耐非生物胁迫转基因水稻的培育——现在和未来

环境胁迫严重降低了作物产量,日益减少的耕地和膨胀的人口对世界粮食安全造成了威胁。长期以来,改善作物的抗逆性一直是农业生产的主要目标。水稻是重要的粮食作物之一,培育具有抗逆性的水稻品种对全球的粮食生产将产生重要影响。在改善水稻的抗逆性方面,转基因比传统方法更有发展潜力。近年来,已有许多抗逆相关基因转入水稻并获得了一些提高抗逆性的转基因植株,文章重点讨论了耐非生物胁迫转基因水稻的研究进展。     

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight. S. Maruthasalam and K. Kalpana have contributed to this article equally.     

水稻对白叶枯病和细菌性条斑病的抗性遗传研究进展

水稻白叶枯病和水稻细菌性条斑病是由稻黄单胞细菌(Xanthomonas oryzae)不同致病变种引起的两种最重要的水稻细菌性病害。发掘和利用抗性基因,培育抗病品种是防治这两种病害的最有效手段之一。本文分别综述了这两种高度相关的病害的抗性遗传研究进展,包括已发掘和利用的主效抗性基因特点及目前国内外对这两种病害的抗性QTL定位研究进展,为水稻抗白叶枯病和细菌性条斑病育种研究提供有用信息。     

Marker-assisted breeding of Chinese elite rice cultivar 9311 for disease resistance to rice blast and bacterial blight and tolerance to submergence

Rice (Oryza sativa L.) is the staple food crop for more than half of the world’s population. The development of hybrid rice is a practical approach to increase rice production. However, rice production was frequently affected by biotic and abiotic stresses. Rice blast and bacterial blight are two major diseases in rice growing regions. Rice plantation is also frequently affected by short-term submergence or seasonal floods in wet seasons and drought in dry seasons. The utilization of natural disease resistance (R) genes and stress tolerance genes in rice breeding is the most economic and efficient way to combat or adapt to these biotic and abiotic stresses. Rice cultivar 9311 is widely planted rice variety, either as inbred rice or the paternal line of two-line hybrid rice. Here, we report the pyramiding of rice blast R gene Pi9, bacterial blight R genes Xa21 and Xa27, and submergence tolerance gene Sub1A in 9311 genetic background through backcrossing and marker-assisted selection. The improved rice line, designated as 49311, theoretically possesses 99.2% genetic background of 9311. 49311 and its hybrid rice, GZ63S/49311, conferred disease resistance to rice blast and bacterial blight and showed tolerance to submergence for over 18 days without significant loss of viability. 49311 and its hybrids had similar agronomic traits and grain quality to 9311 and the control hybrid rice, respectively. The development of 49311 provides an improved paternal line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight and tolerance to submergence.     

Stressed genomics-bringing relief to rice fields

Active research in rice genetics aided by full-genome sequence has generated results to address multiple problems caused by biotic and abiotic stresses. The main challenges are achieving stability of resistance against variable biological agents and defining the genetic basis of traits influenced strongly by genotypexenvironment interactions. As shown in bacterial blight disease, detailed knowledge of host-pathogen interactions has enabled a predictive strategy to combine specific genes to provide durable resistance. Large-effect QTLs conferring tolerance to submergence, salinity, and drought have been identified. Marker-aided incorporation of the submergence tolerance gene into popular rice varieties illustrates how gene discovery can be rapidly converted into useful products. Extensive use of parallel whole-genome expression and mapping analyses is expected to improve our understanding of QTL. To accelerate conversion of discoveries into products, much can be gained by using agronomically proven genotypes and by testing in multiple environments.     

Pyramiding Xa23 and Rxo1 for resistance to two bacterial diseases into an elite indica rice variety using molecular approaches

Rice bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae and bacterial leaf streak (BLS) caused by X. oryzae pv. oryzicola (Xoc) are two important diseases of rice that often outbreak simultaneously and constrain rice production in much of Asia and parts of Africa. Developing resistant cultivars has been the most effective approach to control BB, however, most single resistance genes have limited value in breeding programs because of their narrow-spectrum of resistance to the races of the pathogen. By contrast, there is little progress in breeding varieties resistant to Xoc since BLS resistance in rice was a quantitative trait and so far only a few quantitative resistance loci have been identified. We reported here the development of a high yield elite line, Lu-You-Zhan highly resistant to both BB and BLS by pyramiding Xa23 with a wide-spectrum resistance to BB derived from wild rice and a non-host maize resistance gene, Rxo1, using both marker assisted selection (MAS) and genetic engineering. Our study has provided strong evidence that non-host R genes could be a valuable source of resistance in combating those plant diseases where no single R gene controlling high level of resistance exists and demonstrated that MAS combined with transgenic technologies are an effective strategy to achieve high level of resistance against multiple plant diseases. Y-L Zhou and J-L Xu contributed equally to this work.     

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.     

Transgenic approaches for genetic improvement in groundnut (Arachis hypogaea L.) against major biotic and abiotic stress factors

Cultivated groundnut (Arachis hypogaea L.) is considered as one of the primary oilseed crops and a major fodder for cattle industry in most of the developing countries, owing to its rich source of protein. It is due to its geocarpic nature of growth that the overall yield performance of groundnut is hindered by several biotic and abiotic stress factors. Multidimensional attempts were undertaken to combat these factors by developing superior groundnut varieties, modified with integral mechanism of tolerance/resistance; however this approach proved to be futile, owing to inferior pod and kernel quality. As a superior alternative, biotechnological intervention like transformation of foreign genes, either directly (biolistic) or via Agrobacterium, significantly aided in the development of advanced groundnut genotypes equipped with integral resistance against stresses and enhanced yield attributing traits. Several genes triggered by biotic and abiotic stresses, were detected and some of them were cloned and transformed as major parts of transgenic programmes. Application of modern molecular biological techniques, in designing biotic and abiotic stress tolerant/resistant groundnut varieties that exhibited mechanisms of resistance, relied on the expression of specific genes associated to particular stress. The genetically transformed stress tolerant groundnut varieties possess the potential to be employed as donor parents in traditional breeding programmes for developing varieties that are resilient to fungal, bacterial, and viral diseases, as well as to draught and salinity. The present review emphasizes on the retrospect and prospect of genetic transformation tools, implemented for the enhancement of groundnut varieties against key biotic and abiotic stress factors.     

转基因水稻T—DNA侧翼序列的扩增与分析

利用现有的转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR技术扩增出携带Xa21基因的T－DNA的侧翼序列,对24个有效扩增片段的序列分析结果表明,其中14个侧翼序列是水稻DNA,9个含载体主干序列,1个是外源基因Xa21片段,14个T－DNA侧翼的水稻DNA序列与直接转化法外源基因整合位点的基因组序列具有不同的特点,这些T－DNA在水稻染色体上整合后其两端序列的特点类似于在转基因双子叶植物中观察到的现象,在含主干序列的侧翼序列（37．5％,9／24）,中,载体主干序列是以不同的类型出现的。     

Role of transgenic plants in agriculture and biopharming

At present, environmental degradation and the consistently growing population are two main problems on the planet earth. Fulfilling the needs of this growing population is quite difficult from the limited arable land available on the globe. Although there are legal, social and political barriers to the utilization of biotechnology, advances in this field have substantially improved agriculture and human life to a great extent. One of the vital tools of biotechnology is genetic engineering (GE) which is used to modify plants, animals and microorganisms according to desired needs. In fact, genetic engineering facilitates the transfer of desired characteristics into other plants which is not possible through conventional plant breeding. A variety of crops have been engineered for enhanced resistance to a multitude of stresses such as herbicides, insecticides, viruses and a combination of biotic and abiotic stresses in different crops including rice, mustard, maize, potato, tomato, etc. Apart from the use of GE in agriculture, it is being extensively employed to modify the plants for enhanced production of vaccines, hormones, etc. Vaccines against certain diseases are certainly available in the market, but most of them are very costly. Developing countries cannot afford the disease control through such cost-intensive vaccines. Alternatively, efforts are being made to produce edible vaccines which are cheap and have many advantages over the commercialized vaccines. Transgenic plants generated for this purpose are capable of expressing recombinant proteins including viral and bacterial antigens and antibodies. Common food plants like banana, tomato, rice, carrot, etc. have been used to produce vaccines against certain diseases like hepatitis B, cholera, HIV, etc. Thus, the up- and down-regulation of desired genes which are used for the modification of plants have a marked role in the improvement of genetic crops. In this review, we have comprehensively discussed the role of genetic engineering in generating transgenic lines/cultivars of different crops with improved nutrient quality, biofuel production, enhanced production of vaccines and antibodies, increased resistance against insects, herbicides, diseases and abiotic stresses as well as the safety measures for their commercialization.     

Alien introgression in rice

Rice (Oryza sativa L.) productivity is affected by several biotic and abiotic stresses. The genetic variability for some of these stresses is limited in the cultivated rice germplasm. Moreover, changes in insect biotypes and disease races are a continuing threat to increased rice production. There is thus an urgent need to broaden the rice gene pool by introgressing genes for such traits from diverse sources. The wild species of Oryza representing AA, BB, CC, BBCC, CCDD, EE, FF, GG and HHJJ genomes are an important reservoir of useful genes. However, low crossability and limited recombination between chromosomes of cultivated and wild species limit the transfer of such genes. At IRRI, a series of hybrids and monosomic alien addition lines have been produced through embryo rescue following hybridization between rice and several distantly related species. Cytoplasmic male sterility and genes for resistance to grassy stunt virus and bacterial blight have been transferred from A genome wild species into rice. Similarly, genes for resistance to brown planthopper, bacterial blight and blast have also been introgressed across crossability barriers from distanly related species into rice. Some of the introgressed genes have been mapped via linkage to molecular markers. One of the genes Xa-21 introgressed from O. longistaminata has been cloned and physically mapped on chromosome 11 of rice using BAC library and flourescence in-situ hybridization. RFLP analysis revealed introgression from 11 of the 12 chromosomes of C genome species into rice. Introgression has also been obtained from other distant genomes (EE, FF, GG) into rice and in majority of the cases one or two RFLP markers were introgressed. Reciprocal replacement of RFLP alleles of wild species with the alleles of O. sativa indicates alien gene transfer through crossing over. The rapid recovery of recurrent phenotypes in BC₂ and BC₃ generations from wide crosses is an indication of limited recombination. Further cytogenetic and molecular investigations are required to determine precisely the mechanism of introgression of small chromosome segments from distant genomes in the face of limited homoeologous chromosome pairing. Future research should focus on enhancing recombination between homoeologous chromosomes. Introgression of QTL from wild species should be attempted to increase the yield potential of rice.     

Applications of biotechnology in eggplant

Eggplant (Solanum melongena L.), an economically important vegetable crop in many countries in Asia and Africa, often has insufficient levels of resistance to biotic and abiotic stresses. Genetic resources of eggplant have been assessed for resistance against its most serious diseases and pests (bacterial and fungal wilts, nematodes and shoot and fruit borer). Attempts at crossing eggplant with its wild relatives resulted in limited success due to sexual incompatibilities. However, the ability of eggplant to respond well in tissue culture, notably plant regeneration, has allowed the application of biotechnology, particularly the exploitation of somaclonal variation, haploidisation, somatic hybridisation and genetic transformation for gene transfer. Somaclonal variation has been used to obtain lines with increased resistance to salt and little leaf disease. Traits of resistance against bacterial and fungal wilts have successfully been introduced into the cultivated eggplant through somatic hybridisation. However, most somatic hybrids were sterile when the parental lines were distantly related. In contrast, the use of close relatives as fusion partners or highly asymmetric fusion resulted in the production of fertile hybrids with resistance traits and a morphology close to the cultivated eggplant, thus avoiding the series of backcrosses necessary for introgression of desired traits into eggplant. As far as molecular markers and genetic engineering are concerned, the information available for eggplant is very scanty. Two genetic linkage maps have been established by using RAPD and RFLP markers. In order to analyse the genetic relationships between eggplant and its relatives, some studies based on AFLP and ctDNA analyses have also been conducted. So far only resistance against insects, and parthenocarpic fruit development have successfully been developed in eggplant using Agrobacterium tumefasciens transformation. However, some work on genetic engineering of eggplant for other biotic and abiotic stresses has recently been initiated. This revised version was published online in June 2006 with corrections to the Cover Date.     

农杆菌介导将白叶枯病抗性基因Xa21导入水稻获得转基因植株

利用农杆菌介导的高效遗传转化系统,将白叶枯病抗性基因Xa21转入黄淮稻区主栽品种豫粳6号的胚性愈伤组织,获得转基因植株,GUS染色和PCR分析证明Xa21基因已整合到水稻基因组中,其自交T1代植株经GUS染色和白叶枯病接种鉴定呈现3：1分离,研究为培育抗白叶枯病水稻品种奠定了基础。     

Constitutive heterologous expression of avrXa27 in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains

The vascular pathogen Xanthomonas oryzae pv. oryzae ( Xoo ) and nonvascular pathogen Xanthomonas oryzae pv. oryzicola ( Xoc ) cause bacterial blight (BB) and bacterial leaf streak (BLS) diseases of rice, respectively. We have previously identified the avirulence gene avrXa27 from Xoo PXO99^A, which specifically induces the expression of the rice resistance gene Xa27 , ultimately leading to resistance against BB disease in rice. In this study, we have generated a transgenic rice line (L24) that expresses avrXa27 constitutively under the control of the PR1 promoter, and have examined its role in the host–pathogen interaction. L24 is not more susceptible to BB, indicating that avrXa27 does not contribute to virulence. AvrXa27 retains avirulence activity in L24 and, after crossing with a line containing Xa27 , progeny display phenotypic changes including inhibition of tillering, delay in flowering, stiff leaves, early leaf senescence and activation of pathogenesis-related ( PR ) genes. On challenge with a variety of compatible strains of Xoo and Xoc strain L8, lines with both avrXa27 and Xa27 also show enhanced resistance to bacterial infection. The induction of Xa27 and subsequent inhibition of Xoc growth in Xa27 plants are observed on inoculation with Xoc L8 harbouring avrXa27 . Our results indicate that the heterologous expression of avrXa27 in rice containing Xa27 triggers R gene-specific resistance and, at the same time, confers enhanced resistance to compatible strains of Xoo and Xoc . The expression of AvrXa27 and related proteins in plants has the potential to generate broad resistance in plants.     

Rice siR109944 suppresses plant immunity to sheath blight and impacts multiple agronomic traits by affecting auxin homeostasis

Plant small RNAs (sRNAs) play significant roles in regulating various developmental processes and hormone signalling pathways involved in plant responses to a wide range of biotic and abiotic stresses. However, the functions of sRNAs in response to rice sheath blight remain unclear. We screened rice (Oryza sativa) sRNA expression patterns against Rhizoctonia solani and found that Tourist‐miniature inverted‐repeat transposable element (MITE)‐derived small interfering RNA (siRNA) (here referred to as siR109944) expression was clearly suppressed upon R. solani infection. One potential target of siR109944 is the F‐Box domain and LRR‐containing protein 55 (FBL55), which encode the transport inhibitor response 1 (TIR1)‐like protein. We found that rice had significantly enhanced susceptibility when siR109944 was overexpressed, while FBL55 OE plants showed resistance to R. solani challenge. Additionally, multiple agronomic traits of rice, including root length and flag leaf inclination, were affected by siR109944 expression. Auxin metabolism‐related and signalling pathway‐related genes were differentially expressed in the siR109944 OE and FBL55 OE plants. Importantly, pre‐treatment with auxin enhanced sheath blight resistance by affecting endogenous auxin homeostasis in rice. Furthermore, transgenic Arabidopsis overexpressing siR109944 exhibited early flowering, increased tiller numbers, and increased susceptibility to R. solani. Our results demonstrate that siR109944 has a conserved function in interfering with plant immunity, growth, and development by affecting auxin homeostasis in planta. Thus, siR109944 provides a genetic target for plant breeding in the future. Furthermore, exogenous application of indole‐3‐acetic acid (IAA) or auxin analogues might effectively protect field crops against diseases.     

Recent advances in rice biotechnology--towards genetically superior transgenic rice

Rice biotechnology has made rapid advances since the first transgenic rice plants were produced 15 years ago. Over the past decade, this progress has resulted in the development of high frequency, routine and reproducible genetic transformation protocols for rice. This technology has been applied to produce rice plants that withstand several abiotic stresses, as well as to gain tolerance against various pests and diseases. In addition, quality improving and increased nutritional value traits have also been introduced into rice. Most of these gains were not possible through conventional breeding technologies. Transgenic rice system has been used to understand the process of transformation itself, the integration pattern of transgene as well as to modulate gene expression. Field trials of transgenic rice, especially insect-resistant rice, have recently been performed and several other studies that are prerequisite for safe release of transgenic crops have been initiated. New molecular improvisations such as inducible expression of transgene and selectable marker-free technology will help in producing superior transgenic product. It is also a step towards alleviating public concerns relating to issues of transgenic technology and to gain regulatory approval. Knowledge gained from rice can also be applied to improve other cereals. The completion of the rice genome sequencing together with a rich collection of full-length cDNA resources has opened up a plethora of opportunities, paving the way to integrate data from the large-scale projects to solve specific biological problems.     

Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

Production of transgenic rice with agronomically useful genes: an assessment

Rice is the most important food crop in tropical and subtropical regions of the world. Yield enhancement to increase rice production is one of the essential strategies to meet the demand for food of the growing population. Both abiotic and biotic features limit adversely the productivity of rice growing areas. Conventional breeding has been an effective means for developing high yielding varieties, however; it is associated with its own limitations. It is envisaged that recent trends in biotechnology can contribute to the agronomic improvement of rice in terms of yield and nutritional quality as a supplement to traditional breeding methods. Genetic transformation of rice has demonstrated numerous important opportunities resulting in the genetic improvement of existing elite rice varieties and production of new plant types. Significant advances have been made in the genetic engineering of rice since the first transgenic rice plant production in the late 1980s. Several gene transfer protocols have been employed successfully for the introduction of foreign genes to rice. In more than 60 rice cultivars belonging to indica, japonica, javanica, and elite African cultivars, the protocol has been standardized for transgenic rice production. Selection and use of appropriate promoters, selectable markers, and reporter genes has been helpful for development of efficient protocols for transgenic rice in a number of rice cultivars. The present review is an attempt to assess the current state of development in transgenic rice for the transfer of agronomically useful genes, emphasizing the application and future prospects of transgenic rice production for the genetic improvement of this food crop.