The Study on the Transformation and Selection of Insect-resistant Cotton Harboring Double-gene

采用花粉管通道法，将人工合成的ＧＦＭＣｒｙＩＡ和修饰的ＣｐＴＩ杀虫基因导入棉花优良品种（系）中棉所２３、中棉所２７、中９４０９和中９０系。经田间选择和室内生物鉴定，已获得了转基因抗虫植株。依据生物鉴定结果，对所获得的抗虫材料进行了ＰＣＲ分析，结果证明Ｂｔ和ＣｐＴＩ基因均整合于相应的抗虫材料染色体基因组中，通过自交选育，双价抗虫基因能够传递至ＺＧＫ９７０８抗虫材料Ｒ３代。抗虫鉴定表明，双价抗虫基因能够遗传，后代抗虫性稳定；在获得的Ｒ１代植株中，不同转化体对棉铃虫的抗性存在着差异。     

转Cry1A基因海岛棉的获得和鉴定

进行农杆菌介导转抗虫基因试验，以期选育抗虫性优良的海岛棉新品系(品种)；本实验在海岛棉体细胞胚再生体系的基础上，将携带Cry1A基因的表达载体pBI121通过农杆菌介导法转化到‘新海33号’的胚性愈伤组织中。经培养基筛选、PCR检测、RT-PCR检测、抗虫性鉴定等筛选抗虫后代。经培养基筛选共获得12棵抗性植株。PCR检测结果显示，这12棵抗性植株均能扩增出了大小相符的片段，检测结果呈阳性；RT-PCR结果显示，12棵转基因植株目的基因已经整合入基因组，且能反转录出mRNA；Bt-Cry1Ab/Ac转基因试纸条检测结果表明这12棵转基因植株含有目的蛋白；抗虫性鉴定结果证实了这些转基因植株对棉铃虫幼虫具有一定的抗性。本研究成功将抗虫基因导入至海岛棉的体内，获得了具有抗虫性的植株，其结果对转基因抗虫海岛棉新品种的培育具有一定的参考价值。     

DPC与DTA-6复配对转基因抗虫棉根系功能的调控

以双价转基因抗虫棉sGK321为供试品种,在田间种植条件下研究了DPC与DTA-6复配对抗虫棉根系功能的调控作用。结果表明,与单独应用DPC相比,DPC与DTA-6复配可以更有效地提高转基因抗虫棉的根系活力、增强根系合成氨基酸和细胞分裂素的能力、增强根系吸收和运输NO3-的能力。DPC及其与DTA-6的复配剂均降低了根系对Pi和K的吸收及运输,这可能与抗虫棉的Pi,K体内利用效率提高有关。经韧皮部运向根系的蔗糖增加是DPC与DTA-6复配后改善转基因抗虫棉根系功能的物质基础。     

施钾对不同转基因棉花品种光合特性及产量和品质的影响

 在田间试验条件下,研究了施钾对转Bt+CpTI基因抗虫棉中棉所41、转Bt基因抗虫棉国抗1号和常规棉泗棉3号光合特性和产量及品质的影响。结果表明,施用钾肥能不同程度地提高不同转基因棉花品种的叶绿素含量、叶面积指数(LAI)、叶片净光合速率(P_n)、气孔导度(G_s)、叶绿素荧光动力学参数PSⅡ、最大光化学量子效率(F_v/F_m)、PSⅡ潜在光化学活性(F_v/F_o),从而提高棉花叶片的光合性能。施钾还提高了转基因棉花干物质量及分配到生殖器官的比例,增加了棉花总成铃数、铃重和衣分;施钾处理的中棉所41、国抗1号和泗棉3号的皮棉产量较未施钾处理分别增加5.8%,11.8%和7.0%,棉纤维的主要品质指标也得到改善。转Bt基因抗虫棉国抗1号比转Bt+CpTI基因抗虫棉中棉所41和泗棉3号对钾肥更为敏感。     

Screening techniques and sources of resistance to foliar diseases caused by major necrotrophic fungi in grain legumes

Summary Necrotrophic pathogens of the cool season food legumes (pea, lentil, chickpea, faba bean and lupin) cause wide spread disease and severe crop losses throughout the world. Environmental conditions play an important role in the development and spread of these diseases. Form of inoculum, inoculum concentration and physiological plant growth stage all affect the degree of infection and the amount of crop loss. Measures to control these diseases have relied on identification of resistant germplasm and development of resistant varieties through screening in the field and in controlled environments. Procedures for screening and scoring germplasm and breeding lines for resistance have lacked uniformity among the various programs worldwide. However, this review highlights the most consistent screening and scoring procedures that are simple to use and provide reliable results. Sources of resistance to the major necrotrophic fungi are summarized for each of the cool season food legumes. Marker-assisted selection is underway for Ascochyta blight of pea, lentil and chickpea, and Phomopsis blight of lupin. Other measures such as fungicidal control and cultural control are also reviewed. The emerging genomic information on the model legume, Medicago truncatula, which has various degrees of genetic synteny with the cool season food legumes, has promise for identification of closely linked markers for resistance genes and possibly for eventual map-based cloning of resistance genes. Durable resistance to the necrotrophic pathogens is a common goal of cool season food legume breeders.     

棉花抗红铃虫性对产量性状的影响研究

以不同抗性类型的陆地棉抗虫品种(系)和常规棉品种(系)作亲本配制杂交组合,将亲本和F1代杂交种共20个材料在网室、田间进行抗红铃虫鉴定和棉花抗红铃虫性对棉花产量的影响研究,试验表明,具有外源抗虫基因的抗虫棉皮棉产量高,稳产性好,尤其是抗虫杂交棉具有明显的杂种优势。相关分析结果,棉花种子虫害率与皮棉产量、子棉产量、单株成铃数和霜前花率呈极显著负相关;与衣分和僵瓣黄花率成极显著正相关;与单铃重相关性较低。结果表明,利用外源抗虫基因转导的棉花新材料作为杂交亲本,可以培育出丰产高抗棉花害虫的棉花新品种,尤其适合培育抗红铃虫的杂交棉品种。     

玉米抗虫研究进展

玉米作为一种粮、饲和工业兼用的重要作物,随着生物能源、畜牧业以及生物制药等行业对玉米的需求不断增长,我国玉米需求量日益增长。伴随城镇化步伐的加快,农村劳动力不断减少,田间管理粗放,常年连作,玉米虫害呈现上升趋势,加剧了农药的使用量和降低玉米的产量和品质。为了缓解我国玉米虫害和降低农药的使用,本文归纳了玉米虫害的主要种类、危害,防治办法和优缺点;综述了国内外玉米抗虫鉴定筛选,抗虫基因研究,以及转基因抗虫玉米的研究进展,为我国抗虫玉米遗传育种奠定理论基础。     

棉花品种的抗虫性和抗虫鉴定技术研究

棉花品种抗虫鉴定是棉花品种区域试验中的重要组成部分。棉花品种区域试验中的抗虫鉴定主要对象是红铃虫和棉铃虫。抗虫鉴定方法为网室接虫鉴定,红铃虫的接虫密度是平均每10m2网室接5对成虫;棉铃虫的接虫密度为每10m2网室接虫2对;所接虫源雌雄各占50%。抗红铃虫鉴定指标为种子虫害率;抗棉铃虫鉴定指标为棉株生长点和蕾、花、棉铃受害率。根据棉花受害率将品种的抗虫性分为免疫(I),高抗(HR),抗虫(R),中抗(MR),感虫(S)和高感(HS)共6级。     

利用叶蝉评价转基因玉米对非靶标生物的风险

转基因抗虫作物应用是替代杀虫剂的重要途径。但转基因作物可能存负面效应,特别是对非靶标生物的影响及害虫产生抗性。采用直接和陷阱调查结合方法,连续多年对转Cry1Ac 抗虫基因玉米实验田叶蝉和主要节肢动物数量动态跟踪调查,结果发现叶蝉是玉米田优势非靶标生物种群之一,中期发生比例占刺吸式害虫的28.9%~43.1%,且种群动态与主要非靶标节肢动物群落动态基本一致。又鉴于叶蝉直接刺吸接触到Bt 杀虫蛋白,可直接反映转基因作物对非靶标生物的安全性,因此可利用叶蝉作为转基因抗虫玉米安全性评价指示生物来评价转基因玉米对非靶标生物群落风险,田间测试结果证明此方法是可行的。     

高衣分转Bt+GNA双价基因抗虫棉新种质的选育与利用

本研究对转觑和GNA双价基因后代植株,经南繁加代、抗虫检测、自交、单株和株系选择、纤维品质测定和产量比较试验,选育出双价转基因抗虫棉种质系BGsm16。该种质系高抗棉铃虫等鳞翅目害虫,对蚜虫有一定的抑制作用,抗虫性稳定且不随世代的增加而减弱,衣分高（48．0％左右）,配合力好,结铃性强,通风透光,皮棉产量比受体材料苏棉16号增产11．1％,品质两者相当。与其配置的杂交棉组合衣分高,丰产性好,品质高于对照南农8号,有着很强的杂种优势。转＆和GNA双价基因抗虫棉种质系BGsm16的成功选育,加强转基因棉花的抗虫能力,拓宽了抗虫谱,并有助于延缓害虫对抗虫棉产生抗（耐）性;同时改变了传统对常规抗虫棉衣分低的认识,丰富了抗虫棉种质资源,对选育高衣分杂交抗虫棉有良好的应用前景。     

转基因抗虫棉研究的现状、问题与对策

本文在介绍转基因抗虫棉研究现状的基础上,评述了转基因抗虫棉的使用价值,指出了转基因抗虫棉抗性的持久性,时空性,安全性等问题,从培育转多基因抗虫棉,培育多种类型的抗虫品种轮换种植或混系种植,选用特异启动子或理经因素调控外源基因高效表达,筛选广谱性抗虫基因培育广谱性抗虫棉花和采用农业技术措施改善棉田生态环境延长转基因抗虫棉的使用寿命等方面,提出了提高转基因抗虫棉抗虫性和持久性的几种策略,从而为转基因抗     

Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes

Summary Biotic and abiotic stresses cause significant yield losses in legumes and can significantly affect their productivity. Biotechnology tools such as marker-assisted breeding, tissue culture, in vitro mutagenesis and genetic transformation can contribute to solve or reduce some of these constraints. However, only limited success has been achieved so far. The emergence of “omic” technologies and the establishment of model legume plants such as Medicago truncatula and Lotus japonicus are promising strategies for understanding the molecular genetic basis of stress resistance, which is an important bottleneck for molecular breeding. Understanding the mechanisms that regulate the expression of stress-related genes is a fundamental issue in plant biology and will be necessary for the genetic improvement of legumes. In this review, we describe the current status of biotechnology approaches in relation to biotic and abiotic stresses in legumes and how these useful tools could be used to improve resistance to important constraints affecting legume crops.     

土壤缺钾对棉花钾运转和分配的影响

在盆栽条件下应用86Rb 标记和原子吸收分光光度法,研究了常规棉中棉所12、中棉所35和转基因抗虫棉新棉99B、中棉所41四个品种的钾运转和分配情况.盛花期中棉所35上部叶的86Rb 主要运转到主茎,中部叶的86Rb 主要运转到产量器官.缺钾促进了棉花下部主茎叶和果枝叶中的钾向上部和中部转移,有利于提高钾在植株体内的再利用;而抗虫棉新棉99B和中棉所41下部主茎叶和果枝叶中钾的转运能力较差,可能是它们在生产上后期易早衰的原因之一.产量器官是棉花钾营养的第一大和最终库器官,而主茎则担负着储存和中转的功能.土壤缺钾时,棉花主茎叶和产量器官的钾运转和分配比例提高;另外,种子和纤维的钾浓度随供钾水平的变化在棉株各器官中最小.这些结果说明,钾缺乏时棉株一方面会尽可能维持叶片的功能,从而满足棉株对同化产物的需求;另一方面会利用有限的钾资源优先保证后代的繁衍.     

转cry1C基因抗虫超级粳稻田间目的基因表达及抗螟虫性

为了研究转基因抗虫超级粳稻田间目的基因表达及抗螟虫性,将10个来自于不同独立抗性愈伤组织的转cry1C*基因抗虫超级粳稻品系种植于田间,利用实时荧光定量PCR方法检测孕穗期叶片、茎鞘和幼穗等器官目的基因mRNA,利用酶联免疫吸附(ELISA)法检测孕穗期叶片、茎鞘和幼穗及收获后糙米的Cry1C蛋白,利用田间目测调查二化螟危害的白穗率。结果及分析显示,转基因超级粳稻不同品系及不同器官cry1C*基因田间表达量明显不同,cry1C*基因mRNA表达量叶片＞茎鞘＞幼穗,蛋白质表达量叶片＞茎鞘＞幼穗＞糙米。转基因超级粳稻田间目的基因表达,在mRNA水平和蛋白质水平,不同器官间存在正相关关系,各器官Cry1C蛋白质含量和糙米Bt蛋白质含量呈正相关。在本研究范围内,不论转基因粳稻植株Cry1C蛋白质含量高或低的品系,田间均表现为高抗二化螟。培育转基因抗虫粳稻品种时,注意对目的基因适量表达的抗虫基因型的选择。     

甘蔗新品种苗期抗螟虫性评价

为明确新甘蔗品种对甘蔗螟虫的抗性水平,以期为抗虫品种的推广应用和抗性基因发掘奠定基础,本文采用螟虫自然种群接虫方法,连续2年对10个甘蔗新品种进行了苗期螟害分析与抗螟虫性评价。结果表明,不同甘蔗新品种对螟虫抗性存在显著差异,桂糖29号、桂糖31号等较抗虫,福农39号、德蔗03-83和云蔗05-49等较感虫。部分甘蔗品种抗性会随螟虫选择压力不同而变化,云蔗03-194、云蔗05-51、云蔗06-407等在螟虫虫口密度高时表现为感虫,而在虫口密度低时表现为抗虫。     

Screening techniques and sources of resistance to abiotic stresses in cool-season food legumes

Summary The adaptability and productivity of cool-season food legumes (chickpea, faba bean, lentil, pea) are limited by major abiotic stresses including drought, heat, frost, chilling, waterlogging, salinity and mineral toxicities. The severity of these stresses is unpredictable in field experiments, so field trials are increasingly supplemented with controlled-environment testing and physiological screening. For drought testing, irrigation is used in dry fields and rain-out shelters in damp ones. Carbon isotope discrimination (Δ¹³C) is a well-established screen for drought tolerance in C3 cereal crops which is now being validated for use in grain legumes, but it is relatively expensive per sample and more economical methods include stomatal conductance and canopy temperature. Chickpea lines ICC4958 and FLIP87-59C and faba bean line ILB938 have demonstrated good drought tolerance parameters in different experiments. For frost tolerance, an efficient controlled-environment procedure involves exposing hardened pot-grown plants to sub-zero temperatures. Faba beans Cote d’Or and BPL4628 as well as lentil ILL5865 have demonstrated good freezing tolerance in such tests. Chilling-tolerance tests are more commonly conducted in the field and lentil line ILL1878 as well as derivatives of interspecific crosses between chickpea and its wild relatives have repeatedly shown good results. The timing of chilling is particularly important as temperatures which are not lethal to the plant can greatly disrupt fertilization of flowers. Salinity response can be determined using hydroponic methods with a sand or gravel substrate and rapid, efficient scoring is based on leaf symptoms. Many lines of chickpea, faba bean and lentil have shown good salinity tolerance in a single article but none has become a benchmark. Waterlogging tolerance can be evaluated using paired hydroponic systems, one oxygenated and the other de-oxygenated. The development of lysigenous cavities or aerenchyma in roots, common in warm-season legumes, is reported in pea and lentil but is not well established in chickpea or faba bean. Many stresses are associated with oxidative damage leading to changes in chlorophyll fluorescence, membrane stability and peroxidase levels. An additional factor relevant to the legumes is the response of the symbiotic nitrogen-fixing bacteria to the stress.     

Screening techniques and sources of resistance to nematodes in cool season food legumes

Identification of sources of resistance in cool season legumes to cyst (Heterodera spp.), root-knot (Meloidogyne spp.), and stem nematode (Ditylenchus dipsaci) is generally based on number of cysts on roots, root-knot nematode induced gall index, and stem nematode reproduction in shoot tissue, respectively. Various levels of resistance to cyst nematodes have been detected in chickpea and pea. Resistance has also been identified in chickpea, faba bean, and pea to the root-knot nematodes. Broad based durable sources of resistance to plant parasitic nematodes are required. Basic research is needed to develop transgenic plants with resistance based on hatch stimulants, inhibitors, toxins, or repellents found in antagonistic rhizosphere microorganisms. Selection of genotypes that favor development of beneficial rhizosphere microorganisms or root endophytes that increase the plant resistance to nematode infection deserves attention.     

Drought Stress in Grain Legumes during Reproduction and Grain Filling

Water scarcity is a major constraint limiting grain legume production particularly in the arid and semi‐arid tropics. Different climate models have predicted changes in rainfall distribution and frequent drought spells for the future. Although drought impedes the productivity of grain legumes at all growth stages, its occurrence during reproductive and grain development stages (terminal drought) is more critical and usually results in significant loss in grain yield. However, the extent of yield loss depends on the duration and intensity of the stress. A reduction in the rate of net photosynthesis, and poor grain set and grain development are the principal reasons for terminal drought‐induced loss in grain yield. Insight into the impact and resistance mechanism of terminal drought is required for effective crop improvement programmes aiming to improve resistance to terminal drought in grain legumes. In this article, the impact of terminal drought on leaf development and senescence, light harvesting and carbon fixation, and grain development and grain composition is discussed. The mechanisms of resistance, management options, and innovative breeding and functional genomics strategies to improve resistance to terminal drought in grain legumes are also discussed.     

DTA-6对两种食用豆生理代谢及产量的影响

为探究2-N,N-二乙氨基乙基己酸酯（diethyl aminoethyl hexanoate,DTA-6）对食用豆叶片的生理代谢及产量的调控效应,选用芸豆（英国红）和小豆（龙垦2号）为试验材料,采用大田完全随机试验方法,于芸豆和小豆的初花期叶面喷施DTA-6,以喷施清水为对照（CK）,测定各生育时期叶片光合参数、碳代谢产物、干物质、产量及产量构成因素。结果表明：DTA-6提高了2种食用豆各生育期的SPAD值、盛花期和鼓粒期的净光合速率（P_n）、蒸腾速率（T_r）、气孔导度（G_s）和胞间CO₂浓度（C_i）,显著增加了叶片蔗糖和可溶性糖的含量,促进了叶片淀粉的积累;与CK相比,DTA-6提高了2种食用豆的地上部单株干物质积累量,并且显著提高了芸豆和小豆鼓粒期荚分配率;DTA-6可有效调控2种食用豆的单株荚数、单株粒数和百粒重,从而提高产量,DTA-6处理的芸豆和小豆产量分别较CK增加13.30%和12.91%,增产显著。     

转双价基因棉花对根际土壤酶活性和养分含量的影响

在田间试验条件下,以3种转双价基因棉和常规棉石远321为研究对象,比较分析转双价基因棉和常规棉石远321根际土壤酶活性及养分的变化。结果表明,转双价Cry1Ac+CpTI基因棉sGK321与石远321根际土壤速效磷和铵态氮含量无显著差异,而硝态氮含量则显著高于石远321;转双价Cry1Ac+Cry2Ab基因棉(双Bt抗虫棉)速效磷和铵态氮含量均显著低于石远321,而硝态氮含量与石远321无显著差异;转双价Cry1Ac+Epsps基因棉(抗虫抗除草剂棉)速效磷和硝态氮含量均显著高于石远321,而铵态氮含量显著低于石远321。sGK321棉与石远321根际土壤脲酶、碱性磷酸酶和过氧化氢酶活性均无显著差异;双Bt抗虫棉土壤脲酶活性显著低于石远321,碱性磷酸酶和过氧化氢酶活性与石远321均无显著差异;抗虫抗除草剂棉与石远321土壤脲酶活性无显著差异,碱性磷酸酶活性显著高于石远321,而过氧化氢酶活性显著低于石远321。表明sGK321棉与石远321根际土壤养分(硝态氮除外)含量和酶活性无显著差异,而双Bt抗虫棉和抗虫抗除草剂棉所呈现的差异是因不同品种所致。