棉花黄萎病菌VdSge1基因的敲除与功能分析

为了明确棉花黄萎病菌大丽轮枝菌(Verticillium dahliae Kleb.)的VdSge1基因功能,本研究利用基因同源重组原理,通过构建敲除载体,对棉花黄萎病菌落叶型强致病力菌株V592的VdSge1基因进行了敲除,获得了4个目标基因敲除的突变体;以野生型菌株V592为对照,通过对敲除突变体生物学性状和致病性测定,结果表明,突变体的菌落生长速度、产孢量明显高于野生型,并且丧失对棉花的致病性。利用q RT-PCR对其他基因在突变体中的表达情况进行分析,结果表明,VdSge1基因敲除后,VMK1、VGB和VdGARP1的表达量随之下降,而VDH1和PevD1的表达却明显上升。由此说明,VdSge1基因与大丽轮枝菌的菌落生长速度、产孢量及致病性密切相关,并且可以影响其他一些基因的表达。     

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.     

棉花黄萎病菌分子生物学研究新进展

本文以分子生物学研究手段为纲 ,从RFLP、RAPD、ITS等方面 ,综述了国内外在棉花黄萎病菌种内划分及致病力分化上的研究状况及最新进展     

棉花黄萎病菌(Verticillium dahliae K1eb.)的寄主种类鉴定

1982—1985年对棉花黄萎病菌 Verticillium dahliae 在8科31种栽培植物及31科87种杂草中的寄主范围进行了鉴定试验。查明在31种栽培植物中5种高度感病,8种表现中度或轻微症状,此13种为寄主。水稻未表现症状但分离到病原菌,回接棉花具有致病力。31种中非寄主12种,还有5种须重复鉴定。在87种杂草中,肯定的寄主48种,可疑寄主6种,无症状带菌体5种,非寄主23种,其余5种未定。本项研究首次查明禾本科植物水稻及野燕麦为Verticillium dahliae 的无症状带菌体。     

湿度对蜡蚧轮枝菌KM9803菌株芽生孢子及分生孢子萌发的影响

用小容器空气湿度调节法研究了湿度对蜡蚧轮枝菌KM9803菌株芽生孢子及分生孢子萌发的影响,试验结果表明:在高湿度(RH95%~100%)和相同的营养、时间等条件下该菌株芽生孢子的萌发不如分生孢子,最高萌发率分别为83.5%和93.8%。在湿度66%时二者基本都不萌发。芽生孢子萌发所需湿度在75%以上,分生孢子则在RH85%左右时开始大量萌发。     

大丽轮枝菌毒素诱导表达陆地棉cDNA序列的克隆与定位

 为获得陆地棉黄萎病抗性相关候选基因，利用棉花黄萎病菌大丽轮枝菌V991毒素粗提物诱导耐黄萎病陆地棉品种'中棉12'应激基因的表达。在诱导幼根24 h时间点提取总RNA，PCR法合成双链cDNA，应用抑制消减杂交技术[1]，得到了差异表达的180个克隆。经反Northern blot筛选出15个受毒素粗提物诱导应激表达的阳性cDNA克隆，将其命名为Vdrg（response gene induced by toxin of Verticillium dahliae）。本研究对所得阳性cDNA克隆进行了序列分析，并利用Pima S6 与 Acala Maxxa 海陆杂交四倍体棉花F2群体对Vdrg序列进行了棉花基因组定位。     

茄子砧木与栽培种根的解剖结构比较

利用光学显微技术，对茄子属2种砧木（托鲁巴姆、赤茄）和2个栽培品种（六叶青、七叶）的根部进行了比较解剖学研究，探讨了茄子砧木抗黄萎病的植物体结构机制，结果表明：导管的管孔类型、管孔大小、管壁厚度、导管周围纤维细胞的数量及排列方式、植物体含量细胞的数量等与抗病性密切相关。     

茄子黄萎病菌毒素对茄子叶片及愈伤组织细胞膜透性的影响

本文研究了不同抗病性品种的茄子幼苗在茄子黄萎病菌毒素处理后，其叶片及愈伤组织细胞膜透性的变化与抗病性的关系。结果表明：毒素处理后，茄子叶片及愈伤组织的细胞膜透性变化趋势基本一致，即随处理时间延长和毒素浓度增加，细胞膜透性增大，且感病品种蒙茄4号电导率值高于抗病品种粘毛茄。品种抗病性与细胞膜透性变化呈负相关。     

转几丁质酶和 β-1,3-葡聚糖酶基因提高棉花对枯萎病和黄萎病的抗性

 枯、黄萎病是世界棉花生产中的两大重要病害。传统育种缺乏抗源，几丁质酶和 -1,3-葡聚糖酶是植物防御体系中的两种防卫因子，两者之间存在协同增效作用。据此构建了4个单价和2个双价基因(分别定位于细胞内或细胞外)的植物表达载体，通过花粉管通道法转化棉花，经PCR和Southern杂交检测以及1996 2000年温室及病圃多代筛选鉴定，已培育出对枯、黄萎病抗性提高的转基因棉花株系。将抗病基因导入国产抗虫棉品种GK19中，还获得了兼抗病、虫的转基因优系。     

棉花黄萎病菌致病的分子机理

阐述了大丽轮枝菌(Verticillium dahliae Kleb)产生的细胞壁降解酶、蛋白酶和毒素在致病中的作用及微菌核形成机制,全面分析了基因组学途径、RNA干涉和农杆菌介导的转化等真菌功能基因组学技术在大丽轮枝菌致病分子机理研究上的进展.