Serotonin enrichment of rice endosperm by metabolic engineering

In animals,serotonin is a neurotransmitter and mood regulator.In plants,serotonin functions in energy acquisition,tissue maintenance,delay of senescence,and response to biotic and abiotic stresses.In this study,we examined the effect of serotonin enrichment of rice endosperm on plant growth,endosperm development,and grain quality.To do so,TDCs and T5H were selected as targets for serotonin fortification.Overexpression of TDC1 or TDC3 increased serotonin accumulation relative to overexpression of T5H in rice grain.Transgenic lines of target genes driven by the Gt1 promoter showed better field performance than those driven by the Ubi promoter.Overexpression of T5H showed little effect on plant growth or grain physicochemical quality.In neuronal cell culture assays,serotonin induced neuroprotective action against apoptosis.Breeding of rice cultivars with high serotonin content may be beneficial for health and nutrition.

Division of Free Nuclei in Rice Endosperm and Its Influencing Factors

The division process of endosperm in rice and its influencing factors were investigated. Total dissection, resin embedding and sectioning under light and transmission electron microscopes were used to observe the division patterns of free nuclei and cytological characters of an endosperm. The various division patterns of free nuclei in rice endosperm included mitosis, amitosis, and the mitosis to amitosis change without an interphase. The division velocity of free nuclei in rice endosperm varied with the internucleus distance. Comparing to a cluster, the division velocity increased when the free nuclei were separated from each other. Moderately higher temperatures also enhanced the proliferation and shortened the division cycle of free nuclei in rice endosperm. To a certain extent, nitrogenous fertilizer applied at the booting stage together with a moderate drought treatment accelerated the division of free nuclei as well.

OsPKpa_1 encodes a plastidic pyruvate kinase that affects starch biosynthesis in the rice endosperm

Pyruvate kinase （PK） is a key enzyme in glycolysis and carbon metabolism. Here, we isolated a rice （Oryza sativa） mutant, w59, with a white-core floury endosperm. Map-based cloning of w59 identified a mutation in OsPKpα1, which encodes a plastidic isoform of PK （PKp）. OsPKpα1 localizes to the amyloplast stroma in the developing endosperm, and the mutation of OsPKpα1 in w59 decreases the plastidic PK activity, resulting in dramatic changes to the lipid biosynthesis in seeds. The w59 grains were also characterized by a marked decrease in starch content. Consistent with a decrease in number and size of the w59 amyloplasts, large empty spaces were observed in the central region of the w59 endosperm, at the early grain-filling stage. Moreover, a phylogenetic analysis revealed four potential rice isoforms of OsPKp. We validated the in vitro PK activity of these OsPKps through reconstituting active PKp complexes derived from inactive individual OsPKps, revealing the heteromeric structure of rice PKps, which was further confirmed using a protein- protein interaction analysis. These findings suggest a functional connection between lipid and starch synthesis in rice endosperm amyloplasts.

Endosperm Development in Autotetraploid Rice

By using the laser scanning confocal microscope and plastic （Leica 7022 historesin embedding kit） semi-thin sectioning technique, comparative studies on the development of endosperm were carried out between autotetraploid and diploid rices. About one third of the ovaries in the autotetraploid showed normal endosperm development as those in the diploid. In these ovaries, one of the polar nuclei would fuse with the sperm nucleus, and the primary endosperm nucleus formed and underwent the first division in 4 hours after pollination; the anticlinal wall began to grow centripetally between the free nuclei starting from the wall ingrowths of the embryo sac near the micropylar end, and some of the phragmoplasts formed transformed into periclinal walls. In addition, some of the cell wall situated in the middle of the endosperm appeared to originate from phragmoplasts, whereas others seemed to develop randomly without the obvious formation of phragmoplasts. Cellulose began to accumulate in the wall of aleurone cell layer at 6 days after pollination. The cellulose wall of the cells of the aleurone cell layer appeared to have completely formed within 7 to 8 days after pollination. On the other hand, about two thirds of the ovaries in the autotetraploid showed abnormality in endosperm development with various types, such as non-fertilization, abnormal fertilization, endosperm development-delay and non-synchronization in the development of cellulose wall of cells of the aleurone layer. These abnormalities usually resulted in decreased seed setting in autotetraploid rice.

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-b＇ and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region （SCAR） marker tightly linked with Stv-b＇ and a cleaved amplified polymorphic sequence （CAPS） marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.

The Effect of High Temperature after Anthesis on Rice Quality and Starch Granule Structure of Endosperm

To ascertain the effect mechanism of high temperature after anthesis on rice quality, the experiment was conducted with two rice lines, the heat-tolerant line 996 and heat-sensitive line 4628, with high temperature and optimal temperature in the growth chamber to investigate the effect of high temperature stress after anthesis on rice appearance quality, milling quality, cooking and eating quality and starch granule structure of endo- sperm. The result showed that milled rice rate, head rice rate, amylose content and gel consistency of both lines decreased under high temperature stress after anthesis, while the ratio of grain length to width, chalky rate, chalkiness, protein content increased. Under high temperature treatments, gelatinization temperature, final viscosity, set back and peak time increased, breakdown decreased, Mg content and K content increased, Mg/K ra- tio decreased. Under same treatment, the extent of rice quality of heat tolerant line 996 affected by high temperature was lower than that of heat sensitive line 4628. Under high temperature stress after anthesis, starch granule arranged untightly, most of starch granules existed in the form of a single starch endosperm, refractive index decreased, transparency reduced, and led to the formation of chalk. Under high temperature stress af- ter anthesis, the increase of protein content, the decrease of Mg/K, the changes of rice RVA profile characteristics and starch granule structure of endosperm could be the main reason for the decrease of rice cooking and eating quality and appearance quality.

Studies on the Programmed Cell Death in Rice During Starchy Endosperm Development

Morphological variations of the nucleus in starchy endosperm cell were observed by theelectron-transmisson microscope during endosperm development in rice. Along with thedevelopment of the starchy endosperm, the nuclei of the cells showed chromatin condensation,the typical feature of programmed cell death (PCD). The nuclei also showed nucleusdeformation, disruption of nuclear envelope, nucleoplasm leaking into the cytoplasm andnucleus disintegration resulting in nuclear residue formation. From the nucleus deformationto the nucleus disintegration, the morphological changes of the nucleus were orderlyprogressive. This indicated that the cell death of starchy endosperm in rice wasprogrammed cell death. Evans Blue staining observation showed that the cell death wasinitially detected in the central part of starchy endosperm in rice, then expandedoutward. The activities of superoxide dismutase (SOD) and catalase (CAT) in rice starchyendosperm both descended continuously as development progressed. The analysis of DNA ofrice starchy endosperm did not show the presence of DNA laddering. The above resultsshowed that the cell death of starchy endosperm in rice was a special form of PCD.

Chalkiness Characters and Scanning Electron Microscope Observation of Rice Grain Endosperm of Japonica Varieties in Southern Henan

Chalkiness characters affect not only the grain appearance,milling,eating and cooking qualities but also the grain nutritional quality in rice,thus it is one of the most important traits in rice. It is very important for us to investigate the relation of the chalkiness formation and the development of endosperm structure and starch granule of different rice varieties. Here,we have investigated the chalkiness characters such as chalkiness rate,chalkiness degree and chalkiness area in 15 japonica rice varieties from southern Henan. Furthermore,the endosperm structure and starch granules of rice grain were also observed with scanning electron microscope. The results showed that the 15 japonica varieties have a significantly linear relationship between the chalkiness rate and chalkiness degree. Among the varieties,the biggest difference is the chalkiness rate,the second is the chalkiness area,and the last is the chalkiness degree. Moreover,there is a certain correlation between the distribution of starch granules,the arrangement of endosperm cells and the occurrence of grain chalkiness in the different rice varieties. For the same variety,the starch granules of chalky and non-chalky grains have obvious difference,while the starch granules from the transparent part of chalky rice and non-chalky rice do not have significant difference. The results would provide useful references for the improvement of grain quality in rice.

A Cleaved Amplified Polymorphic Sequence Marker to Detect Variation in Wx Locus Conditioning Translucent Endosperm in Rice

The translucent endosperm trait in a japonica rice variety ＇Kantou 194＇ is controlled by a Wx-mq gene which is allelic to Wx locus by genetic analysis and allelic test. The amylose content analysis showed that an intermediate amylose content between those of glutinous and non-glutinous rice existed in endosperm of homozygous Wx-mq genotype. The slight changes of amylose content in different varieties and F1 grains with an identical Wx-mq genotype might be influenced by dissimilar genetic background. To identify the Wx-mq genotype simply and rapidly, a cleaved amplified polymorphic sequence （CAPS） marker was designed. The result from the molecular detection indicated that it could be used for marker-assisted selection for low amylose content varieties in rice breeding.

Phenotype of Rice Floury Endosperm Mutant flo7 and Fine Mapping of Mutated Gene

The major storage substance in rice endosperm is starch, which accounts for 80% of dry matter weight. In this study, rice mutant flo7, selected from the progeny of Nipponbare＇s tissue culture, displayed floury and opaque endosperm. Compared with its corresponding wild type （WT） Nipponbare, the mutant rio7 produced longer, narrower, thinner and lighter grains. The levels of glucose, fructose and sucrose in the mutant flo7 endosperm were higher than those in the WT endosperm, whereas the protein content was not affected. With respect to both amylose content and gel consistency, the mutant flo7 was lower than WT, but its alkali value was higher. Scanning electron microscopic examinations showed that the endosperm of the mutant flo7 contained irregular, loosely packed and compound starch granules. Genetic analysis indicated that the mutant phenotype was determined by a single recessive nuclear gene The flo7 locus was mapped to a region on the long arm of chromosome 12, within a 95.1 kb interval defined by the markers C2-11 and C5-15. There are 13 open reading frames in the mapping interval. Transcription profiling of the developing grains showed that a number of genes involved in starch synthesis were affected differently in the mutant flo7.

Quantitative Trait Loci for Grain Chalkiness and Endosperm Transparency Detected in Three Recombinant Inbred Line Populations of Indica Rice

Quantitative trait loci（QTL） for percentage of chalky grain,degree of chalkiness,and endosperm transparency were detected using 3 recombinant inbred line populations derived from crosses between parental lines of commercial three-line hybrids of indica rice.Two of the populations showed great variations on heading date,and the other had a short range of heading date variation.A total of 40 QTLs were detected and fell into 15 regions of 10 chromosomes,of which 5 regions were detected for 1 or more same traits over different populations,2 were detected for different traits in different populations,3 were detected for 2 or all the 3 traits in a single population,and 5 were detected for a single trait in a single population.Most of these QTLs have been reported previously,but a region located on the long arm of chromosome 10 showing significant effects in all the 3 populations has not been reported before.It was shown that a number of gene cloned,including the Wx and Alk for the physiochemical property of rice grain,and GW2,GS3 and GW5 for grain weight and grain size,could have played important roles for the genetic control of grain chalkiness in rice,but there are many more QTLs exerting stable effects for rice chalkiness over different genetic backgrounds.It is worth paying more attentions to these regions which harbor QTL such as the qPCG5.2/qDC5.2/qET5.2 and qPCG10/qDC10/qET10 detected in our study.Our results also showed that the use of segregating populations having high-uniform heading date could greatly increase the efficiency of the identification of QTL responsible for traits that are subjected to great environmental influence.

高温胁迫下水稻胚乳磷酸化与泛素化蛋白质组学联合分析

水稻胚乳的代谢依赖于多个翻译后修饰信号网络的精确调控,包括磷酸化和泛素化修饰。本研究通过磷酸化与泛素化蛋白质组学的联合分析,发现水稻胚乳中有143个蛋白质同时具有磷酸化和泛素化位点,这为研究磷酸化和泛素化修饰之间的串扰机制提供了组学基础。一方面,磷酸化修饰通过影响泛素-蛋白酶体系统中的关键蛋白来调控泛素化修饰;另一方面,泛素化修饰通过影响蛋白激酶和蛋白磷酸酶来间接调控植物体内的磷酸化信号网络。高温胁迫下,磷酸化和泛素化修饰协同调控酚类化合物的合成通路,并最终影响酚类化合物清除活性氧的能力以及植物在逆境中的抗性。多种淀粉代谢相关酶同时具有磷酸化和泛素化修饰,并且有18对具有不同修饰类型的位点出现在同一蛋白质的相邻位置,揭示了不同修饰之间的串扰机制以及高温胁迫中磷酸化与泛素化修饰对淀粉代谢的协同调节作用。

H5N1亚型禽流感病毒HA蛋白在水稻胚乳中的表达及其纯化

为制备H5N1亚型禽流感病毒HA蛋白并评估其免疫原性,利用水稻表达系统表达H5N1亚型禽流感病毒重组HA蛋白。首先构建了重组植物表达载体pCAMBIA1300-HA,该载体具有GT13特有启动子、信号肽SP和终止子,有利于外源基因在水稻中表达。之后将重组质粒pCAMBIA1300-HA通过电转化法导入根癌农杆菌EHA105中,筛选出阳性菌落并侵染水稻愈伤组织。经过暗培养、潮霉素筛选、分化、生根、成苗,采用CTAB法提取水稻叶片DNA,PCR鉴定结果显示,HA基因已插入到水稻基因组中,重组HA基因大小为4660 bp。将阳性植株移植到大田中,4个月后收获水稻种子,提取水稻胚乳蛋白,Western blot鉴定结果表明,HA蛋白在水稻胚乳中成功表达。重组HA蛋白通过Q阴离子层析、疏水层析和凝胶过滤层析三步分离纯化,其纯度达到90%以上。最后将纯化后的重组HA蛋白与ISA 50V佐剂混合并乳化制成疫苗,免疫小鼠,小鼠血清具有较高的特异性抗体水平,表明重组HA蛋白免疫原性较好。综上,成功构建了H5N1亚型禽流感病毒HA重组蛋白的水稻表达系统,并分离纯化获得了高纯度和免疫原性良好的重组HA蛋白。

Analysis of Low Amylose and Processability Fractured Endosperms Derived from Somatic Variation

Recently, improving eating quality and processing properties has become one of the most important objectives in japonica rice breeding programs in Korea. This study was carried out to determine the agronomy and physicochemical characteristics of the opaque endosperm rice "S-21-3-8" regenerated from seed-derived callus culture of a rice cultivar, "Ilpum". S3 generation of opaque endosperm mutants selected from pedigree breeding was used for analysis of agronomic and physicochemical traits. Genetic segregation was observed at the highest frequency among opaque endosperm mutants, being present in 85.7% (12/14 lines) of the entire opaque lines. However, the major agricultural characteristics and grain traits of "S-21-3-8" were similar to those of a donor cultivar, "Ilpum". "S-21-3-8" showed significantly lower (10.6%) amylose than those (17.7%) of "Ilpum" in brown rice, while the protein levels were similar to those of the donor plant. The grain hardness of "S-21-3-8" (1.67 kgf/mm2) was lower than that of "Ilpum" (1.97 kgf/mm2), resulting in a high flour-milling percentage. The loosely packed starch granules of "S-21-3-8" in the opaque endosperm were demonstrated by SEM analysis of cross-sectioned rice grains. The opaque endosperm mutants that were of somaclonal variations in the tissue culture will lead to improved eating quality and processing properties of rice.

水稻肽转运蛋白基因OsPtr1的功能分析

【目的】利用T-DNA插入突变的方法揭示候选基因P0421H01.23(命名为OsPtr1,Peptite 24-48Transporter1)的功能表达。【方法】提取水稻明恢86成花后12 d的胚乳RNA,反转录成cDNA后,进行OsPtr1基因扩增,以胚乳特异表达Gt1为启动子,构建OsPtr1基因植物过表达载体,用农杆菌介导法将OsPtr1基因导入水稻品种日本晴,对其后代及突变体w9101进行氮代谢物测定分析;同时,构建绿色荧光蛋白基因GFP和OsPtr1基因融合表达载体,用基因枪转入洋葱表皮细胞,培养24~48 h后,用荧光共聚焦电子显微镜观察OsPtr1基因亚细胞定位。【结果】OsPtr1基因编码蛋白定位于质膜上,该基因与氮类物质的运输有关,OsPtr1功能缺失突变体种子的氨基酸和蛋白质等含氮物质积累效率降低,过表达植株种子的氨基酸和蛋白质等含氮物质含量增加。【结论】OsPtr1基因参与水稻氮类物质的跨膜运输。

Optical Sum Frequency Generation Image of Rice Grains

We observed optical sum frequency generation (SFG) images of cross-sections of glutinous rice grains, in order to test a possibility of the SFG microscopy as a tool for monitoring polysaccharide species in rice grains. The SFG response in the CH vibration range was the most intense in the crush cell layer at the edge of the endosperm adjacent to the embryo probably due to optical reflection and scattering effectby the rugged dielectric structure of the crush cell layer. The SFG spectra as a function of the infrared wavelength depended on the measurement position in the endosperm. The SFG results were compared with those by Raman and infrared spectroscopies for the same samples.

水稻调控淀粉合成基因的研究进展

水稻是全球重要的粮食作物之一,近年来随着人们生活质量提升,对稻米的品质逐渐重视。胚乳是稻米的主要组成成分,为种子萌发和种胚发育提供能量,其中淀粉含量约占水稻种子干物质积累的80%,研究淀粉生物合成的分子机制对水稻品质改良具有重要理论意义与应用价值。虽然淀粉的基本合成途径已比较清晰,但是大田条件下淀粉的合成是一个受遗传和环境条件共同决定的复杂生物学过程。由于表型鉴定相对困难,很难通过QTL等方法对稻米品质的影响因子进行图位克隆。通过物理化学诱变获得的淀粉合成缺陷突变体多为单基因控制,这些突变体由于淀粉颗粒形态改变或填充不紧密,通常表现为胚乳粉质的表型,加代纯合后构建群体可对突变基因进行图位克隆。近年来,利用突变体克隆的新调控因子逐渐增多,参与合成的路径多样化,充实完善了淀粉合成的调控网络。本研究通过对近年来的此类突变体进行概括综述,探讨影响淀粉合成的不同调控因子类型及代谢通路,以期对水稻品质改良提供参考。

极良食味粳型软米胚乳成分特征分析

中国稻作目前已经由高产型逐渐向质量效益型转变,对于消费者来说,稻米食味是稻米品质的核心。软米总体具有较高的食味,但软米品种间还存在食味差异,遴选食味更加优良的软米品种具有重要意义。采用稻米食味感官品尝、饭食味计及快速黏度分析仪分析大米样品的食味特性,并比较大米样品胚乳性状组成及米饭显微特征,以明确几个软米新材料的食味特性。结果表明:软米感官品尝食味综合评价显著优于其他材料,100分制评分在90分以上,达到极良水平。其快速黏度分析仪特征值的峰值黏度、崩解值显著高,最低黏度、最终黏度和消减值、峰值时间显著低;胚乳性状表现为直链淀粉含量、支链淀粉长链显著低,其上限可能分别在13%、15%,清蛋白含量显著高,甜味氨基酸和苦味氨基酸总含量偏高,样品间可溶性糖含量差异显著,但极良食味品种没有表现出显著多。极良食味品种米饭显微图像自表及里,表现出充分糊化的网孔状特征,而不良食味品种则表现为没有充分糊化的熔岩状特点。多元回归分析表明,食味性状是由胚乳特性综合影响导致。综上,本研究的软米材料食味明显优良,具有显著的快速黏度分析仪特征值和胚乳成分含量特征。

胚乳蛋白质对稻米品质影响的研究进展

蛋白质是稻米胚乳中仅次于淀粉的第二大贮藏物质,蛋白质含量、组分、形态以及分布特征对稻米品质均具有重要影响。本文梳理了胚乳蛋白质与稻米外观品质、加工品质、营养品质和蒸煮食味品质关系的研究进展,并对未来研究方向提出了展望。结果可为进一步解析胚乳蛋白质在稻米品质形成中的作用和稻米品质改良提供参考。

植物表达外源蛋白研究进展及展望

植物为宿主表达外源蛋白的系统称之为分子农场,通过农杆菌将外源基因导入植物进行表达具有高效、安全、廉价的优点,特别是植物具备一系列翻译后修饰功能,因此该系统能弥补原核表达系统的缺陷。本综述首先介绍了近些年在烟草叶片瞬时表达和水稻胚乳组织特异性表达上取得的进展,特别是一些利用分子农场进行医用蛋白表达、药物合成、疫苗制备等典型案例。在优化生物反应器、提高表达效率策略上,本综述重点探讨了蛋白翻译后水平上的调控,包括蛋白酶抑制剂的作用、糖基化修饰环节以及分子伴侣共表达等对外源蛋白表达的影响。最后,围绕外源蛋白大量囤积于内质网可能引发内质网胁迫的问题,展望了通过优化内质网环境来提高外源蛋白表达效率的可行性。