水稻褐飞虱抗性基因研究进展

随着全球性气候变暖以及稻作区耕作栽培措施的改变,稻褐飞虱已成为严重危害水稻产量的害虫之一.综述当前水稻褐飞虱抗性基因的定位、克隆以及抗性机制的最新研究进展,并对抗褐飞虱水稻育种的利用现状作报道.     

水稻抗褐飞虱基因研究和利用现状

稻褐飞虱(Nilaparvata lugens Stal)是一种单食性水稻害虫,根据其对水稻品种的危害,可分为生物型1、2、3、4等类型.它对亚洲各国水稻生产造成极大危害.实践证明,利用抗虫品种是防治稻褐飞虱最经济有效的方法.本文综述了稻褐飞虱的生物学特征和分布、生物类型,着重介绍抗稻褐飞虱基因的研究和利用现状,并对今后开展抗稻褐飞虱基因研究提出几点建议.     

Monitoring on Insecticide Resistance of the Brown Planthopper and the White Backed Planthopper in Asia

Annual change in insecticide susceptibility of the long-range migrating rice insects, the brown planthopper (BPH) and the white backed planthopper (WBPH), monitored by topical application was reviewed covering temperate and tropical Asia. Monitoring in Japan revealed consistent increase in topical LD₅₀ of BPH for various insecticides during the period between 1967 and 1983 with small peak in 1979 and abrupt increase occurred in 1984 and 1985 with the highest peak ever observed, and then recovered to the 1979 level and remain unchanged to the present. The WBPH showed no obvious change until 1984 when marked increase in LD₃₀, especially for the organoosphates occurred and gradual increase still continues. In the tropics, recent data on BPH obtained from Vietnam, Thailand and Malaysia shows apparent development of insecticide resistance with remarkable increase in LD₅₀ as compared to the late 1970's data. Present level of insecticide susceptibility is close to those in temperate Asia. WBPH from those tropical zone also showed remarkable increase in LD₅₀ during the last two decades.     

Sterols and Asparagine in the Rice Plant,Endogenous Factors Related to Resistance against the Brown Planthopper (Nilaparvata lugens)

The garlic-derived antibacterial principle, alk(en)yl sulfinate compounds, has long been considered as very short-lived substance. However, there are some data showing a rather more stable nature of allicin. We determined here the thermostability of allicin by a systematic analyses employing chemical quantification and an antibacterial activity assay. Allicin in an aqueous extract of garlic was degraded stoichiometrically in proportion to the temperature; we estimated the half-life of allicin to be about a year at 4 °C (from 1.8 mg/ml to 0.9 mg/ml) and 32 d at 15 °C, but only 1 d at 37 °C (from 2.0 mg/ml to 1.0 mg/ml). The half-life values for antibacterial activity showed a similar trend in results: 63 d or more at 4 °C for both antibacterial activities, 14 d for anti-staphylococcal activity, and 26 d for anti-escherichia activity at 15 °C, but only 1.2 d and 1.9 d for the respective activities at 37 °C. Such antibacterial activities were attributable to the major allicin, allyl 2-propenylthiosulfinate. Surprisingly, the decline in the quantity of allicin was not accompanied by its degradation; instead, allicin became a larger molecule, ajoene, which was 3-times larger than allicin.     

利用普通野生稻褐飞虱抗性改良水稻恢复系的研究

褐飞虱是我国水稻主要害虫之一,挖掘和利用稻飞虱抗性基因,选育抗稻飞虱的水稻品种是目前防治褐飞虱最科学、安全的一种措施。本研究利用具有高抗褐飞虱显性基因的抗源材料HS204作为抗源供体,以恢复系明恢65、582、MR等作为轮回亲本,通过回交,苗期群体鉴定和成株期农艺性状选择,成功地将高抗褐飞虱基因转育到轮回亲本中,获得45份抗性纯合株系,同时其配合力、米质等其他农艺性状也得到了相应改善。     

Transgenic indica Rice Expressing ns-LTP-Like Protein Shows Enhanced Resistance to Both Fungal and Bacterial Pathogens

Antimicrobial peptides (AMPs) from plant seeds, known to inhibit pathogen growth have a great potential in developing transgenic plants resistant to disease. Some of the nonspecific-lipid transfer proteins (ns-LTP) that facilitate in vitro transport of lipids, show antimicrobial activity in vitro. Rice seeds also contain ns-LTPs; however, these genes are expressed weakly in seedlings. We have transformed Pusa Basmati 1, an elite indica rice cultivar, with the gene for Ace-AMP1 from Allium cepa, coding for an effective antimicrobial protein homologous to ns-LTPs. The gene for Ace-AMP1 was cloned under an inducible rice phenylalanine ammonia-lyase (PAL) or a constitutive maize ubiquitin (UbI) promoter. Ace-AMP1 was expressed in transgenic lines and secreted in the apoplastic space. Protein extracts from leaves of transgenic plants inhibited three major rice pathogens, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae, in vitro. Enhanced resistance against these pathogens was observed in in planta assays, and the degree of resistance correlating with the levels of Ace-AMP1 with an average increase in resistance to blast, sheath blight, and bacterial leaf blight disease by 86%, 67%, and 82%, respectively. Importantly, transgenic rice plants, with stable integration and expression of Ace-AMP1, retained their agronomic characteristics while displaying enhanced resistance to both fungal and bacterial pathogens.     

紧穗野生稻的褐飞虱抗性导入栽培稻的研究

栽培品种的远缘野生种O.eichingeri (2n=24,CC染色体组)是褐飞虱的重要抗源。为了将原产乌干达的O.eichingeri两个编号材料的褐飞虱抗性导入栽培稻02428中,利用胚培养技术获得了两个组合的F.杂种,可交配力分别为0.36％和1.62％。所得F.杂种生长旺盛,分蘖力强,但高度不育,其花粉母细胞中期Ⅰ二价体数为0～4个,平均1.33～1.37。F_1植株用02428回交及套袋自交产生的BC_1F_1和F_2植株形态相似,染色体组均为AAC,花粉母细胞中期Ⅰ染色体构型为(12.02～12.18)Ⅰ (11.67～11.89)Ⅱ (0.04～0.19)Ⅲ,均表现完全不育。进一步检查了42个BC_1F_2植株和9个BC_2F_1植株的染色体数目,其变幅为24～38,从中筛选到2n=25及2n=24的植株各21个,其中5个整倍体植株对褐飞虱表现抗,说明两份紧穗野生稻载有抗性基因的染色体片段已成功转入02428中。本研究还发现一些株高及每穗粒数等明显超亲的材料,这可能与染色体组A与C上某些基因的互作有关。     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

Transgenic indica Rice Variety Pusa Basmati 1 Constitutively Expressing a Rice Chitinase Gene Exhibits Enhanced Resistance to Rhizoctonia solani

Agrobacterium-mediated transformation of an elite indica rice variety, Pusa Basmati 1, was performed using LBA4404 (pSB1, pMKU-RF2) that harbours a rice chitinase gene (chi11) under the control of the maize ubiquitin (Ubi1) promoter-intron. Right border (gus) and left border (hph) flanking sequences and the transgene (chi11) in the middle of the T-DNA were used as probes in Southern analysis. Out of eleven independent T0 plants regenerated, three had single copy T-DNA insertions and eight had multiple T-DNA insertions. Nine T₀ plants carried the complete T-DNA with the chitinase transgene. Two T₀ plants did not carry chi11, though they had other T-DNA portions. Three plants harbouring single copy insertions and one plant harbouring two inserted copies were analyzed in detail. A segregation ratio of 3:1, reflecting T-DNA insertion at a single locus, was observed in the progeny of all the four T₀ plants. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase at high levels. Bioassays of T₁ plants indicated enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, in comparison to control plants. A homozygous transgenic line was established from one T₀ line, which exhibited the maximum resistance to R. solani.     

Transgenic Lines of Oryza Sativa (Basmati 2000) Expressing Xa21 Demonstrate Enhanced Resistance to Bacterial Blight

An efficient transformation system for rice was established by co-cultivating calli, derived from 21-day-old scutellum, with Agrobacterium tumefaciens cells (OD600 = 0.04), maintained on filter paper, moistened with 4 mL of a medium, and supplemented with 400 μM acetosyringone, for a period of 2 days. Presence of the transgene was confirmed by polymerase chain reaction, and stable integration and copy number of the transgene were determined by Southern blot analysis. Among seven plants analyzed, six possessed single T-DNA integration events, while one plant was found to have two integrated copies of the T-DNA. A total of 45 T₀ plants were grown in the greenhouse to obtain the T₁ generation. T₁ plants evaluated for presence of the transgene and for response to inoculation with the bacterial leaf blight pathogen, Xanthomonas oryzae pv. Oryzae, exhibited Mendelian segregation (3:1) for the transgene as well as enhanced resistance to bacterial blight     

水稻与褐飞虱的相互作用研究进展

褐飞虱(Nilapavata lugens,brown planthopper,BPH)是水稻生产中最重要的害虫.水稻与褐飞虱互作机制的研究为培育新的水稻品种做出了贡献.本文综述了栽培稻和野生稻中分离定位的抗褐飞虱基因,以及褐飞虱唾液蛋白、水稻和褐飞虱代谢物和褐飞虱共生菌在水稻-褐飞虱互作关系中的作用.目前从栽培稻和野生稻中鉴定了 40个抗褐飞虱基因,褐飞虱取食的信号转导开启水稻抗性基因的表达和防御机制的改变,包括筛管封闭、次生代谢产物的产生,以及蛋白酶抑制剂的诱导等.褐飞虱的唾液蛋白以及体内存在多种共生微生物对褐飞虱的生长发育和适应抗性都起到一定作用,但目前提出的水稻-褐飞虱分子相互作用的模型仍有许多方面有待研究.     

水稻抗性转基因研究进展

随着水稻遗传转化技术的发展,通过基因工程手段获得抗病虫草害及逆境水稻品种成为可能。本文综述了近年来国内外水稻抗性转基因的技术、方法和策略等研究现状、发展趋势、存在的问题及解决方法,对水稻抗性转基因在水稻育种上的应用也作了评价。     

水稻抗褐飞虱基因bph2的SSR定位和标记辅助选择

利用综合性状较好对褐飞虱敏感的粳稻恢复系C418为父本,以含有bph2基因的抗褐飞虱品种ASD7为母本构建了包含134个F23家系的群体,利用苗期鉴定法对F2：3家系进行抗性鉴定：用SSR标记技术,将bph2基因定位在第12染色体长臂上,标记RM7102和RM463之间,其遗传距离分别为7．6cM和7．2cM。在进行表型选择的同时,利用与bph2基因连锁的SSR标记RM7102和RM463对BC1F1和BC2F1进行了标记辅助选择,选择效率分别为89．9％和91．2％,为培育高抗褐飞虱水稻品种奠定了基础。     

水稻对褐飞虱抗性相关蛋白的双向电泳分析

以药用野生稻 (Oryzaofficinalis)的转育后代B5 (高抗褐飞虱 (NilaparvatalugensSt l) )与感虫品种明恢 6 3(OryzasativaL .)为亲本 ,构建了一个重组自交系群体。通过抗褐飞虱鉴定 ,筛选出极端抗虫株系和极端感虫株系 ,运用分群分析法 (bulkedsegregantanalysis ,BSA)分别建成了极端抗虫集团 (resistantbulk)和极端感虫集团 (susceptiblebulk)的蛋白质池。利用双向电泳技术 ,分别分析了极端抗虫集团和极端感虫集团受虫害与未受虫害的秧苗蛋白质的变化。结果发现 ,虫害 48h后 ,感虫集团的一个分子量为 40kD的蛋白质P40 (pI=6 .3)的表达明显减弱甚至消失 ,而在抗虫集团中 ,P40的表达未受影响。与褐飞虱为害后抗虫株系和感虫株系不同的生理反应相联系 ,推测P40与水稻受褐飞虱虫害后引起的应答反应相关     

水稻对褐飞虱抗性相关蛋白的双向电泳分析

以药用野生稻(Oryza officinalis)的转育后代B5(高抗褐飞虱(Nilaparvata lugens Stl))与感虫品种明恢63 (Oryza sativa L.)为亲本,构建了一个重组自交系群体.通过抗褐飞虱鉴定,筛选出极端抗虫株系和极端感虫株系,运用分群分析法(bulked segregant analysis,BSA)分别建成了极端抗虫集团(resistant bulk)和极端感虫集团(susceptible bulk)的蛋白质池.利用双向电泳技术,分别分析了极端抗虫集团和极端感虫集团受虫害与未受虫害的秧苗蛋白质的变化.结果发现,虫害48 h后,感虫集团的一个分子量为40 kD的蛋白质P40 (pI=6.3)的表达明显减弱甚至消失,而在抗虫集团中,P40的表达未受影响.与褐飞虱为害后抗虫株系和感虫株系不同的生理反应相联系,推测P40与水稻受褐飞虱虫害后引起的应答反应相关.     

Selection of Plant Cell Lines with Enhanced Chilling Resistance

Cell cultures of Nicotiana sylvestris and Capsicum annuum, bothwithout and following exposure to the mutagen, EMS, have beensubmitted to chilling for 21 days at –3°C and +5°Crespectively and the cell lines derived from the surviving cellstested for their subsequent resistance to the chilling treatment.Some of the cell lines when again submitted to the chillingstress showed no enhanced survival, others retained their resistanceafter an extended period of growth at 24°C. The applicationof the mutagen promoted the isolation of such stable resistantcell lines. Studies of the response of the respiratory activityof isolated mitochondria to temperature from a resistant andfrom a sensitive cell line of C. annuum revealed a differencesimilar to that previously reported from studies on isolatedmitochondria from chilling-sensitive and chilling-resistantplants of various species.     

小粒野生稻导入系对稻飞虱的抗性鉴定与分析

稻飞虱是水稻生产最严重的害虫之一。野生稻拥有丰富的抗虫基因资源,导入系是鉴定和利用野生稻有利基因的有效途径。本研究通过对371份小粒野生稻导入系进行抗褐飞虱和白背飞虱接虫鉴定,分别筛选出了11份抗、72份中抗褐飞虱的材料和7份抗、45份中抗白背飞虱的材料,其中有5份材料兼抗褐飞虱和白背飞虱,这是从小粒野生稻中鉴定出抗白背飞虱材料的首次报道。通过对2份抗性导入系材料与感虫亲本杂交构建的F1和F2群体的抗虫鉴定和分析表明:K41对褐飞虱和白背飞虱的抗性受2对显性抗虫基因通过互补作用所控制;P114对褐飞虱和白背飞虱的抗性都是由1对主效的隐性基因控制。这些结果必将有利于小粒野生稻抗稻飞虱的基因定位和育种利用。