基因枪介导的转PYL5基因小麦的获得与鉴定

【目的】通过基因枪介导共转化,获得转拟南芥抗旱基因PYL5的小麦植株,为转基因小麦的抗旱功能研究奠定基础。【方法】以小麦品种西农889、绵阳19和宁春16为供试材料,通过基因枪介导法将诱导型启动子Rd29A启动的PYL5基因和筛选标记基因Bar共转化到小麦幼胚愈伤组织中,经过除草剂PPT(Phosphinothricin)筛选和愈伤组织分化,获得再生植株。根据目标基因PYL5和Bar基因序列分别设计特异引物,对移栽成活的小麦T0代再生植株进行基因特异性PCR检测。【结果】采用基因枪分别轰击西农889的1800个、绵阳19的800个和宁春16的800个幼胚愈伤组织,经过筛选和分化分别获得了9,5和14株小麦再生植株;对转基因小麦T0代再生植株的基因特异性PCR检测结果表明,西农889、绵阳19和宁春16 Bar基因的转化率分别为0.280%,0.500%和0.750%,PYL5基因的转化率分别为0.110%,0.125%和0.500%,Bar和PYL5基因的共转化率分别为0.110%,0.125%和0.500%。【结论】PYL5基因成功转入到了小麦品种西农889、绵阳19和宁春16中。     

小偃6号成株期高温抗条锈性遗传分析

为揭示小偃6号抗病机制和培育持久抗病品种,采用常规杂交分析方法,在小麦抽穗期利用小麦条锈菌小种CYR30、CYR32和Su11-4对小偃6号、铭贤169及其杂交F1、F2、F2∶3接种,平均气温达到21℃时对小偃6号进行了抗条锈性调查和遗传分析。结果显示,接种CYR30、CYR32时,F1代表现高感,F2代群体中抗感分离比例符合1 R∶15 S的理论比例。接种Su11-4时,F1代表现高抗,F2代群体中抗感分离比例符合3R∶1S的理论比例。研究表明小偃6号对CYR30、CYR32的抗病性均由2对隐性基因累加作用控制,对Su11-4的抗病性由1对显性基因控制。     

Combining Phytate/Ca2+ Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca²⁺ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00–4.99 and 6.50–7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.     

Genetic Analysis and Molecular Mapping of an All-Stage Stripe Rust Resistance Gene in Triticum aestivum-Haynaldia villosa Translocation Line V3

Triticum aestivum-Hayaldia villosa translocation line V3 has shown effective all-stage resistance to the seven dominant pathotypes of Puccinia striiforms f.sp.tritici prevalent in China.To elucidate the genetic basis of the resistance,the segregating populations were developed from the cross between V3 and susceptible genotype Mingxian 169,seedlings of the parents and F 2 progeny were tested with six prevalent pathotypes,including CYR29,CYR31,CYR32-6,CYR33,Sun11-4,and Sun11-11,F 1 plants and F 3 lines were also inoculated with Sun11-11 to confirm the result further.The genetic studied results showed that the resistance of V3 against CYR29 was conferred by two dominant genes,independently,one dominant gene and one recessive gene conferring independently or a single dominant gene to confer resistance to CYR31,two complementary dominant genes conferring resistance to both CYR32-6 and Sun11-4,two independently dominant genes or three dominant genes（two of the genes show cumulative effect） conferring resistance to CYR33,a single dominant gene for resistance to Sun11-11.Resistance gene analog polymorphism（RGAP） and simple-sequence repeat（SSR） techniques were used to identify molecular markers linked to the single dominant gene（temporarily designated as YrV3） for resistance to Sun11-11.A linkage map of 2 RGAP and 7 SSR markers was constructed for the dominant gene using data from 221 F 2 plants and their derived F 2：3 lines tested with Sun11-11 in the greenhouse.Amplification of the complete set of nulli-tetrasomic lines of Chinese Spring with a RGAP marker RG1 mapped the gene on the chromosome 1B,and then the linked 7 SSR markers located this gene on the long arm of chromosome 1B.The linkage map spanned a genetic distance of 25.0 cM,the SSR markers Xgwm124 and Xcfa2147 closely linked to YrV3 with genetic distances of 3.0 and 3.8 cM,respectively.Based on the linkage map,it concluded that the resistance gene YrV3 was located on chromosome arm 1BL.Given chromosomal location,the reaction patterns and pedigree analysis,YrV3 should be a novel gene for resistance to stripe rust in wheat.These closely linked markers should be useful in stacking genes from different sources for wheat breeding and diversification of resistance genes against stripe rust.     

关中西部小麦条锈菌贵农22致病类群的监测

正小麦条锈病是由条形柄锈菌小麦专化型(Puccinia striiformis West.f.sp.tritici Eriks.Henn.)引起的气传病害,是我国和全世界最重要的病害之一。一般年份可造成小麦减产10%~30%,流行年份减产50%~60%,甚至绝收。小麦条锈病是大区流行性病害。陕西地处越夏菌源基地和黄淮海冬麦区的地理接合地域,是条锈病流行的重要"桥梁"地带~([1])。早期研究认为甘     

Hyperspectral detection of rice damaged by rice leaf folder (Cnaphalocrocis medinalis)

The reflectance from rice (Oryza sativa L.) leaves and canopy damaged by rice leaf folder (RLF), Cnaphalocrocis medinalis (Guenée) was studied at the booting stage in order to establish a monitoring method for RLF based on hyperspectral data. The results showed that reflectance from rice leaves significantly decreased in the green (530–570 nm) and near infrared (700–1000 nm) regions, and significantly increased in the blue (450–520 nm) and red (580–700 nm) regions as the leaf-roll rate of rice increased. Reflectance from rice canopy significantly decreased in the spectral regions from 737 to 1000 nm as the infestation scale of RLF increased, and the most correlation appeared at 938 nm. Seven spectral regions 503–521, 526–545, 550–568, 581–606, 688–699, 703–715, and 722–770 nm at leaf-level, and one region 747–754 nm at canopy-level were found to be sensitive bands to exhibit the damage severity in rice by RLF. The position of the red edge peak remarkably moved to blue region, and the amplitude and area of the red edge significantly decreased when rice leaves were severely infected by RLF. Thirty-eight spectral indices at leaf-level and 29 indices at canopy-level were found to be sensitive to leaf-roll rate and infestation scale in rice, respectively. The linear regression models were built to detect the leaf-roll rate (0.0–1.0) and infestation scale (0–5) in rice using leaf- and canopy-level reflectance data. The root mean square error of the model was only 0.059 and 0.22 for the leaf-roll rate and infestation scale, respectively. These results suggested that the hyperspectral reflectance was potential to detect RLF damage severity in rice.     

苹果黑腐皮壳菌CAP超家族蛋白基因鉴定及毒性功能分析

【目的】CAP（Cysteine-rich secretory protein,Antigen 5 and Pathogenesis related protein 1）超家族蛋白广泛存在于真菌、细菌、动植物等物种,并且参与病原菌的致病过程。本研究旨在鉴定苹果树腐烂病致病菌——苹果黑腐皮壳菌（Valsa mali）的CAP超家族基因并明确CAP超家族基因在病菌致病方面的作用。【方法】通过BLASTP在苹果黑腐皮壳菌全基因组中检索具有CAP保守结构域的基因;利用特异性引物对鉴定到的CAP基因进行PCR扩增和凝胶电泳检测;使用生物信息学软件和在线数据库进行蛋白序列特征和系统发育分析;利用RT-qPCR分析基因在病菌侵染过程中的表达模式;使用特异性引物扩增CAP基因上、下游片段并从PDL2质粒上扩增筛选标记基因;利用Double-joint PCR构建基因敲除盒并通过PEG介导的原生质转化技术进行基因敲除和基因回补;以遗传霉素（G418）抗性为筛选标记并利用4对引物PCR检测获得基因敲除突变体;以潮霉素（HPH）抗性为筛选标记获得基因回补菌株;通过对菌株进行培养皿内生长试验明确基因对病菌营养生长的影响;通过离体苹果枝条接种试验分析该病菌CAP超家族基因的毒性功能。【结果】在苹果黑腐皮壳菌中鉴定到3个具有CAP保守结构域的基因,分别命名为VmPR1a、VmPR1b和VmPR1c。序列特征分析发现,3个CAP蛋白均包含4个保守区：N端信号肽、N端延伸区（NTE）、CAP保守功能域和C端延伸区（CTE）。系统发育分析显示,3个CAP蛋白聚于不同的进化支,VmPR1a聚于clade2进化支并与粗糙链孢霉（Neurospora crassa）CAP蛋白进化关系较近;VmPR1b聚于clade3且与镰孢菌属（Fusarium spp.）CAP蛋白的进化关系更近;VmPR1c聚于clade1,并且也与粗糙链孢霉的CAP蛋白进化关系较近。RT-qPCR分析结果显示,VmPR1a、VmPR1b和VmPR1c在病菌侵染早期（6 h和12 h）均显著上调表达。利用PEG遗传转化技术获得VmPR1a、VmPR1b和VmPR1c敲除突变体（ΔVmPR1a-7/23、ΔVmPR1b-20/31和ΔVmPR1c-26/40）;营养生长观察发现,所有敲除突变体生长表型与野生型菌株03-8均无明显差异;致病力检测发现,ΔVmPR1b-20/31致病力较野生型无明显变化,而ΔVmPR1a-7/23和ΔVmPR1c-26/40致病力较野生型显著下降。将VmPR1a和VmPR1c分别回补至ΔVmPR1a和ΔVmPR1c,回补菌株（VmPR1a/C和VmPR1c/C）致病力恢复至野生型水平。【结论】苹果黑腐皮壳菌中存在3个CAP超家族基因（VmPR1a、VmPR1b和VmPR1c）,其中VmPR1a和VmPR1c是苹果黑腐皮壳菌重要的毒性因子。     

苹果树腐烂病菌小G蛋白VmRab7基因的功能分析

苹果树腐烂病菌（Valsa mali）引起的真菌病害严重制约着苹果经济产业的发展,深入研究其致病机制,对该病害的防控十分重要。初步鉴定了小G蛋白VmRab7的功能,为解析苹果树腐烂病菌的致病机理和病害防控提供理论依据。采用同源重组的方法对VmRAB7进行基因敲除,获得ΔVmrab7敲除突变体;通过菌落直径的测量,分析VmRab7对菌丝生长速率的影响;通过离体枝条和叶片接种,分析VmRab7对致病力的影响;通过透射电镜观察,分析ΔVmrab7突变体的自噬体及液泡形态。研究发现,VmRAB7基因的缺失使菌丝生长速率降低了约40%,致病力降低了约50%,突变体丧失了产孢能力;ΔVmrab7敲除突变体具有小且多的碎片化液泡,液泡中无球状的自噬体结构。因此,VmRab7调控着苹果树腐烂病菌的营养生长、产孢、致病力、液泡融合和细胞自噬过程。     

碳同位素分辨率与小麦光合生理指标的关系

为了解碳同位素分辨率(Δ)与小麦光合生理指标的关系,以洛旱6号和西农389的154个F4代株系中的24个高Δ株系和24个低Δ株系及亲本为供试材料,分析了陕西杨凌和永寿两种不同雨养环境条件下小麦籽粒Δ与灌浆期叶片光合生理指标的相关性,并对高、低Δ材料间光合生理指标和产量进行了比较分析。结果表明,在降雨较多的杨凌地区,高Δ株系表现出高的籽粒产量;在降雨较少、相对干旱的永寿地区,高、低Δ株系间籽粒产量差异不显著,但低Δ株系表现出低的气孔导度。在杨凌生态条件下,灌浆中期籽粒Δ与叶片蒸腾速率、光合速率、气孔导度和Ci/Ca生理指标的相关性在小麦整个灌浆期最为显著;在永寿生态条件下,灌浆中期籽粒Δ与蒸腾速率和Ci/Ca均相关显著。说环境条件影响小麦籽粒Δ与光合生理指标的关系。     

Investigation of Host Responses of Different Potato Genotypes at Tissue, Cellular and Subcellular Levels After Infection with Phytophthora infestans

In histological and cytological investigations, the infection process of Phytophthora infestans, the late blight pathogen, was comparatively studied in several potato cultivars and somatic hybrid genotypes and their parents using fluorescence microscopy and electron microscopy methods. The results showed that germination of zoospores of P. infestans and frequency of invading by infection hyphae did not differ among the cultivar-pathogen interactions, but, extension of hyphae in host cells markedly differed among these genotypes. In the susceptible genotypes the pathogen grew rapidly inter- and intracellularly, 12 h after inoculation (hai), and some digital like haustoria were formed and the cytoplasm of the host cells became disorganized. In the resistance genotypes, the pathogen was restricted to the site of initial penetration, although some hyphae could penetrate the epidermal cell, however, the host cells produced resistance responses, such as formed wall appositions when in contact with hyphae, and no haustoria like structures were found. In the somatic hybride genotypes, the host response was different according to their parents as shown by transmission electron microscopy. In the hybrid genotype 1508/2, like in the wild species S. bulbocastanum, no hyphae were found in host cells. In the other genotypes, hyphae of P. infestans spread intercellularly and formed haustoria, but the cytoplasm of hyphae and haustoria was disorganized and host cell resistant responses often appeared, such as, host cells were disorganized and necrotic and cell wall apposition were observed.