粒用高粱glutelin基因的表达与进化分析

高粱glutelin是一种储藏蛋白,存在于水稻、玉米等禾谷类作物的籽粒中,是不溶于纯水、中性的盐溶液或酒精,但溶于稀碱或稀酸的蛋白质总称。为进一步研究高粱glutelin功能,改善籽粒品质提供基础,通过BLAST Phytozome数据库获得高粱glutelin的DNA和氨基酸序列数据,结合生物信息学方法鉴定到SbGLUA1(So⁃bic.001G143700)、SbGLUA2(Sobic.009G017600)、SbGLUB4(Sobic.009G007100)基因,并对高粱glutelin基因家族信息、基因结构、系统进化树、glutelin不同基因型各组织表达以及glutelin蛋白质理化性质、跨膜、二级结构、三级结构、蛋白互作关系进行分析。结果表明,glutelin蛋白大多不是分泌性蛋白,是亲水蛋白且流动性较差,SbGLUB4的二级结构主要是无规则卷曲和α-螺旋。glutelin蛋白结构域分析结果显示,大多蛋白较为保守;高粱glutelin与小麦谷蛋白同源性较高。SbGLUB4为种子专性表达,且随种子成熟度程度的增加而增加;各基因表达在品种间存在差异,IS22204较Laohanye略高;SbGLUA2在种子、穗等多个器官表达。STRING预测互作蛋白显示,SbGLUB4与多个种子储藏蛋白结构基因及调节基因(如MYB和bHLH)互作,而SbGLUA2与DNA解旋酶、重组酶等互作,推测可能是其功能发生了变化。

Micro-heterogeneity of Storage Glutelin in Rice Seed

The storage glutelin in rice ( Oryza sativa L.) seeds could be separated at least into more than 13 acidic and 19 basic polypeptides by two dimensional electrophoresis. Rice glutelin might be mainly controlled by about six major genes according to the expression of glutelin polypeptides. The acidic polypeptide of glutelin could be clearly separated into two groups by peptide mapping and N-terminal amino acid sequence analysis. These two groups were just in accord with the GluA and GluB subfamilies exactly.

Fine mapping and marker-assisted selection (MAS) of a low glutelin content gene in rice

Rice with low glutelin content is suitable as functional food for patients affected with diabetes and kidney failure. The fine mapping of the gene(s) responsible for low glutelin content will provide information regarding the distribution of glutelin related genes in rice genome and will generate markers for the selection of low glutelin rice varieties. Following an SDS-PAGE screen of rice germplasm from Taihu Valley of China, Japonica selection W3660 is identified to be a novel mutant characterized with low glutelin content. For fine mapping the mutant gene for low glutelin content, F2 and F3 populations were derived from a cross between W3660 and Jingrennuo. SDS-PAGE analysis of the total endosperm protein showed that the low glutelin content trait was controlled by a single dominant nuclear gene. Genetic mapping, using SSRs, located this gene to chromosome 2, in the region between SSR2-001/SSR2-004 and RM1358. The dis- tances of the two markers to the target gene were 1.1 cM and 3.8 cM respectively. By semi-quantitative RT-PCR analysis, the transcripts of GluB4/GluB5 genes located within the region do not change. However, GluB5 gene located proximal to SSR2-001/SSR2-004 was specifically reduced. SSR profiles of seven Japonica varieties were compared with that of W3660 for loci in the relevant genetic region. The markers SSR2-004 and RM1358 were used for marker- assisted selection. The selection efficiencies of SSR2-004 and RM1358 were 96.8% and 92.7% respectively. This provides a standard starting point for the breeding of low glutelin content rice varieties in China.

Status and Perspectives on the Researches of Rice Glutelin Mutants

Rice (Oryza sativa L. ) is one of the model plants for genomics research. As the raising of functional rice breeding for special usage, glutelin mutants play a more and more important role in the functional rice breeding as well as eukaryotic gene expression and regulation research materials. For example, the rice cultivar special for the patients suffering from kidney disease and diabetes could be developed from the rice glutelin mutants. In this paper, current researches on characterization, mutation mechanism and breeding usage of various rice glutelin mutants, especially the low glutelin content cultivars, were all discussed with perspectives on the trends of the glutelin mutant researches in the era of post-genomics.

Brassinosteroid and gibberellin coordinate rice seed germination and embryo growth by regulating glutelin mobilization

Seed germination is the beginning of a new lifecycle,and involves many complex physiological and biochemical reactions including seed reserve mobilization in the endosperm and nutrient transport and reuse in the embryo.Although glutelin is a dominant storage protein in rice,its contribution to seed germination and its regulatory mechanisms are mostly unknown.Gibberellin (GA) and brassinosteroid (BR),two major growth-promoting phytohormones,also play positive roles in controlling seed germination.However,how GA and BR interact and coordinate seed germination and facilitate glutelin mobilization remains unclear.In the present study,biochemical and physiological analyses of seed germination indicated that both GA and BR promote seed germination and post-germination growth.Exogenous application of GA restored germination defects caused by BR deficiency or insensitivity.Proteomic and q RT-PCR results showed that the expression of several glutelin proteins and their encoding genes was induced by BR and GA in the embryo.Expression assays suggested that the increased accumulation of glutelin protein in the embryo was due to the accelerated degradation of glutelin by a cysteine proteinase (REP-1) in the endosperm.The breakdown of glutelin in the endosperm showed a strict positive correspondence with the length of the shoot.The GluA2 mutation led to reduced degradation rate of glutelin and defects in seed germination,and the promotion effect of GA on seed germination was weakened in the glua2mutant.In vitro culture assay of rice embryos showed that glutelin mobilization functioned downstream of the GA and BR pathways to promote shoot elongation.These findings suggest a mechanism that mediates crosstalk between BR and GA in co-regulating rice seed germination and embryo growth.

Development of Simple Functional Markers for Low Glutelin Content Gene 1 (Lgc1) in Rice (Oryza sativa)

Rice with low glutelin content is suitable as functional food for patients affected by kidney failure. Low glutelincontent gene Lgc1 in rice has a 3.5-kb deletion between two highly similar glutelin genes GluB4 and GluB5, which locates on the short arm of chromosome 2. To improve the selection efficiency in low glutelin-content rice breeding, two molecular markers designated as InDel-Lgc1-1 and InDel-Lgc1-2 were developed to detect the low glutelin-content gene Lgc1. A double PCR detection indicated that combined use of the two markers could easily distinguish the genotypes of Lgc1 from different rice varieties. Therefore, as a simple and low-cost technique, the molecular marker could be widely used to identify different varieties with Lgc1 gene and applied in marker-assisted selection of low glutelin-content rice.

Charactering protein fraction concentrations as influenced by nitrogen application in low-glutelin rice cultivars

To optimize both grain yield and quality of low-glutelin rice cultivars under N-fertilizer strategies, two-year field experiments involving three low-glutelin rice cultivars（W1240, W1721, W025） and an ordinary rice cultivar（H9405） with five N treatments were carried out to determine the effects of N application rate and genotype on protein fractions contents and Glutelin/Prolamin ratio（Glu/Pro）. The difference of protein fraction concentrations affected by N application rate existed in genotypes. Ordinary rice cultivar had a larger increase in glutlein concentration affected by N application rate than low-glutelin rice cultivars did. Glutelin in H9405 had a increase of 30.6 and 41.0% under the N4 treatment（360 kg N ha^（–1）） when compared with N0 treatment（no fertilizer N） in 2010 and 2011 respectively, while all the low-glutelin rice cultivars showed relatively smaller increases for two years. Variance analysis showed no significant effect of N application rate on glutelin in W1240 and W025 while the effects on albumin, globulin and prolamin were significant in low-glutelin rice. What＇s more, N treatment had no significant i nfluence on Glu/Pro ratios in low-glutelin rice cultivars while a significant increase in Glu/Pro ratio was observed in ordinary rice cultivar. So low-gultelin rice cultivars showed a different pattern from ordinary rice cultivars when influenced by N application rate.

碱性蛋白酶水解时间对玉米谷蛋白理化性质及抗氧化活性的影响

为探究碱性蛋白酶(alcalase)水解对玉米谷蛋白理化性质及抗氧化活性的影响,对不同水解时间获得的玉米谷蛋白水解物的表面疏水性、二硫键含量、热性能、分子质量分布、抗氧化活性等进行了评价。结果表明:碱性蛋白酶水解对玉米谷蛋白的性质有显著影响,分子质量和二硫键含量显著降低(P<0.05),与玉米谷蛋白相比其水解物具有较高的溶解性、起泡性、乳化性和热稳定性。此外,玉米谷蛋白水解产物具有良好的抗氧化性能,当水解时间为150 min时玉米谷蛋白水解物的DPPH自由基清除为(54.46±1.43)%,羟自由基(·OH)清除率为(74.06±1.49)%,Fe^(2+)螯合力为(86.69±1.35)%。

利用基因编辑技术创制低谷蛋白水稻种质

水稻(Oryza sativa L.)是重要的粮食作物,蛋白质是稻米的第二大营养物质。肾脏病人通常需要限制蛋白质的摄入量以减轻肾脏的负担和控制病情进展。稻米蛋白中谷蛋白含量最高,且易被人体消化。本研究以LGC-1作为供体亲本培育出的粳稻品种为转基因受体,利用CRISPR/Cas9介导的基因编辑技术一次性敲除谷蛋白A(Glutelin A,GluA)亚家族编码基因GluA1、GluA2和GluA3,获得了不含转基因元件的谷蛋白含量约为1.8%的低谷蛋白种质,并对所得种质的稻米品质和农艺性状进行了全面评估。所得低谷蛋白种质外观品质较好,相比于受体品种垩白度降低、糙米率和精米率提高。本研究为创制低谷蛋白水稻品种提供了一种高效、简便的方法,为培育适合肾病患者的功能性水稻品种提供了一种新的遗传材料。

果胶复合提升米谷蛋白界面特性及Pickering乳液的贮藏稳定性

米谷蛋白(Rice Glutelin,RG)是一种常用食品乳化剂,但在中性pH值下,RG易聚集成疏水性大尺寸颗粒,导致其稳定性差、界面活性弱。该研究通过果胶静电复合修饰米谷蛋白,探究两者复合比例对颗粒的尺寸、接触角、界面吸附性能和界面膜结构的影响,在此基础上,以复合颗粒为界面稳定剂制备Pickering乳液,考察其粒径分布、微观结构、流变学性质及储藏稳定性。结果表明,当米谷蛋白与果胶质量比为1:1时,复合颗粒平均粒径为675.4 nm,接触角为64.23°,油-水界面张力从36.55 mN/m降低至13.00 mN/m,界面膜以弹性为主,弹性模量达46.07 mN/m,由此复合颗粒稳定的Pickering乳液具有最小的粒径(d4,3~31.1μm)、最高的表观黏度和储能模量。因此,果胶复合可以显著降低米谷蛋白的尺寸,并赋予其合适的表面亲疏水性,进而提升其降低界面张力和增加界面膜黏弹特性的能力,有利于制备高稳定Pickering乳液,为拓展米谷蛋白在食品乳液体系中的应用提供了新策略。

核桃谷蛋白多肽及其肽锌螯合物的分离纯化、鉴定与结合位点分析

为提高核桃的附加值,以核桃粕为原料,将核桃谷蛋白多肽与锌进行螯合,利用扫描电子显微镜、傅里叶变换红外光谱、X射线衍射光谱等分析手段表征多肽与锌离子的螯合特征,并通过超滤、凝胶柱层析、液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)技术筛选出与锌结合能力较高的肽段,结合分子对接技术探究二者的具体结合位点和结合能力。结果表明,核桃谷蛋白多肽与锌离子发生了螯合反应,螯合前后多肽的表面微观结构和结晶程度均有显著差异;经超滤、凝胶柱层析分离后,F41组分螯合能力最强,为(117.43±1.99)mg/g;LC-MS/MS结合虚拟筛选得到5条无毒无潜在致敏性且具有潜在生物活性的肽段;利用分子对接技术模拟分析肽锌螯合物的结合过程,发现五肽Phe-Asp-Ala-Asp-Phe(FDADF)可与锌离子形成结构稳定的复合物,通过配位键、氢键和疏水相互作用结合,结合能最低,为-7.27 kcal/mol,结合位点主要为天冬氨酸侧链的羧基氧原子(Asp4:O-Zn)。本研究可为肽锌螯合物的开发应用提供理论依据。

Rapid improvement of rice eating and cooking quality through gene editing toward glutelin as target

Rice eating and cooking quality(ECQ)is a major concern of breeders and consumers,determining market competitiveness worldwide.Rice grain protein content(GPC)is negatively related to ECQ,making it possible to improve ECQ by manipulating GPC.However,GPC is genetically complex and sensitive to environmental conditions;therefore,little progress has been made in traditional breeding for ECQ.Here,we report that CRISPR/Cas9-mediated knockout of genes encoding the grain storage protein glutelin rapidly produced lines with downregulated GPC and improved ECQ.Our finding provides a new strategy for improving rice ECQ.

酶解法制备核桃谷蛋白-1 ACE抑制肽的工艺优化

为获得优质的核桃蛋白血管紧张素转化酶(ACE)抑制肽的制备原料及优化其制备工艺,采用连续提取法从脱脂核桃粕中依次分离出清蛋白、球蛋白、醇溶蛋白、谷蛋白-1和谷蛋白-25种组分蛋白,测定5种组分蛋白的占比及ACE抑制率,以ACE抑制率最大的组分蛋白作为原料采用酶解法制备ACE抑制肽,在筛选出最适酶解用酶基础上,采用单因素试验与响应面试验优化酶解制备核桃蛋白ACE抑制肽的工艺。结果表明:5种组分蛋白中谷蛋白-1占比仅次于谷蛋白-2,且其ACE抑制率最高,以核桃谷蛋白-1为原料,在筛选出胃蛋白酶作为酶解用酶基础上,经工艺优化得到最优的酶解法制备核桃谷蛋白-1 ACE抑制肽的工艺条件为酶解温度46℃、酶解时间6 h、酶用量4.2%(以底物质量计)、酶解pH 1.6,在该条件下所得核桃谷蛋白-1酶解液的ACE抑制率为(50.08±2.34)%。因此,核桃谷蛋白-1经胃蛋白酶酶解可生产ACE抑制活性较高的核桃多肽。

限制性酶解、pH值和离子强度对油棕粕谷蛋白-1乳化性能的影响

该试验利用限制性酶解技术对油棕粕谷蛋白-1(oil palm expeller gultelin-1,OPEG-1)进行改性,得到水解度分别为6.28%、17.35%和22.67%的3种酶解产物。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfatepolyacrylamide gel electrophoresis,SDS-PAGE)试验表明,OPEG-1的分子量范围为18.8~51.5 kDa,由6个亚基构成;随着水解度的增大,OPEG-1分子量减小。限制性酶解也显著降低OPEG-1的表面疏水性,且其表面疏水性与水解度呈负相关关系。水解度为6.28%~17.35%的限制性酶解显著提高OPEG-1的乳化性,而水解度为22.67%时,OPEG-1的乳化性显著降低。水解度为6.28%~17.35%的限制性酶解显著提高OPEG-1在不同pH值的乳化性;而水解度为17.35%的限制性酶解,显著提高OPEG-1在离子强度为0.1~0.2 mol/L时的乳化性。同时,在pH2.0~4.0时,OPEG-1及其酶解物的乳化性随pH值的增大而上升;在离子强度为0~0.1 mol/L时,增加离子强度可显著提高OPEG-1及其酶解物的乳化性,乳化稳定性略有提升;而离子强度为0.2~0.6 mol/L时,OPEG-1及其酶解物的乳化性与乳化稳定性均随离子强度的增大而减小。结果表明,限制性酶解能够改善OPEG-1的乳化性能,拓宽其在食品工业中的应用。

利用分子标记辅助选择培育优良食味、低谷蛋白香粳稻新品系

【目的】选育低谷蛋白新品种是功能性水稻育种的一个重要方向。为满足肾脏病患者对人体健康和稻米品质的需求,迫切需要在育种中加强对功能性和食味品质的协同改良。【方法】以具有低直链淀粉含量基因Wxmp和香味基因fgr的优良食味粳稻品种南粳46与含低谷蛋白基因Lgc1的日本粳稻品种LGC-1杂交、回交,利用与目标基因共分离的分子标记进行跟踪检测并结合田间选择,在BC2F6获得5个新品系。以亲本南粳46和LGC-1为对照,对新品系的农艺、产量、品质性状进行分析。【结果】与LGC-1相比,新品系的谷蛋白含量和可吸收蛋白含量相当,食味品质得到显著提升,且综合性状优良,表现出较高的产量潜力,适宜在江苏不同稻区进行种植。【结论】分子标记辅助选择作为一种快速、准确、有效的方法,与常规育种技术紧密结合,能够显著提高优质、高产、低谷蛋白水稻品种的选育效率。

低谷蛋白半糯型粳稻营养品质与蒸煮食味品质特征分析

【目的】旨在明确低谷蛋白半糯型粳稻品种(系)与半糯型粳稻和普通粳稻营养和蒸煮食味品质性状的差异及各指标间的关系,为健康优质粳稻的选育提供科学依据。【方法】以低谷蛋白半糯型粳稻品系为材料,半糯型粳稻和普通粳稻品种为对照,在相同环境和栽培条件下种植3种不同类型的粳稻品种共16个,测定并分析稻米总蛋白质及各组分含量的差异,同时分析直链淀粉含量、糊化温度、胶稠度等淀粉理化特征的差异,并进一步分析稻米营养与蒸煮食味品质的相关性。【结果】低谷蛋白半糯型粳稻的谷蛋白含量显著低于半糯型粳稻与普通粳稻,醇溶蛋白和清蛋白含量显著高于半糯型粳稻和普通粳稻。低谷蛋白半糯型粳稻的崩解值显著低于半糯型粳稻和普通粳稻,半糯型粳稻的胶稠度与峰值黏度显著高于普通粳稻与低谷蛋白粳稻;低谷蛋白半糯型粳稻的外观、黏度、平衡度、食味值和普通粳稻无显著差异,但显著低于半糯型粳稻。相关性分析结果显示,总蛋白和醇溶蛋白含量与峰值黏度、崩解值、外观、黏度、平衡度、食味值显著负相关,与消减值、硬度显著正相关;而谷蛋白含量的规律则刚好与之相反。【结论】3种不同类型粳稻品种在谷蛋白含量、直链淀粉含量和崩解值上存在差异显著,其中低谷蛋白半糯型粳稻品种具有较低的谷蛋白含量,较高的醇溶蛋白;半糯型粳稻品种具有较低的蛋白质含量、直链淀粉含量、消减值和回复值,较高的胶稠度和崩解值。4个蛋白组分中,醇溶蛋白对米饭食味品质的负影响最大,与米饭的外观、黏度和平衡度极显著负相关。

油牡丹籽粕蛋白质提取工艺优化及其抗氧化活性研究

以经超临界CO_(2)萃取提油后的油牡丹籽粕为原料,研究油牡丹籽粕蛋白质提取工艺、氨基酸组成和抗氧化活性。通过Osborne分级法分离出四种蛋白质,并对清蛋白、醇溶蛋白、谷蛋白及球蛋白提取工艺进行初步优化,采用响应面法分析油牡丹籽粕主要蛋白质清蛋白最佳提取条件,分析四种蛋白质的氨基酸组成。在此基础上,通过DPPH及ABTS自由基清除率评价四种蛋白的抗氧化效果。研究结果表明:油牡丹籽粕清蛋白、球蛋白、醇溶蛋白、谷蛋白的最佳提取条件为粉碎度100目,提取温度依次为45、40、50、30℃,提取时间依次为86、100、100、60 min,清蛋白提取率为47.02%。此外,四种蛋白质均为完全蛋白质具有较强的DPPH自由基清除能力及ABTS自由基清除率能力。油牡丹籽粕蛋白是优质蛋白质资源,本研究为油牡丹籽粕综合利用奠定理论基础。

挤压处理对淀粉-米谷蛋白复合体系回生特性及流变特性的影响

大米淀粉回生老化是限制其应用的主要原因,而挤压协同米谷蛋白处理对其复合体系回生特性的抑制具有明显效果。为明确挤压处理对大米淀粉/米谷蛋白混合体系回生特性的影响,本研究考察了螺杆转速、挤压温度、物料水分质量分数及大米淀粉/米谷蛋白物料比(m/m)对混合体系回生特性的影响规律,并利用快速黏度分析仪、差示扫描量热仪、X射线衍射仪、傅里叶变换红外光谱仪、低场核磁共振仪、扫描电子显微镜、激光扫描共聚焦显微镜以及流变仪分析挤压处理延缓混合体系回生的作用机制。结果表明:当螺杆转速为300 r/min、挤压温度为90℃、物料水分质量分数为34%、大米淀粉/米谷蛋白物料比为91∶9时,挤压处理延缓混合体系回生的效果最显著。与未挤压的混合物相比,挤压处理后混合体系的峰值黏度、崩解值、回生值均显著下降(P<0.05),分别由(3005.00±25.00)、(1193.00±6.00)、(164.33±3.51)cP降低至(429.00±5.00)、(213.00±3.00)、(27.05±0.14)cP,相对结晶度由(20.18±0.13)%下降至(2.17±0.43)%,吸热峰消失,峰值强度比值(1047 cm^(−1)/1022 cm^(−1))显著降低至0.43±0.08(P<0.05),微观结构更加均匀致密、保水性增强、剪切应力下降、滞后环面积增大、储能模量G′及损耗模量G″降低,表现出较好的抗回生能力。本研究结果为挤压处理在延缓淀粉/蛋白混合体系回生中的应用提供理论参考。

Protamex酶水解对玉米谷蛋白泡沫性质及结构特性的影响

采用Protamex酶对玉米谷蛋白进行水解,研究不同水解时间对玉米谷蛋白的泡沫性质、表面张力、理化性质、拉曼光谱及静态流变学性质等的影响。结果表明,Protamex酶水解显著改善玉米谷蛋白的溶解性和泡沫性质,在120 min水解物的起泡力最大,是原玉米谷蛋白的2.8倍左右,泡沫稳定性表现良好。此时水解物的表面张力最低、表观黏度最高,所形成泡沫的微观形态细腻均匀、蛋白膜较厚。随着水解时间延长,玉米谷蛋白水解物的平均粒径持续减小、内源荧光强度和表面疏水性逐渐增加,而表面净电荷呈先减小后增加的变化趋势。拉曼光谱分析结果表明适当水解使α-螺旋含量减少、无规卷曲和β-转角含量增高、酪氨酸残基峰强比(I_(850)/I_(830))增强、色氨酸残基峰强比(I_(760))降低,但对β-折叠含量影响较小;长时间水解使无规卷曲含量显著增加,酪氨酸残基峰强比(I_(850)/I_(830))降低。因此,通过限制性水解作用可改变玉米谷蛋白的结构及界面性质、改善其泡沫性质,提高玉米蛋白在食品领域中的潜在利用率。

米谷蛋白对挤压淀粉复合物结构及性质的影响

以大米淀粉及米谷蛋白为原料,通过双螺杆挤压技术制备了不同比例的大米淀粉/米谷蛋白复合物,利用FTIR、1HNMR、XRD、DSC、快速黏度计、低场核磁(LF-NMR)、流变仪和SEM对复合物的形成机制、结构变化、理化性质以及微观形态进行了表征。结果表明,大米淀粉发生降解、α-1,6-糖苷键断裂,分支化减弱,并与米谷蛋白发生化学相互作用(包括美拉德反应),形成均匀致密的片层微观结构,从而增强对水分子的束缚能力。挤压后复合物表现为较低的热稳定性、结晶特性、糊化特性以及黏弹性。这些特性随着米谷蛋白添加量的提高得到改善,尤其在米谷蛋白添加量(以淀粉质量为基准,下同)为13%时,复合物的相对结晶度由2.10%(米谷蛋白添加量为9%)增加至3.51%(米谷蛋白添加量为13%),峰值黏度增加至376.51 Cp(1000 c P=1 Pa·s),崩解值提升为362.20 Cp,回生值增加至87.14 Cp,剪切应力随之增大,储能模量与损耗模量明显升高。