Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization (FISH) using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells (PMCs). It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-α-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.     

Centromere inactivation in a dicentric rice chromosome during sexual reproduction

T0135 is a variant selected from the progeny of a rice line telotrisomic for the short arm of chromosome 11 （2n＋IIS＇）. Fluores- cent in situ hybridization （FISH） results indicated that T0135 contained two telocentric chromosomes, which have two centro- mere-specific molecular markers （5S rDNA） for chromosome 11; thus T0135 is a newly-described rice chromosome variant with two dicentric chromosomes, named 22＋11L-＋11L＇＋I IS.11S-＋I 1S-11S. （22 represents the 22 chromosomes excluding chromo- some 11 in the rice genome, ＂-＂ represents the centromere）. To investigate the genetic stability of the rice dicentric chromosomes during sexual reproduction, we observed the chromosome types in the progeny. Ninety-four percent of the progeny had the same chromosome type as the parental line. This result indicates that the dicentric chromosomes are mostly stable during mitosis and meiosis. Immunofluorescence analysis for centromere specific histone H3 （CENH3） revealed that only one centromere is active and the other centromere is inactivated in the rice dicentric chromosomes.     

Construction of chimeric inducible promoters by elicitors of rice fungal blast pathogen and their expression in transgenic rice

The promoter fragments of wheatGstA1 and potatoGst1 have been amplified by PCR, cloned and fused respectively to the minimal promoter sequence of rice actin gene (Act1)) and its 5′ untranslated leader sequence together withGUS. The constructs with 2 chimeric promoters (WGA and PGA) have been transferred into rice in order to analyze their inducibility patterns in transgenic rice plants. The results show that: WGA and PGA are both inducible by elicitors ofPyricularia oryzae in transgenic rice cells; the intron I of riceAct1 gene is important for the heterogenic expression of monocot and dicot promoter elements in rice; and theAct1 minimal promoter and its 5′ untranslated leader sequence produced low level background expression in rice.     

转基因水稻对赤霉素敏感性的初步研究

以水稻品种中花11及其转基因株系为材料,研究它们在苗期对GA3的反应程度.结果表明:用100mg/L的GA3在二叶期对各植株进行喷雾,5d后各个株系苗高都比以喷水的对照组显著增长.在充分考虑起始株高和试验期间对照正常生长动态变化的基础上提出了比净伸长率(specificnetelongateratio,GA3处理的净伸长率和对照净伸长率之比)的概念,并建议根据比净伸长率分析各株系对GA3的敏感性.应用该方法发现2份对GA3钝感的材料.文章报道这一结果并探讨了造成用比净伸长率与常用的“株高增长率”分析水稻品种/株系对GA3敏感性差异的原因.     

Review and prospect of transgenic rice research

Rice is one of the most important crops as the staple food for more than half of the world＇s population. Rice improvement has achieved remarkable success in the past half-century, with the yield doubled in most parts of the world and even tripled in certain regions, which has contributed greatly to food security globally. Rapid population growth and economic development pose a constantly increased food requirement. However, rice yield has been hovering in the past decade, which is mainly caused by the absence of novel breeding technologies, reduction of genetic diversity of rice cultivars, and serious yield loss due to increasingly severe occurrences of insects, diseases, and abiotic stresses. To address these challenges, Chinese scientists proposed a novel rice breeding goal of developing Green Super Rice to improve rice varieties and realize the sustainable development of agriculture, by focusing on the following 5 classes of traits： insect and disease resistance, drought-tolerance, nutrient-use effi- ciency, quality and yield potential. As a modern breeding approach, transgenic strategy will play an important role in realizing the goal of Green Super Rice. Presently, many transgenic studies of rice have been conducted, and most of target traits are consistent with the goal of Green Super Rice. In this paper, we firstly review technical advances of rice transformation, and then outline the main progress in transgenic rice research with respect to the most important traits： insect and disease-resistance, drought-tolerance, nutrient-use efficiency, quality, yield potential and herbicide-tolerance. The prospects of developing transgenic rice are also discussed.     

Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

The gene of trichosanthin has been transferred into rice plants through agrobacterium method. The single copy insertion and the expression of foreign gene have been proved in regenerated plants. In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressingGUS gene as control have been evaluated. The differences such as the time of disease symptom observed, the number of infected plants and damaged leaves, the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant. The transgenic plants with trichosanthin gene grew faster than the plants withGUS gene, even when humidity environment was removed. The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control. In addition, no damage caused by the expression of trichosanthin gene in transgenic plants has been observed.     

Transformation of japonica rice with RHL gene and salt tolerance of the transgenic rice plant

Overexpression of the yeast HAL2 gene increases salt tolerance of yeast and plant. Rice HAL2-like (RHL) gene was introduced into a japonica rice cultivar HJ19 with Agrobacterium tumefaciens-mediated transformation. Transgenic plants in R0 generation were selected on the principle of GUS-positive, RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R1 generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R1 generation showed that the RHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

Overexpression of glutamine synthetases confers transgenic rice herbicide resistance

Glutamine synthetase (GS, E.C.6.3.1.2) is a key enzyme involved in the assimilation of inorganic nitrogen in higher plants and gram-negative microorganisms. GS is the targeting enzyme of a herbicide phosphinothricin (PPT) or Basta. In order to generate PPT-resistant transgenic rice via overexpression of GS, we constructed a plant expression vector p2GS harboring two different isoenzymes GS1 and GS2 cDNAs under the control of constitutive promoters of rice Act1 and maize Ubiquitin(Ubi) genes. The p2GS was introduced into rice genome by Agrobacterium-mediated transformation and confirmed by PCR and Southern blot hybridization. GS-transgene expression was first detected by Northern blot analyses. Results from Basta test indicated that GS-transgenic plants can tolerate as high as 0.3% Basta solution. In addition, our results also demonstrated that GS overexpression conferred transformed rice calli PPT resistance. Thus, GS cassette can serve as a selective marker gene instead of bar cassette for selection of PPT transformants.     

Preparation of 3D graphene-based architectures and their applications in supercapacitors

Three dimensional (3D) graphene-based architectures such as 3D graphene-based hydrogels, aerogels, foams, and sponges have attracted huge attention owing to the combination of the structural interconnectivities and the outstanding properties of graphene which offer these interesting structures with low density, high porosity, large surface area, stable mechanical properties, fast mass and electron transport. They have been extensively studied for a wide range of applications including capacitors, batteries, sensors, catalyst, etc. There are several reviews focusing on the 3D graphene-based architectures and their applications. In this work, we only summarise the latest development on the preparation of 3D graphene-based architectures and their applications in supercapacitors, with emphasis on the preparation strategies.     

Effect of HS2 and HS3 elements on erythroid-specific expression in transgenic mice

The expression plasmids CMV/GFP, HS2ALL, HS3ALL and HS23ALL were selected to investigate the effect of HS2 and HS3 element on erythroid-specific expression in transgenic mice. These plasmids were digested with restriction enzymes and purified. And five DNA fragments, CMV/GFP, HS2/GFP, CMV/HS2/GFP, HS23/GFP and HS3/GFP were obtained. After purification, the above DNA fragments were microinjected into the pre-nuclei of the mice fertilized eggs and transgenic mice were generated, with an integration rate of 10.89%. The green fluorescence protein(GFP) expression in many transgenic mouse tissues was determined by FACS analysis. The results showed that the HS2 and 1.7 kb of β-globin gene promoter were sufficient for the erythroid-specific expression of β-globin gene. The GFP expression of different recombinant constructs was also analyzed in blood of all the transgenic mice with FACS. The results indicated that HS2 and HS3 had the same enhancement activity on the regulation of β-globin gene expression. Moreover, these two elements showed a significant synergistic effect on gene expression at the transgenic mouse level, although the GFP expression varied largely among different transgenic mouse litters.     

水稻雄性不育突变体OsMS121的遗传及定位分析

通过射线诱变粳稻9522种子获得一株水稻雄性不育突变体OsMS121．遗传分析的结果显示突变体是单基因隐性突变．细胞学观察发现突变体花粉的萌发孔在发育过程中出现异常．萌发孔的塞子体积较小,且畸形．萌发孔的孢粉素层与野生型相比较为稀疏;环状突起不明显,结构松散,呈颗粒状．用图位克隆的方法将该基因定位在水稻第二条染色体分子标记R2M16—2和R2M18—1之间约200KB范围内．这些结果为该基因的克隆及其在花粉发育中的功能研究奠定了基础。     

转Bt基因水稻对稻田节肢动物群落的影响

以转crylAb/crylAc基因水稻汕优63(以下简称汕优63/Bt水稻)为材料,亲本汕优63(以下简称汕优63/CK水稻)为对照,在田间自然条件下研究了汕优63/Bt水稻对稻田节肢动物群落的影响.结果表明,白背飞虱Sogatella furcifera Horváth和黑尾叶蝉Nephotettix cincticeps Ubler为汕优63/Bt水稻上优势种.在整个调查期间,汕优63/Bt水稻田害虫和天敌亚群落物种丰富度、均匀度和多样性指数都高于汕优63/CK,而群落优势集中性低于汕优63/CK,但差异都不显著.汕优63/Bt水稻田害虫和天敌亚群落物种丰富度、均匀度和多样性指数消长动态变化均较汕优63/CK水稻平缓.本研究结果初步表明汕优63/Bt水稻田节肢动物群落的稳定性高于常规稻田,利于发挥自然天敌的调控作用.     

水稻S5-ORF3蛋白质的进化分析

生殖隔离在形成新物种和物种同一性的保持中有重要作用,因而在水稻优化育种中有重要的意义。S5位点是一个已克隆的控制水稻生殖隔离的基因,它产生的S5-ORF3,ORF4和ORF5蛋白质共同调控着indica-japonica杂交后代的育性,特别是S5-ORF3蛋白质在打破水稻亚种indica-japonica之间的生殖隔离与促进物种间的基因交流中发挥重要作用。针对S5-ORF3的进化分析对于研究它的功能和起源很重要,但目前尚无报道。本文基于序列和进化分析,提出S5-ORF3为一种定位在内质网中的新的HSP70家族蛋白,但缺乏HSP70的C端的多肽结合结构域,推测S5-ORF3可能通过影响alpha-淀粉酶的合成来作用于内质网压力;找到了ORF3的19条同源蛋白,并用极大似然算法构建了可靠的进化树,发现水稻的S5-ORF3与节节麦的luminal-binding蛋白进化关系最为密切;作了置信度100%的S5-ORF3蛋白质的三维结构预测,预测了居中的配体绑定位点;发现了水稻的S5-ORF3与其他HSP70蛋白相比独特的基序,为进一步研究S5-ORF3的作用机理和演化历史提供了线索和数据支持。     

Inheritance and expression of multiple disease and insect resistance genes in transgenic rice

2—3 anti-fungal disease genes are coinserted with hygromycin phosphotransferase in the same vector. Two insecticidal genes and PPT acetyl transferase genes are placed in another one. The vectors are co-delivered to rice embryonic cellus tissue at a molar ratio of 1︰1 using the particle gun method. 55 independent regenerated lines have been obtained through screening for hygromycin resistance. Of these, 70% transgenic plants harbor 6—7 foreign genes. The genes on the same vectors are always co-delivered to rice plant. Northern blot analysis has indicated that the multiple foreign genes give stable expression. In the 6 transgenic plants carrying 6—7 foreign genes, multiple foreign genes tend to integrate in 1 or 2 genetic loci. Progeny segregation is consistent with Mendel’s 3︰1 segregation law. 8 homozygous R₁ transgenic plants harboring 2—3 anti-fungal and 2 insecticidal genes are selected from large number of transgenic progeny screening for hygromycin and Basta resistance.     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS) alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin α from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3′ end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS} alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin a from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3' end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

Expression analysis ofgdcsP promoter from C3-C4 intermediate plantFlaveria anomala in transgenic rice

ThegdcsP promoter isolated from C₃-C₄ intermediate plantFlaveria anomala was fused to the β-glucuronidase (GUS) gene. The chimeric gene was inserted into the binary vector pBin19 and introduced into the rice (Oryza sativa L.) cv. 8706 byAgrobacteriummediated gene transfer. GUS activity can be detected in leaf, leaf sheath, stem and root tissues via fluorometric GUS assay. However, no GUS activity was found in mature endosperm. Histochemical localization revealed that GUS expression was exclusively restricted to vascular tissues in transgenic plants. This promoter also showed spatial-temporal expression patterns that GUS expression declined significantly with the maturity of plants. These expression patterns make thegdcsP promoter extremely valuable in the applied biotechnology that needs target gene expression restricted to vascular tissues.     

FISH analysis of the integration patterns in transgenic rice co-transformed by microprojectile bombardment

Using multi-color fluorescencein situ hybridization (FISH), we localized transferredbarnase-ps1 andpHctinG DNA sequences onto chromosomes of two transgenic rice plants, named Q12 and Q13, both of which were produced by micro-projectile bombardment. In both Q12 and Q13, each detected cell showed 2–3 signal spots on their chromosomes respectively. The signals of bothbarnase-ps1 andpHctinG were mostly detected in the adjacent chromosomal sites in which their signals were overlapped and could be recognized by the signal color on the metaphase chromosomes. Fiber FISH further demonstrated that the multiple copies in each of the two DNA sequences distributed adjacently on the DNA fiber in Q13. Combined with the results of Southern hybridization, the possible integration patterns in transgenic rice co-transformed by micro-projectile bombardment have been discussed.     

暗紫贝母植被分布格局的数值分析

通过相似系数法对暗紫贝母分布的群落进行了数值分类，确定了暗紫贝母分布的群落类型和群落特征．报道了暗紫贝母的生态分布、植被的种类组成及其区系分布．为川贝母的野生抚育和药材生产保护区的建立提出了依据．