The hypocholesterolemic activity of transgenic rice seed accumulating lactostatin, a bioactive peptide derived from bovine milk β-lactoglobulin

Lactostatin is a novel pentapeptide (IIAEK) derived from bovine milk β-lactoglobulin with greater hypocholesterolemic activity than β-sitosterol, the drug commonly used to treat hypercholesterolemia. We developed transgenic rice expressing lactostatin as a fusion protein with seed storage protein (SSP) glutelins under the control of three different endosperm-specific promoters. Lactostatin accumulated in transgenic rice seed at approximately 1.6 mg/g seeds (dry seeds) without any apparent influence on seed traits such as endogenous SSP expression levels or alterations in the intracellular structures of endosperm cells. Short-term (three day) oral administration of the glutelin fraction containing lactostatin (namely three times of 300 mg/kg body weight/day) extracted from transgenic rice seeds resulted in hypocholesterolemic activity in rats; namely, the serum low-density-lipoprotein (LDL) cholesterol level was significantly reduced accompanied by a significant increase in beneficial serum high-density-lipoprotein (HDL) cholesterol.     

Nitrogen Fertilizer Increases Seed Protein and Milling Quality of Rice

Rice grain breakage during milling is a problem in many parts of Asia. It has been suggested that nitrogen (N) fertilizer can improve the milling quality of rice. Therefore, this study investigates effects of N fertilization on grain N concentration, endosperm storage protein distribution, and milling quality of rice. Four Thai extra long grain commercial rice cultivars (KDML105, KLG1, PTT1, and CNT1) were grown at Chiang Mai University in the wet season of 2001 with 0 or 120 kg of N/ha at flowering. Anatomical sections showed that there was more storage protein accumulated in the lateral regions of polished grain of high N concentration than in grain of low N concentration. Percent (%) unbroken rice was positively correlated with relative abundance of storage protein in the lateral region of the endosperm in all cultivars. Applying N increased head rice N concentration in all cultivars, whereas % unbroken rice was increased in KLG1 and CNT1. KDML105 cultivar, on the other hand, already had high % unbroken rice and more abundant storage protein in the lateral region with the grain of low N concentration. It is hypothesized that high density of storage protein in the lateral region of the endosperm provides resilience and lessens grain breakage during milling. The additional protein may increase hardness in rice grains and thus could make the rice more resistant to breakage during milling. Furthermore, N fertilization may enhance the nutritional quality of rice grain by increasing the glutelin content, which is rich in lysine.     

Characterization of Oat Endoproteinases that Hydrolyze Oat Globulins

During the germination of oats, the major seed storage proteins (globulins) are hydrolyzed by endoproteinases. We have used two methods to characterize these endoproteinases. A qualitative PAGE method that used oat globulins as gel‐incorporated substrates was used to determine which enzymes hydrolyzed the globulins. The proteolytic hydrolysis products were studied by hydrolyzing the globulins in vitro with the endoproteinases and analyzing the products by SDS‐PAGE. Class‐specific proteinase inhibitors were used to show that the globulin hydrolyzing enzymes were cysteine‐class proteinases. The proteinases were active at pH 3.8. Using the gel analysis method, a little activity was present at the beginning of seed germination, but the major activity only appeared on the sixth day of germination. Extracts from four‐day germinated oats contained cysteine proteinases that hydrolyzed the globulins in vitro to form a polypeptide of intermediate size (MW ≈34,500). Cysteine proteases from an eight‐day germinated sample totally hydrolyzed the globulins in <1 hr. Very little hydrolysis occurred at pH 6.2, the pH of germinated oats endosperm tissue. The fact that hydrolysis occurred quickly at pH 3.8 implies that there is probably pH compartmentalization within the endosperm, with some areas of the seed having a low pH value where the globulins can be degraded.     

Influence of Rice Proteins on Eating Quality of Cooked Rice and on Aroma and Flavor of Sake

An improved method for the extraction of storage proteins from rice endosperm under conditions safe for producing food was developed. The contribution of the protein extracts to the eating quality of cooked rice and to the aroma and flavor of sake was examined. Sensory analysis was performed to evaluate the eating quality of cooked rice enriched with the protein extracts. Prolamin‐enrichment increased the hardness of cooked rice, and glutelin‐enrichment degraded the appearance of cooked rice. Physical analyses confirmed that prolamin‐enrichment changed, whereas the glutelin‐enrichment did not change the physical properties of cooked rice. Light and scanning electron microscopy of rice protein extracts revealed small particles of the prolamin extract and larger aggregated particles of the glutelin extract; these features remained after heating. The aroma and flavor of sake were negatively affected by the addition of the protein extracts. Especially, addition of prolamin significantly lowered the evaluation score of the aroma obtained by sensory analysis.     

Ca2+对苯丙烯酸胁迫下黄瓜种子萌发特性的影响

以黄瓜（Cucumis Sativus L.）种子为材料,研究钙浸种对苯丙烯酸（CA）胁迫下黄瓜种子萌发的影响及其机理。结果表明,CA对黄瓜种子萌发有明显的抑制作用,且抑制程度随CA浓度（0.25~1.0mmol·L-1）的增加而提高。CA胁迫下,黄瓜种子的发芽势、发芽率下降,膜结构受损,透性增大,呼吸速率、α-淀粉酶和蛋白酶活性降低,淀粉、蛋白质消耗率下降,钙浸种（500 mg·L-1CaCl2）可以缓解CA对种子萌发的抑制作用,表现为发芽率提高,膜透性降低,呼吸速率、α-淀粉酶、蛋白酶活性以及淀粉和蛋白质消耗率提高。     

Degradation of gluten by proteases from dry and germinating wheat (Triticum durum) seeds: An in vitro approach to storage protein mobilization

Vital gluten was used as an ideal substrate to investigate the role of some proteases in storage protein degradation. Aspartic proteinase and carboxypeptidase were identified as endogenous enzymes adsorbed on gluten and their optimum pH values determined. SDS-PAGE of soluble products released by gluten digestion revealed that the activity of these proteases plays a minor role in protein mobilization, whereas cysteine proteinase, purified from wheat seeds at the fourth day of germination, is extremely effective, producing a remarkable protein degradation in short times. Synergistic effects of aspartic and cysteine proteinase were not observed. Spin labeling of the sulfhydryl groups of gluten proteins enabled a comparative EPR investigation of the consequences of proteolytic degradation on gluten elasticity. It was found that storage protein mobilization brings a loss of elasticity to the polymeric network of gluten, which is particularly marked when the hydrolysis is performed by cysteine proteinase.     

海藻糖对转C4型PEPC水稻种子萌发耐旱性的影响

为揭示海藻糖参与高表达转玉米C₄型磷酸烯醇式丙酮酸羧化酶(PEPC)基因(C₄-PEPC)水稻(Oryza sativa)(简称PC)在干旱胁迫下种子萌发的生理机制,本研究以PC及其未转基因野生型受体Kitaake(简称WT)种子为材料,研究外施不同浓度海藻糖联合干旱处理下,其种子活力、萌发过程中可溶性糖和脯氨酸含量、α-淀粉酶活性,以及α-淀粉酶、PEPC、糖信号、部分海藻糖相关基因的表达。结果表明,干旱处理均显著抑制了两材料的发芽率,并且抑制了芽的生长;外施低浓度海藻糖可缓解干旱对种子萌发的抑制,与WT相比,PC对海藻糖更加敏感,当海藻糖浓度为0.5 mmol·L^-1时已表现出明显的缓解效应,且较10 mmol·L^-1海藻糖对WT的缓解效果更佳。外施0.5 mmol·L^-1海藻糖联合干旱处理可以维持水稻的种子活力、种子内渗透调节物质含量以及α-淀粉酶活性,尤其有益于PC。外施0.5 mmol·L^-1海藻糖通过上调PC内依赖钙信号的CBL1-OsSnRK3.1/3.23基因的表达,激活OsK1a-OsMYBS1/2-OsAmy3/8途径;同时诱导OsTPP1/7合成海藻糖,上调蔗糖和葡萄糖含量,进而增强糖代谢,维持种子萌发。此外,海藻糖也上调了SAPK8/9/10和C₄-PEPC的转录水平,使PC在芽期表现耐旱。本研究结果为改善直播稻田的发芽率和“C₄稻”的创制提供了新线索。     

Amino acid variation in the 10 kDa Oryza prolamin seed storage protein

The deduced amino acid variability for the 10 kDa prolamin was determined for 16 Oryza species, both cultivated (rice) and wild. Prolamin, a seed storage protein and site of nitrogen and sulfur accumulation, is sequestered in the subaleurone layer of the starchy endosperm for use during seedling germination. The 10 kDa prolamin amino acid distribution for the cultivated species (O. sativa and O. glaberrima) was determined and compared to those of wild and, hitherto unknown, noncultivated Oryza species. Four wild species (O. granulata, O. australiensis, O. brachyantha, and O. meyeriana) exhibited the greatest residue heterogeneity in both the signal and mature peptide regions. A breakdown of the essential amino acid variance among three Central/South American and one African endemic wild species is also presented and compared with those of rice.     

Identification and variation of glutelin alpha polypeptides in the genus Oryza assessed by two-dimensional electrophoresis and step-by-step immunodetection

To obtain fundamental information for nutritional improvement of rice (Oryza sativa) seed proteins, the alpha polypeptides of the major storage protein glutelin varied over the genus Oryza were qualitatively and quantitatively characterized with unique methods. The polypeptides were maximally separated by two-dimensional electrophoresis (2D-PAGE) composed of nonequilibrium pH gradient gel electrophoresis (NEPHGE) and higher temperature SDS-PAGE. Then the subunit for each polypeptide spot was identified with the sequential immunodetection called a step-by-step detection method, making use of highly subunit-specific antibodies. The comparative analysis showed considerable variation in the accumulation level of A-type and B-type glutelin subunits and found unknown glutelin subunits that were unable to be identified with the antibodies used. Wild species accumulating a high amount of lysine-rich B-type glutelin subunits and unknown unique subunits were identified as they might play a crucial role in nutritional quality improvement of the cultivated rice.     

Bowman-Brik胰蛋白酶抑制剂在水稻叶片生长和种子萌发过程中的表达

Bowman-Birk胰蛋白酶抑制剂(BBTI)是一类富含半胱氨酸的丝氨酸蛋白酶抑制剂,广泛分布于豆科、禾本科和菊科植物中,在蛋白质存储和植物防御中具有重要作用。水稻(Oryza sativa L.ssp.indica)BBTI家族具有13个成员,其中BBTI-9和BBTI-10都为假基因。其余BBTI成员都存在数量不同的保守Bowman-Birk结构域。为了解BBTI的表达信息,本研究主要通过设计多肽序列的方法,制备了11个BBTI的多克隆抗体,利用Western blot技术检测其在叶片生长发育和种子萌发过程中的表达。结果表明BBTI在叶片生长过程中呈现不同的时空特异性表达,BBTI-1、-2、-11、-12和-13伴随叶片生长含量增加,BBTI-5、-6和-8在苗期表达含量高,而BBTI-3、-4、-7在苗早期1cm时表达量达到峰值,以修饰或多聚体的形式在叶片生长过程中表达。在种子萌发过程中发现BBTI-8在种子中表达量较高,萌发后2h降到最低,24h后一条分子量略小的降解带开始表达,其余抗体在种子萌发阶段未发现有明显的变化。本研究还统计了苗期叶片、分蘖期叶片和萌发期的种子的MPSStags数据,试图比较转录与蛋白质水平对应关系。本研究结果证明,BBTI蛋白质在水稻生长发育过程中存在差异表达,为深入了解其功能提供了重要线索。     

Effect of acetic acid added to cooking water on the dissolution of proteins and activation of protease in rice

The effect of acetic acid on the dissolution of proteins in rice was studied to elucidate the mechanism for the textural change induced by the acid by chemical and SDS-PAGE analyses of the rice proteins in the soaking solution. More proteins were extracted with 0.2 M acetic acid (pH 2.7) than with water (pH 6.8). The effect of acetic acid on the protein dissolution increased with increasing temperature. Immunoblotting confirmed that, when rice was soaked in acetic acid, glutelin was dissolved into the soaking solution and degraded by aspartic proteinase. Aspartic proteinase degraded glutelin much more than it did albumin and globulin. It was found that the combined amount of albumin and globulin dissolved into the acetic acid solution was much larger than that of glutelin, despite the smaller amounts present of albumin and globulin than of glutelin. Metal ions were extracted more with acetic acid than with water. In addition, carboxypeptidase was activated under the acidic condition and resulted in an increase in the amount of free amino acids. The main effect of acetic acid on the dissolution of rice proteins was enhancement of the solubility of albumin, globulin, and glutelin, the effect of proteases being minor.     

盐胁迫下两种基因型冬小麦萌发过程中钠离子吸收及对碳、氮代谢的影响

采用两种基因型冬小麦种子为试验材料,研究了盐胁迫对小麦种子萌发过程中Na 的吸收和累积及其对C、N代谢的影响及外源Ca(NO3)2和CaCl2对小麦萌发过程中盐害的缓解效应。结果表明,150mmol/L NaCl胁迫明显抑制小麦种子的萌发。种子萌发过程中,"小偃6号"的耐盐性优于"陕229"。外源钙对两种小麦盐胁迫效应的缓解作用不同。6mmol/L钙离子可明显减轻"陕229"的盐害作用,对"小偃6号"作用不大。盐胁迫对种子萌发过程中的蛋白酶和淀粉酶活性具有明显抑制作用。盐胁迫下,"小偃6号"种子的蛋白酶和淀粉酶活性明显高于"陕229"。钙离子对盐胁迫下小麦萌发过程胚乳内蛋白酶的活性有较大调节作用,对淀粉酶活性影响较小。盐胁迫下小麦种子中Na 含量显著增加,K/Na比显著降低。钙离子对盐胁迫下小麦种子中Na 累积的影响较大,盐胁迫和外源钙处理对K 含量影响较小。     

Antioxidants,free radicals,storage proteins,and proteolytic activities in wheat (Triticum durum) seeds during accelerated aging

Accelerated aging was performed by incubation of wheat seeds at 40 degrees C and 100% relative humidity for 3, 4, 6, 10, and 14 days. The effects of the treatment on seed germinability and on several biochemical characteristics of flour (carotenoids, free radical and protein contents, and proteolytic activity) and gluten (free radical content and flexibility) were evaluated. A decrease of germinability was found during aging, the germination being completely inhibited after 14 days. The lutein content decreased gradually, without going to zero, while that of free radicals increased. A reduction of soluble proteins and a degradation of glutenins and gliadins were observed, associated with a substantial increase of protease activity and a decrease in gluten flexibility. The results were discussed in reference to those previously obtained by natural aging of wheat seeds of the same species and cultivar.     

水稻近等基因导入系芽期抗旱性鉴定及抗旱指标筛选

为筛选水稻抗旱性鉴定指标并科学鉴定水稻抗旱性,本研究以蒸馏水为对照,采用质量体积比为5％、10％、15％、20％的PEG-6000溶液处理6份川香29B近等基因导入系进行芽期抗旱鉴定,研究模拟干旱对水稻育种材料芽期种子萌发的影响,并探讨芽期抗旱性鉴定指标及筛选抗旱材料.结果表明,轻度水分胁迫对种子发芽抑制作用较小,部分...     

Proteolytic Enzymes in Germinating Rye Grains

The proteolytic activities during rye (Secale cereale L. ‘Humbolt’) grain germination were monitored using in‐solution methods and one‐ and two‐dimensional PAGE with gels that contained incorporated substrate proteins. The total proteolytic activity increased during the first three days of germination, but not after that. The proteinase activity was measured at pH 3.8, 6.0, and 8.0 in the presence and absence of class‐specific proteinase inhibitors. This indicated that enzymes from all four proteinase classes were present during the germination process. Germinated rye grain contained mainly aspartic and cysteine proteinase activities that are especially active at pH 3.8. Serine‐ and metallo‐proteinases were less abundant. Overall, the pattern of hydrolysis was very similar to that observed during barley and wheat germination.     

Effect of Excess Nickel on Starch Mobilization in Germinating Rice Grains

The effect of excess nickel (as NiSO₄) on starch mobilization of rice (Oryza sativa L.) grains or dehulled rice grains during germination was investigated. Excess NiSO₄ had no effect on starch content and α-amylase activity in endosperm of germinating rice grains or germinating dehulled rice grains. Evidence is provided to show that the hull is a barrier against influx of Ni²⁺ to endosperm; endosperm per se is less effective in Ni²⁺ uptake; and α-amylase extracted from the endosperm of germinating rice grains is highly resistant to excess Ni²⁺.     

Effects of supplemental‐nitrogen on the quality of rice proteins

A study was conducted on the effect of supplemental nitrogen (N) (20 hg/ha) applied as a foliar spray or to the soil on seed production, protein percentage, and protein fractions of rice. Plants were grown in a greenhouse over two different periods of time, i.e., August 1988 to January 1989 (Period I), and December 1988 to April 1989 (Period II). Nitrogen was applied to the leaves 10 and 20 days after anthesis (DAA), and to the soil at anthesis and at 15 DAA. Average temperature was 28.7°C during Period I and 32°C during Period II, corresponding to 18.7 and 22.0 growing degree‐day/day (GDD/day), respectively. The difference in GDD/day reduced the plant cycle from 130 days during Period I to 109 days during Period II. Plants grown during Period II had larger numbers of spikelets, a higher percentage of “full grown grains”;, and higher grain weight. Although percentage crude protein was about the same for the two periods, prolamin content was increased and the albumin+globulin fraction was decreased during Period II, but with no difference in glutelin content. The increase in number of spikelets, percent full grains, and grain weight appeared to result in a greater energy demand for plants grown during Period II. This may explain the increase in prolamins, since prolamin synthesis requires less energy than globulin or albumin synthesis. There was a simultaneous decrease in albumin and globulin synthesis during Period II. The content of glutelins, which represent the major reserve proteins in rice grains, was constant during both periods.     

籽粒黄熟度对杂交水稻种子活力的影响

为明确籽粒黄熟度对杂交水稻种子活力的影响,探究杂交水稻高活力种子适宜收获期的感官指标,于2016-2017年以深两优1813、隆两优1813、Y两优1128、创两优华占和Ⅱ优838为材料进行杂交水稻制种,通过不同收获时期形成籽粒黄熟度不同的群体,系统分析了不同籽粒黄熟度群体间种子活力、水分、千粒重及内含物的差异。结果表明,在不同品种间,籽粒黄熟度的增加速率存在明显差异。随着籽粒黄熟度的增加,种子活力先增加后趋于稳定;种子水分先降低后趋于稳定;种子千粒重逐渐增加,但差异不显著;种子的直链淀粉、总淀粉、蛋白质及可溶性糖含量均先增加后趋于稳定。当籽粒的黄熟度为75%~90%时,种子发芽率、发芽势、发芽指数和活力指数均处于高值水平,且差异不显著;种子水分和千粒重均未达到显著差异。相关性分析表明,籽粒黄熟度与种子发芽率、活力指数呈极显著正相关,相关系数分别为0.870 3^**和0.664 0^**;与种子水分呈极显著负相关(R²=-0.842 4^**),与种子千粒重相关性不显著(R²=0.197 2)。籽粒黄熟度与种子内含物均无显著相关性,但种子发芽势、发芽指数和活力指数均与种子内含物达到显著相关。综上,籽粒黄熟度为75%~90%可以作为杂交水稻高活力种子生产适宜收获期的感官指标。本研究结果为杂交水稻高活力种子生产提供了理论参考。     

Changes in Germination and Physiochemical Properties of Transgenic cry1Ab/cry1Ac Gene Rice During Long‐Term Storage

The changes in germination, free fatty acid (FFA), and viscosity of the transgenic Bacillus thuringiensis (Bt) rice expressing the cry1Ab/cry1Ac gene and its associated Bt protein were observed at three‐month intervals during 24 months of storage at ambient temperature and relative humidity. After nine months of storage, FFA of transgenic Bt rice was significantly lower than that of nontransgenic rice, while germination was significantly higher. Rates of change of FFA and germination in transgenic rice were lower than in nontransgenic rice. The content of Bt protein in the husk and in the brown rice both decreased 80% during 24 months of storage. Our observations suggest that transgenic Bt rice used in this study had long‐term storability.