水稻菠菜紫黄质脱环氧化酶基因的克隆遗传转化及其特性的研究

自发现叶黄素循环具有热耗散的作用后它被引起广泛的关注目前普遍认为叶黄素循环的色素定位于天线色素蛋白复合体上在跨膜质子梯度pH形成后玉米黄质Z和环氧玉米黄质A能够从叶绿素中吸收过多的激发能并以热能的形式耗散到体外从而保护光合器官免受强光的破坏紫黄质脱环氧化酶VDE是叶黄素循环的关键酶在较低的pH条件下它能在数分钟内将紫黄质V转变为Z和A本论文从水稻和菠菜中克隆了编码VDE酶的基因并通过转基因植物进一步研究了叶黄素循环在热耗散方面的作用主要获得了以下结果 首次从两个水稻亚种籼稻和粳稻中克隆了Rvde基因分别命名为iRvde和jRvde的全长cDNA序列分别长1647bp和1887bp两者开放阅读框的同源性为98%与其它已知vde基因的同源性在60以上推导两者均编码446个氨基酸其中转运肽序列长98个氨基酸两者成熟蛋白的氨基酸序列完全相同与已知VDE成熟蛋白的同源性在75%以上其中与小麦的同源性最高达87.4 通过PCR扩增获得了Rvde基因的核基因组DNA序列在它们的编码区中含有4个内含子其长度在jRvde中分别为105bp327bp81bp和69bp而iRvde基因的第2个内含子长425bp与jRvde的第2个内含子差别较大内含子的AT含量为6063%其两端为典型的GT/AG结构 构建了Rvde基因的原核表达载体pET-Rvde在0.4mmol/L IPTG的诱导下该基因能在大肠杆菌BL21(DE3)中大量表达SDS-PAGE和Western杂交表明表达蛋白的分子量约为 43 kDa随着IPTG诱导时间的延长蛋白量逐渐增加诱导4h后它占大肠杆菌总蛋白的25左右吸收光谱差值A502-540随反应的进行逐渐增大反应体系总色素的HPLC分析表明V逐渐降低而Z刚好相反说明表达的蛋白具有与活体VDE酶相同的功能能在体外将V转变为A和Z 从菠菜中克隆了Svde基因并构建了该基因的反义抑制植物表达载体pCB-antiSvde用根癌农杆菌介导法转化烟草获得了大量的转基因植株再生的愈伤组织经GUS染色后呈蓝色PCR扩增潮霉素抗性基因hpt和Svde基因结果显示在转基因植株T0和T1代中都分别扩增出1.0 kb和1.4 kb的目的片段而在未转化的对照植株中没有扩增转基因植株的T0代种子在潮霉素培养基上的萌发数与未萌发数的比值为3:1符合单基因的孟德尔分离规律从T1代转基因植株中筛选出抑制程度较强的一个株系A29Southern杂交结果表明外源Svde基因已整合到烟草的基因组中并且只有一个插入位点通过冻融法从该植株的类囊体中提取VDE酶其酶活性为3.2是对照植株的45.7表明VDE酶受到了抑制荧光动力学及HPLC测定结果显示强光处理后在转基因植株中Z和A的形成较少非光化学淬灭NPQ值较对照低Fv/Fm的下降较对照快表明转基因植株的热耗散能力下降进而说明叶黄素循环具有热耗散的功能 同时还建立了根癌农杆菌介导的水稻遗传转化体系并初步作了转化Svde基因的试验另外还建立了一种适合于筛选转基因植株的DNA微量提取法此方法操作快捷方便一个人在一天内能制备50多个样品100mg的植物鲜样平均可获得40µg的DNA提取的DNA可直接用于PCR反应酶切分析及Southern分析

The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia : Anura): a new gene order in the vertebrate mtDNA

The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNA(Thr), tRNA(Pro), tRNA(Leu) ((CUN))) a tandem duplication of tRNA(Mer) gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNA(Thr)/tRNA(Pro)/tRNA(Leu)/tRNA(Phe). The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNAM(Met) and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNA(Met) were first observed in the vertebrate mitochondrial genomes. (c) 2004 Elsevier B.V. All rights reserved.

The ADH1B Arg47His polymorphism in East Asian populations and expansion of rice domestication in history

Background: The emergence of agriculture about 10,000 years ago marks a dramatic change in human evolutionary history. The diet shift in agriculture societies might have a great impact on the genetic makeup of Neolithic human populations. The regionally restricted enrichment of the class I alcohol dehydrogenase sequence polymorphism (ADH1BArg47His) in southern China and the adjacent areas suggests Darwinian positive selection on this genetic locus during Neolithic time though the driving force is yet to be disclosed. Results: We studied a total of 38 populations (2,275 individuals) including Han Chinese, Tibetan and other ethnic populations across China. The geographic distribution of the ADH1B*47His allele in these populations indicates a clear east-to-west cline, and it is dominant in south-eastern populations but rare in Tibetan populations. The molecular dating suggests that the emergence of the ADH1B*47His allele occurred about 10,000 similar to 7,000 years ago. Conclusion: We present genetic evidence of selection on the ADH1BArg47His polymorphism caused by the emergence and expansion of rice domestication in East Asia. The geographic distribution of the ADH1B*47His allele in East Asia is consistent with the unearthed culture relic sites of rice domestication in China. The estimated origin time of ADH1B*47His allele in those populations coincides with the time of origin and expansion of Neolithic agriculture in southern China.

Growth and yield of GIFT (Oreochromis niloticus) and Thai silver barb (Barbodes gonionotus Bleeker) in rice fields and their effects on the yield of rice

(GIFT) (Oreochromis niloticus) and Silver barb (Barbodes gonionotus) in rice fields and their effects on the yield of rice was carried out in nine experimental rice plots. Three treatments viz., treatment-1 with 0. niloticus (T1), treatment-2 with B. gonionotus (T2) and treatment- 3 was kept as control (T3, without fish) were used in this study. Fertilizers such as, Urea (178 kg ha-1), T.S.P (125 kg ha-1) and M.P. (67 kg ha-1) were applied in each treatment. The fishes were stocked @ 6250 ha·1 and the experiment was continued for a period of 107 days. The values of water quality parameters such as, water temperature, dissolved oxygen, pH and chlorophyll-a were found within suitable level. Between the two species, higher specific growth rate was recorded in 0. niloticus than that of B. gonionotus. But B. gonionotus showed much higher survival (72%) than that of 0. niloticus (35%). Similar to survival, higher production (244 kg ha-1) and income (Tk. 6399 ha-1) were recorded in B. gonionotus than those of 0. niloticus (142.8 kg ha'1 and Tk. 2137 ha-1). Significant differences (price grain and straw were observed between the treatments with fish and without fish.

Growth performance, recovery rate and fish yield of GIFT strain at various water depths under rice-fish culture systems

Effect of water depth on recovery rate, growth performance and fish yield of GIFT in the rice-fish production systems was studies in experimental plots of 123 m2 with a pond refuge of I meter deep which covered 10% of the total land area. Mortality rate of fish was very low ranging from 0.81-1.63%. However, at harvest, recovery rate ranged from 76.69-82.93% with the highest recovery at 11-15 em of water depth. Significantly the highest absolute growth (99.97) and specific growth rate (2.42%) were found at 21-25 cm water depth. The same treatment also produced significantly higher fish yield (909.76 kg/ha) although statistically similar to the fish yield (862.60 kg/ha) obtained at ll-15 em of water depth. Results also suggested that higher water depth can produce bigger fish but no significant effects of water depth was found on fish yield in the treatments 11-15 cm and 21-25 cm water depths of this experiment.

Performance of Amblypharyngodon mola with Barbodes gonionotus, Cyprinus carpio and Macrobrachium rosenbergii in rice-fish culture system

An experiment was conducted to assess the performance of mola (Amblyphmyngodon mola) in rice fish culture system with freshwater prawn Macrobrachium rosenbergii), Thai silver barb (Bm·bades ganianotus) and common carp (Cyprinus cmpia) for a period of 4 months at the Agronomy Field Laboratory, Bangladesh Agricultural University, Mymensingh, Bangladesh. Four treatments viz., treatment-I (T1) with A. mala and M. rosenbergii; treatmen t-II (T2) with A. mala, M. rosenbergii and B. gonianatus ; treatmentIII (T3) with A. mala, 1Vf. rosenbergii and C. cmpia, and treatment- IV (T4) as control (without fish) were used in triplicate. All treatments were equally fertilized with urea (200 kg/ha), TSP (150 kg/ha) and MP (75 kg/ha). The mean values of water quality parameters viz., temperature, dissolved oxygen, pH, nitrate-nitrogen showed a very small variations among different treatments, but phosphate-phosphorus and chlorophyll-a were relatively higher in T4 without fish (i.e., control). The fish production of 480.5 kg/ha in T3 was significantly higher than that of 355.6 kg/ha T2 and 223.8 kg/ha in T 1• The values of soil organic matter, total-nitrogen, phosphorus and potassium at harvest were significantly (P<0.05) higher in the treatments with fish than without fish, but pH did not show any significant differences. The yield of rice grain and straw was also obtained significantly (P<0.05) higher in the treatments with fish. The increase in grain was higher over the control by 11.81%, 9.41% and 14.76% and that in straw was by 9.83%, 4.77% and 13.29% in Tl> T2, and T3 respectively.

Culture of Amblypharyngodon mola in rice fields alone and in combination with Barbodes gonionotus and Cyprinus carpio

A rice-fish culture experiment with four treatments viz., T 1 with Amblyphmyngadan mala alone, T 2 with A. mala and Barbodes gonionotus, T3 with A. mala and Cyprinis carpio and T4 as control (without fish) was carried out in the rice fields during April through August1999. The recovery rate of A. mala were 42%, 37% and 42% in treatments 1, 2 and 3 respectively and the same recorded for B. gonionotus and C. cmpio were 62% and 55% respectively. Among the three species of fish, B. gonionotus showed much higher recovery rate than both of A. mala and C. carpio. The production of A. nwla was 12.50 kg/ha/3 months in monoculture, and 7.92 kglha/3 months and 8.86 kglha/3 months in combination with B. gonionotus and C. carpio, respectively. The production of B. gonionotus in T2 was 169.29 kg/ha/3 months and C. cmpio in T 3 was 252.92 kg/ha/3 months. The total fish production was 12.50 kg/ha/3 months, 175.21 kg/ha/3 months and 261.88 kg/ha/3 months in Tp T2 and T3, respectively. The highest yields of rice grain (5.78 ton/ha) and straw (7.83 ton/ha) were recorded in T3 and the lowest of the same was in T4 (grain 4.96 ton/ha and straw 6.62 ton/ha). Rice yield increased by about 12.10% in T1, 13.30% in T2 and 16.33% in T3 in context to T4, rice-alone culture. The results demonstrated that the culture of fish in rice fields had profound beneficial impact on the production of rice grain and straw.

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy

Background. The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. Methods. Seven baboons receiv

Shifting rice farming to fish culture in some selected areas of Mymensingh, Bangladesh: the process, conflicts and impacts

The evolutionary process of converting low-lying paddy fields into fish farms and its impact on agrarian communities in some selected areas of Mymensingh district were studied. This study was conducted through participatory rural appraisal (PRA) covering 12 villages from each of selected upazillas viz. Fulpur and Haluaghat of Mymensing [sic] district. A total of 12 PRA sessions were conducted where 90 farmers participated during 29 July to 26 August 2004. It is seen that the use of low-lying paddy fields was mostly confined to Broadcast Aman (B. Aman) rice production until 1960s. With the introduction of modern rice farming technology, the farmers started to produce Boro rice in Rabi season and B. Aman rice in Kharif season. With the passage of time, aquaculture technologies have been evolved and the farmers realized that fish farming is more profitable than rice cultivation, and then they started to utilize their paddy fields for alternate rice-fish farming and rice-cum-fish farming. Now a days, aquaculture based crop production system is in practice in more than 25% of the low-lying paddy fields. Conversion of rice fields in to fish ponds has brought up a change in the livelihood patterns of the rural farmers. The areas where the farmers involved themselves in the new production systems were fingerling collection, transportation and marketing of fry and fingerlings. During 1960s to 1970s, a few people used to culture fish in the permanent ponds for their own consumption, the species produced were rohu, catla, mrigal, ghainna, long whiskered catfish, freshwater shark (boal), snake head (shol) etc. Small fishes like climbing perch, stinging catfish, walking catfish, barb, minnows etc. were available in the rice fields during monsoon season. In 1980s to mid 1990s, some rice fields were converted into fish ponds and the people started to produce fish for commercial purposes. When rice-fish farming became profitable, a large number of people started converting their rice fields in to rice-fish culture ponds. Culture of some exotic fishes like silver carp, tilapia, grass carp, silver barb etc. also started in the paddy fields. Higher income from fish farming contributed positively in improving the housing, sanitation and education system in the study areas. It is seen that the medium and medium high lands were only used for alternate rice fish farming. The net income was high in any fish based cropping system that motivated the farmers to introduce fish based cropping system in the low-lying inland areas. As a result, the regional as well as communal income disparities occurred. However, the extraction of ground water became common during the dry period as the water was used for both rice and fish farming. Mass conversion of paddy fields into rice-fish culture ponds caused water logging in the study areas. In most cases, the participated farmers mentioned that they could be easily benefited by producing fish with T. Aman or only fish during the monsoon season. They agreed that this was an impressive technology to them and they could generate employment opportunities throughout the year. Finally, the social, economic and technical problems which are acting as constraints to rapid expansion of fish production system were reported from the interviewee.

Adoption, productivity and profitability of rice-fish and fingerling culture in three selected districts of Bangladesh

The study aimed to investigate the productivity and profitability of rice-fish farming. It also determined the factors affecting rice-fish and fingerling production in Bangladesh. Among the total respondents about 55% were involved in rice-fish farming, 37% in pond fish culture and rest 9% in fish nursery. Integrated rice-fish farming contributed about 83% of the annual farm income in Comilla while it was about 20% in both Kishoregonj and Chandpur districts. Among the different farming systems integrated rice-fish culture and rearing fingerlings in the nursery pond were more profitable at the farm level. Farmers earned a net return of Tk. 49,714/ha from integrated fish culture with boro rice while the net return of boro rice-alone was only Tk. 11,179/ha. This indicated that farmer could earn 218% higher net return from integrated rice-fish farming than that of boro rice only farming. Moreover, integrated rice-fish culture also reduced pesticides cost to about 77% and weeding cost to about 51% in boro season. Fingerling nursing earned highest net returns among the three technologies.

A characterization of community fish refuge typologies in rice field fisheries ecosystems

In rural Cambodia, fish is a source of food and income to millions of people. However, there has been a real threat to fish populations in natural wetlands due to the degradation of aquatic biodiversity and habitat, illegal fishing, increase of population and demand for fish, and the use of harmful pesticides for agriculture. The Rice Field Fisheries Enhancement Project (RFFEP) seeks to rebuild and protect the fish populations through innovative methods. The project works with communities to sustainably strengthen the rice field fisheries near their villages by improving protected habitats called "community fish refuges". This handbook characterizes rice field fisheries that are connected to community fish refuges. Community fish refuges are designated fish conservation areas promoted by the Fisheries Administration of the Royal Cambodian Government. It also examines the characteristics of rain-fed rice field ecosystems that are connected to community fish refuges in order to further refine descriptive criteria and better understand potential benefits and management strategies.

Rapid evolution in a pair of recent duplicate segments of rice

Gene duplication has been considered the most important way of generating genetic novelties. The subsequent evolution right after gene duplication is critical for new function to occur. Here we analyzed the evolutionary pattern for a recently duplicated s

Duplication and independent selection of cell-wall invertase genes GIF1 and OsCIN1 during rice evolution and domestication

Background: Various evolutionary models have been proposed to interpret the fate of paralogous duplicates, which provides substrates on which evolution selection could act. In particular, domestication, as a special selection, has played important role in crop cultivation with divergence of many genes controlling important agronomic traits. Recent studies have indicated that a pair of duplicate genes was often sub-functionalized from their ancestral functions held by the parental genes. We previously demonstrated that the rice cell-wall invertase (CWI) gene GIF1 that plays an important role in the grain-filling process was most likely subjected to domestication selection in the promoter region. Here, we report that GIF1 and another CWI gene OsCIN1 constitute a pair of duplicate genes with differentiated expression and function through independent selection. Results: Through synteny analysis, we show that GIF1 and another cell-wall invertase gene OsCIN1 were paralogues derived from a segmental duplication originated during genome duplication of grasses. Results based on analyses of population genetics and gene phylogenetic tree of 25 cultivars and 25 wild rice sequences demonstrated that OsCIN1 was also artificially selected during rice domestication with a fixed mutation in the coding region, in contrast to GIF1 that was selected in the promoter region. GIF1 and OsCIN1 have evolved into different expression patterns and probable different kinetics parameters of enzymatic activity with the latter displaying less enzymatic activity. Overexpression of GIF1 and OsCIN1 also resulted in different phenotypes, suggesting that OsCIN1 might regulate other unrecognized biological process. Conclusion: How gene duplication and divergence contribute to genetic novelty and morphological adaptation has been an interesting issue to geneticists and biologists. Our discovery that the duplicated pair of GIF1 and OsCIN1 has experiencedsub-functionalization implies that selection could act independently on each duplicate towards different functional specificity, which provides a vivid example for evolution of genetic novelties in a model crop. Our results also further support the established hypothesis that gene duplication with sub-functionalization could be one solution for genetic adaptive conflict.

Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome

Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in similar to 33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.