φ_PSII (r² = 0.015) nor ETR (r² = 0.010) was affected by Ψ_PD, despite exceptionally low Ψ_PD values (?1.1 MPa) causing a 71.7 % decline in P_N relative to values predicted for well‐watered G. hirsutum leaves at Ψ_PD = ?0.3 MPa. Further, P_N was strongly influenced by g_s, whereas ETR and Φ_PSII were not. We conclude that photosynthetic electron flow through photosystem II is insensitive to water deficit in field‐grown G. hirsutum.