Genetic engineering of rice to resist rice tungro disease

Rice tungro disease (RTD), caused by the co-infection of rice tungro bacilliform virus (RTBV) and rice tungro spherical virus, is one of the most important viral diseases of rice in South and Southeast Asia. The disease remains one of the major threats to sustainable rice production in many countries. The lack of resistance genes to RTBV—the causal agent of tungro disease—makes it even more difficult to manage RTD. In this review, we summarize previous and current research efforts to genetically engineer rice in order to increase the crop’s resistance to tungro disease, including the use of pathogen-derived resistance and of host genes that confer RTD resistance and/or that restrict feeding by the insect vector. The prospects of developing rice cultivars with durable resistance to RTD are also discussed.     

田埂留草控制稻飞虱效果及对捕食性天敌多样性影响

为探讨稻田田埂保留杂草控制稻飞虱的效果及对捕食性天敌多样性的影响,以稻田田埂周年性保留杂草为处理,以稻田田埂不保留杂草为对照,对处理区和对照区稻田稻飞虱成若虫、捕食性天敌种类和数量进行系统调查和分析。结果表明,在调查期内(6—10月),田埂留草稻田百丛稻株稻飞虱数量为(891.11±133.12)头,较非留草稻田减少35.31%;田埂留草稻田捕食性天敌隶属5目23科35种,而田埂非留草稻田隶属5目21科33种;田埂非留草稻田捕食性天敌优势种为食虫沟瘤蛛Ummeliata insecticepsBoes.etStr.、拟水狼蛛Pirata subpiraticus Boes.etStr.和黑肩绿盲蝽Cyrtorrhinus livdipennis Reuter,而田埂留草后优势种为食虫沟瘤蛛U.insecticeps、草间小黑蛛Erigonidium graminicolum(Sundevall)、拟水狼蛛和拟环纹狼蛛Lycosa pseudoamulata(Bose.etStr.);田埂留草稻田捕食性天敌的个体数量、丰富度、多样性指数分别为(128.89±13.52)、(33.67±0.50)、(4.53±0.04)头/百丛,较田埂非留草稻田分别增加31.96%、25.73%、5.59%,而优势度指数和均匀性指数没有显著变化。研究结果为发展稻田景观多样化控害提供依据。     

产大米蛋白水解酶的菌株筛选、酶学性质及制备大米寡肽

【目的】旨在分离、筛选并鉴定具有大米蛋白降解作用的菌株及其关键蛋白酶,为高效制备大米寡肽提供制备酶及最优制备条件。【方法】以"水解圈"为评价指标,从粮食仓库附近土壤筛选获得具有降解大米蛋白能力的菌株;通过16S rRNA序列分析确定菌株归属;利用单因素实验获得最佳氮源并初步分析酶学性质;利用HPLC检测寡肽得率,并对制备条件进一步优化。【结果】经鉴定具有大米蛋白降解作用的菌株为沙雷氏菌(Serratia sp. JWG-D15),以大米蛋白为氮源培养菌株JWG-D15,蛋白酶So PRO产量最高,其最适温度40℃,最适pH 8.0;在加酶量20 U/mg,大米蛋白浓度40 mg/mL,40℃,4 h条件下寡肽得率最高72.38%。【结论】以大米蛋白为氮源培养菌株JWG-D15,蛋白酶So PRO产量最高;蛋白酶So PRO制得的大米寡肽,其得率是目前行业最高。本研究既丰富了大米寡肽的制备用酶的种类,又为深入大米寡肽产业化提供一定理论基础。     

Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.     

Characterization of rice mutants with enhanced susceptibility to rice blast

As a first step towards identifying genes involving in the signal transduction pathways mediating rice blast resistance, we isolated 3 mutants lines that showed enhanced susceptibility to rice blast KJ105 (91-033) from a T-DNA insertion library of the japonica rice cultivar, Hwayeong. Since none of the susceptible phenotypes co-segregated with the T-DNA insertion we adapted a map-based cloning strategy to isolate the gene(s) responsible for the enhanced susceptibility of the Hwayeong mutants. A genetic mapping population was produced by crossing the resistant wild type Hwayeong with the susceptible cultivar, Nagdong. Chi-square analysis of the F2 segregating population indicated that resistance in Hwayeong was controlled by a single major gene that we tentatively named Pi-hy. Randomly selected susceptible plants in the F2 population were used to build an initial map of Pi-hy. The SSLP marker RM2265 on chromosome 2 was closely linked to resistance. High resolution mapping using 105 F2 plants revealed that the resistance gene was tightly linked, or identical, to Pib, a resistance gene with a nucleotide binding sequence and leucine-rich repeats (NB-LRR) previously isolated. Sequence analysis of the Pib locus amplified from three susceptible mutants revealed lesions within this gene, demonstrating that the Pi-hy gene is Pib. The Pib mutations in 1D-22-10-13, 1D-54-16-8, and 1C-143-16-1 were, respectively, a missense mutation in the conserved NB domain 3, a nonsense mutation in the 5th LRR, and a nonsense mutation in the C terminus following the LRRs that causes a small deletion of the C terminus. These findings provide evidence that NB domain 3 and the C terminus are required for full activity of the plant R gene. They also suggest that alterations of the resistance gene can cause major differences in pathogen specificity by affecting interactions with an avirulence factor.     

Occurrence of D-serine in rice and characterization of rice serine racemase

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg²⁺ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg²⁺, and this structural change probably regulates the two enzymatic activities.     

Dynamic viscoelasticity of protease-treated rice batters for gluten-free rice bread making

Papain (cysteine protease), subtilisin (Protin SD-AY10, serine protease), and bacillolysin (Protin SD-NY10, metallo protease) increased the specific volume of gluten-free rice breads by 19–63% compared to untreated bread. In contrast, Newlase F (aspartyl protease) did not expand the volume of the rice bread. In a rheological analysis, the viscoelastic properties of the gluten-free rice batters also depended on the protease categories. Principal component analysis (PCA) analysis suggested that the storage and loss moduli (G′ and G″, respectively) at 35 °C, and the maximum values of G′ and G″, were important factors in the volume expansion. Judging from the PCA of the viscoelastic parameters of the rice batters, papain and Protin SD-AY10 improved the viscoelasticity for gluten-free rice bread making, and Protin SD-NY effectively expanded the gluten-free rice bread. The rheological properties differed between Protin SD-NY and the other protease treatments.     

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.     

Proteomic changes in rice leaves during development of field-grown rice plants

Of the numerous factors affecting rice yield, how solar radiation is transformed into biomass through rice leaves is the most important. We have analyzed proteomic changes in rice leaves collected from six different developing stages (vegetative to ripening). We studied protein expression profiles of rice leaves by running two-dimensional gel electrophoresis. Differential protein expression among the six phases were analyzed by image analysis, which allowed the identification of 49 significantly different gel spots. The spots were further verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, in which 89.8% of them were confirmed to be rice proteins. Finally, we confirmed some of the interesting rice proteins by immunoblotting. Three major conclusions can be drawn from these experimental results. (i) Protein expression in rice leaves, at least for high or middle abundance proteins, is attenuated during growth (especially some chloroplast proteins). However, the change is slow and the expression profiles are relatively stable during rice development. (ii) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major protein in rice leaves, is expressed at constant levels at different growth stages. Interestingly, a high ratio of degradation of the RuBisCO large subunit was found in all samples. This was confirmed by two approaches, mass spectrometry and immunoblotting. The degraded fragments are similar to other digested products of RuBisCO mediated by free radials. (iii) The expression of antioxidant proteins such as superoxide dismutase and peroxidase decline at the early ripening stage.     

Plant regeneration from protoplasts of Indica type rice and CMS rice

Cell suspensions were established from 12 rice genotypes including Indica type, Japonica type and cytoplasmic male sterile (cms) rice lines of both types. 133 green plants were regenerated from protoplasts isolated from an Indica type rice and 15 plants were obtained from a Japonica type cms rice. A cell suspension, from which protoplasts can be isolated (about 10⁶/ml) but from which cell division has not yet been induced, has been established from the most widely used Indica type cms rice which contains Wild Abortive (WA) cytoplasm.     

辽宁地区水稻资源抗稻瘟病基因的检测分析

为了明确辽宁地区水稻资源中抗稻瘟病基因的分布情况及抗病效应,选取辽宁地区水稻资源176份,鉴定了抗稻瘟病基因pi21、Pi36、Pi37、Pita、Pid2、Pid3、Pi5及Pib在这些材料中的分布情况,并接种鉴定了这些材料对稻瘟病的抗性。结果表明:176份供试材料中,83份对稻瘟病表现抗病,栽培稻、杂草稻及农家种中抗病品种所占的比率分别为41.48%、1.14%及4.54%。抗稻瘟病基因pi21、Pi36和Pi37在所有参试材料中均未检测到,且分别有74份、49份、47份、52份及89份材料携带Pita、Pid2、Pid3、Pi5及Pib的抗病等位基因。抗病基因绝大部分分布在栽培种中,农家种和杂草稻中分布较少。不含有抗稻瘟病基因和只携带单个抗病基因的材料对稻瘟病的抗性均较差,而抗病基因聚合可不同程度提高材料的抗性。经检测,不含有本试验鉴定的pi21等8个已克隆抗病基因的材料共32份,其中表现抗病的占21.87%;只携带1个抗稻瘟病基因的材料为52份,表现抗病的占17.31%;携带2个抗稻瘟病基因的材料为39份,表现抗病的占69.23%,其中以携带Pita+Pi5的材料最多(14份),且均表现抗病;携带3个抗稻瘟病基因的材料为31份,表现抗病的占77.42%,以携带Pita+Pid3+Pi5的材料抗性最强;携带4个抗稻瘟病基因的水稻材料22份,表现抗病的占72.73%,携带5个抗病基因的水稻材料未检测到。     

A field study of pollen-mediated gene flow from Mediterranean GM rice to conventional rice and the red rice weed

The objective of this study was to assess the frequency of pollen-mediated gene flow from a transgenic rice line, harbouring the gusA and the bar genes encoding respectively, -glucuronidase and phosphinothricin acetyl transferase as markers, to the red rice weed and conventional rice in the Spanish japonica cultivar Senia. A circular field trial design was set up to investigate the influence of the wind on the frequency of pollination of red rice and conventional rice recipient plants with the transgenic pollen. Frequencies of gene flow based on detection of herbicide resistant, GUS positive seedlings among seed progenies of recipient plants averaged over all wind directions were 0.036 ± 0.006% and 0.086 ± 0.007 for red rice and conventional rice, respectively. However, for both red rice and conventional rice, a clear asymmetric distribution was observed with pollination frequency favoured in plants placed under the local prevailing winds. Southern analyses confirmed the hemizygous status and the origin of the transgenes in progenies of surviving, GUS positive plants. Gene flow detected in conventional rice planted at 1, 2, 5 and 10 m distance revealed a clear decrease with increasing distance which was less dramatic under the prevailing wind direction. Consequences of these findings for containment of gene flow from transgenic rice crops to the red rice weed are discussed. The precise determination of the local wind conditions at flowering time and pollination day time appear to be of primary importance for setting up suitable isolation distances.     

水稻OsLecRK1基因介导的抗褐飞虱功能分析

褐飞虱Nilaparvata lugens St(a)l是对水稻最具破坏性的害虫之一,OsLecRK1是水稻Bph3基因簇中对褐飞虱抗性贡献最大的基因.本文对RHTd(含Bph3)等材料进行了褐飞虱抗性评价,克隆并构建了OsLecRK1过量表达突变体水稻,利用该突变体分析了OsLecRK1基因对褐飞虱若虫存活率、若虫发育历期等生物学参数的影响.结果 表明,含Bph3基因水稻RHTd对褐飞虱的抗性明显地强于含Bph1基因水稻Mudgo和bph2基因水稻ASD7,RHTd水稻的褐飞虱受害指数仅为Mudgo和ASD7水稻的53.5％和24.1％.过量表达OsLecRK1基因能显著地增加水稻对褐飞虱的驱避性和抗生性,褐飞虱雌成虫偏好于在野生型水稻上产卵;突变体水稻上的褐飞虱若虫存活率显著地降低,仅为野生型水稻上若虫存活率的75.2％ ～81.8％,且若虫发育历期显著地延长,羽化率和初羽化雌成虫体重均显著地降低;此外,褐飞虱在突变体水稻上取食分泌的蜜露量只有野生型上的40.3％ ～ 60.9％,褐飞虱单雌产卵量只为野生型51％ ～61.2％,卵孵化率只有野生型的52.2％～56.7％,均显著地减少.结果 表明,含Bph3基因水稻RHTd对褐飞虱的抗性明显地高于分别含Bph1、bph2的水稻Mudgo和ASD7;水稻Bph3基因座的OsLecRK1单个基因过量表达即可显著增加水稻对褐飞虱的抗性,OsLecRK1协同影响褐飞虱的多个生物学参数降低褐飞虱的适合度.     

Carbon footprints of rice production in five typical rice districts in China

Five provinces located in the five main rice-growing regions in China were selected as study areas, which were Jiangsu, Heilongjiang, Sichuan, Guangdong and Hunan province respectively in the middle and lower reaches of the Yangtze River, northern, southwest, southern and central rice districts. Carbon footprints of rice production in these five provinces were calculated through the life cycle assessment method using governmental statistical data, industrial standards and relevant technical data separately. Material and energy consumptions were estimated, key stages of energy consumptions and carbon emissions were identified as well. Moreover, improving measurements had been suggested correspondingly. The results indicated that: the energy consumptions of rice production in these five provinces ranked as following (high to low): Guangdong, Heilongjiang, Hunan, Sichuan and Jiangsu. The carbon footprints of rice production were 2504.20 kg carbon dioxide equation per ton rice (kgCO₂-eq./t) (Guangdong province), 2326.47 kgCO₂-eq./t (Hunan province), 1889.97 kgCO₂-eq./t (Heilongjiang province), 1538.90 kgCO₂-eq./t (Sichuan province) and 1344.92 kgCO₂-eq./t (Jiangsu province) respectively. Reducing the quantities of urea and using the intermittent irrigation method could decrease energy consumption as well as carbon footprint.     

Deep rice root systems reduce methane emissions in rice paddies

Plant and Soil - Phosphorus (P) mobility depends on the availability of water in soil, both of which are limited resources for crop production. We studied the mechanisms governing tolerance to...     

Induced volatiles in elicitor-treated and rice blast fungus-inoculated rice leaves

The volatiles released from elicitor (copper chloride, jasmonic acid, UV, L-methionine and chitosan oligomer)-treated and rice blast fungus-inoculated rice leaves were collected by the solid-phase microextraction technique and analyzed by GC-MS. (Z)-3-Hexen-1-ol, monoterpenes, methyl salicylate, and sesquiterpenes were identified as elicitor-induced volatiles by a comparison of their GC retention times and mass spectra with those of authentic compounds. The different elicitors resulted in some qualitative and quantitative differences in the production of volatiles. Monoterpenes and sesquiterpenes were identified as the rice blast fungus-induced volatiles.