转磷酸甘露糖变位酶基因提高水稻维生素C含量

维生素C(VC)是人体健康所必需的营养元素。人类由于缺乏VC合成途径中的最后一种酶(L-古洛糖酸内酯氧化酶),自身不能合成VC。水稻是重要的粮食作物,增加水稻种子中VC含量,能够提高其营养价值。磷酸甘露糖变位酶(PMM)是VC合成通路中一种重要的酶,催化甘露糖-6-磷酸到甘露糖-1-磷酸的转变。将水稻PMM基因(OsPMM)构建在双右边界双元载体pMNDRBBin6上,并用种子特异表达的启动子BX14驱动其表达。通过农杆菌介导的转化系统,OsPMM基因被转入粳型三系恢复系C418中。通过分子检测,在T_2代筛选到了无选择标记的转基因植株。对OsPMM基因在转基因植株中的表达进行分析,发现OsPMM基因在转基因水稻种子内的表达水平明显提高,相应地,转基因系种子中的VC含量也提高了25%~50%。     

Transgenic Expression of the Recombinant Phytase in Rice （Oryza sativa）

In many plants, phytic acid (phytate, 1, 2, 3, 4, 5, 6-hexakisphosphate) is one of the main storage forms of phosphate. About 80% of phosphorus (P) in cereal plants, including rice is stored as phytic acid [1-2]. P in phytic acid can’t be utilized by monogastric animals including human, while it was estimated that only 1/3 of the total P in most of the vegetal feedstuff could be efficiently utilized by the livestock. Therefore, for animal feed with P supplementation is expected to meet the d…     

乔松根特异启动子PmPgPR10驱动Na+/H+逆转运蛋白提高转基因水稻的耐盐性

 为获得抗盐水稻,将小麦液泡膜Na+/H+逆向转运蛋白基因TaNHX2与乔松(Pinus griffithii)根诱导型特异表达启动子PmPgPR10融合（PmPgPR10∷TaNHX2）并转化水稻,以研究PmPgPR10启动子对TaNHX2基因表达的影响以及转基因植物的耐盐性。PCR、Southern和实时PCR试验结果表明,PmPgPR10∷TaNHX2基因已通过农杆菌介导法整合进水稻基因组,而且外源基因已在受体细胞中正确表达。在盐胁迫处理时,转PmPgPR10基因植株的耐盐性以及外源基因的表达量显著高于对照植株,说明PmPgPR10启动子可以调控TaNHX2基因在根中特异表达。为了进一步分析转基因植株耐盐机理, 比较了日本晴和转基因T3代植株中液泡腺苷三磷酸酶（V ATPase）和液泡焦磷酸酶（V PPase）活性,发现转PmPgPR10∷TaNHX2基因水稻的V ATPase和V PPase酶活性显著高于非转基因对照,说明V ATPase和V PPase活性提高在转TaNHX2基因水稻耐盐性过程中发挥重要作用。在盐胁迫处理时,V ATPase和V PPase活性只能在转基因植株的根中但不能在叶片中被检测到,进一步说明PmPgPR10启动子在根中特异性表达。因此,PmPgPR10具有在根中增强下游TaNHX2基因表达,并显著提高转基因植株耐盐性的能力。     

转基因水稻中重组植酸酶的表达

为通过在转基因植株中表达植酸酶降解植酸来提高水稻中无机磷利用率,构建了由玉米泛素基因Ubi启动子控制的植酸酶基因植物表达载体,并以来源于水稻未成熟胚的愈伤组织作为转化受体,经农杆菌介导法将植酸酶基因导入水稻中,共获得15个独立的转基因株系。对转基因水稻总DNA的PCR和Southern 杂交分析证明目的基因已整合入转基因水稻植株基因组中,并能稳定遗传。对部分转基因水稻未成熟种子总RNA进行RT PCR分析,表明导入的植酸酶基因能够在转基因水稻种子中正常表达。无机磷含量分析表明含目的基因的转基因水稻种子及其后代叶片中的无机磷含量较未转化植株均有了明显的提高。     

拟南芥热激启动子（HSP18.2）在水稻中的启动效率探讨

从拟南芥中克隆热激蛋白基因HSP18.2的启动子区域,构建了由HSP18.2启动子引导的GUS融合基因,并经农杆菌介导导入籼稻愈伤组织中。对热激和常温条件下转基因籼稻愈伤组织中GUS活性的比较测定表明,HSP18.2启动子在热激条件下可驱动GUS报告基因在转基因愈伤组织中高效表达,而常温条件下GUS活性在检测水平以下,证明HSP18.2启动子在籼稻中对基因的调控十分灵敏。     

大麦醇溶蛋白启动子HorD的克隆及其种子特异性表达分析

种子特异性启动子的克隆和功能分析不仅有助于阐明作物种子发育和胚乳特异性基因的表达调控机制，也是进行作物品质改良和种子生物反应器在内的植物基因工程改造的基础。本研究从大麦品种Golden pormise中克隆到大麦胚乳特异性启动子HorD，生物信息学分析表明，其具备启动子的基本元件，如A-box、TATA-box、CAAT-box等，此外还含有胚乳特异性表达所需要的元件Prolamin-box。为验证HorD启动子的表达特性，以pU1300载体为骨架，将HorD启动子和新型冠状病毒（SARA-CoV-2）刺突蛋白基因LPS通过双酶切连接的方式构建表达载体phorD-LPS，并利用农杆菌介导的遗传转化试验验证其种子表达特性。结果表明，HorD启动子能驱动LPS基因在转基因大麦各组织中表达，但在根、茎、叶及颖壳中表达量较低，在种子中表达量较高。这说明HorD启动子可以驱动外源目的基因在大麦种子中特异高效表达。     

Sesame oleosin and prepro-2S albumin expressed as a fusion polypeptide in transgenic rice were split, processed and separately assembled into oil bodies and protein bodies

A recombinant polypeptide containing the precursor protein of a sesame storage protein, 2S albumin, fused to the C-terminus of a sesame oleosin was expressed in transgenic rice seeds under the control of a rice glutelin promoter. The recombinant polypeptide of 32 kDa, equivalent to the resultant molecular mass of sesame oleosin (15 kDa) and prepro-2S albumin (17 kDa), was detected in the endoplasmic reticulum fraction of maturing transgenic rice seeds, but not in the purified oil bodies or the soluble extract of transgenic seeds. However, sesame oleosin presumably fused with a 2 kDa C-terminal appendix originating from the signal sequence of prepro-2S albumin, was found in the purified oil bodies, and mature sesame 2S albumin apparently processed into two subunits (9 and 4 kDa) linked by disulfide bonds was detected in extracts of transgenic seeds. Immunogold labeling revealed that the sesame oleosin and 2S albumin were separately located in oil bodies and protein bodies of embryo cells of transgenic rice seeds. While sesame 2S albumin was also detected in protein bodies of endosperm cells of transgenic seeds, the co-expressed sesame oleosin, probably degraded due to the lack of oil bodies in this tissue, and was not detected. The results provide a new technique for introducing two recombinant polypeptides separately into rice oil bodies and protein bodies from one expression construct.     

Sesame oleosin and prepro-2S albumin expressed as a fusion polypeptide in transgenic rice were split,processed and separately assembled into oil bodies and protein bodies

A recombinant polypeptide containing the precursor protein of a sesame storage protein, 2S albumin, fused to the C-terminus of a sesame oleosin was expressed in transgenic rice seeds under the control of a rice glutelin promoter. The recombinant polypeptide of 32 kDa, equivalent to the resultant molecular mass of sesame oleosin (15 kDa) and prepro-2S albumin (17 kDa), was detected in the endoplasmic reticulum fraction of maturing transgenic rice seeds, but not in the purified oil bodies or the soluble extract of transgenic seeds. However, sesame oleosin presumably fused with a 2 kDa C-terminal appendix originating from the signal sequence of prepro-2S albumin, was found in the purified oil bodies, and mature sesame 2S albumin apparently processed into two subunits (9 and 4 kDa) linked by disulfide bonds was detected in extracts of transgenic seeds. Immunogold labeling revealed that the sesame oleosin and 2S albumin were separately located in oil bodies and protein bodies of embryo cells of transgenic rice seeds. While sesame 2S albumin was also detected in protein bodies of endosperm cells of transgenic seeds, the co-expressed sesame oleosin, probably degraded due to the lack of oil bodies in this tissue, and was not detected. The results provide a new technique for introducing two recombinant polypeptides separately into rice oil bodies and protein bodies from one expression construct.     

外源木聚糖酶基因atx在水稻中的表达

 为通过在转基因植株中表达木聚糖酶来提高木聚糖酶的生产效率,将具有较高热稳定性和催化活性的杂合木聚糖酶基因atx连接到双元表达载体pCAMBIA1301上,成功构建了木聚糖酶植物表达载体atx Ru3ep 1301。然后以水稻成熟胚的愈伤组织作为转化受体,采用农杆菌介导法将木聚糖酶基因导入水稻(中花11)中。经过潮霉素抗性检测和PCR鉴定,证实目的基因已经整合到转基因水稻基因组中。RT PCR分析结果显示,外源木聚糖酶基因能够在CaMV 35S启动子的引导下在转基因水稻中正常转录。转基因水稻能够正常生长和繁殖。木聚糖酶活性分析表明,转基因植株最高木聚糖酶活性约为4.37 U/g（鲜叶片）。因此,利用转基因水稻生产木聚糖酶将会是一种经济、有效的方法。     

水稻异淀粉酶基因ISA1及其启动子的表达特性分析

 利用实时定量RT-PCR方法研究了水稻异淀粉酶基因ISA1在各组织及不同发育阶段籽粒中的表达模式,结果表明该基因只在发育籽粒中表达。同时,克隆了ISA1基因起始密码子上游1.1 kb和2.1 kb两个不同长度的启动子片段,并分别与GUS报告基因融合,经农杆菌介导转入水稻中。对转基因水稻植株中GUS活性的定性与定量结果表明,2.1 kb的ISA1启动子具有很好的胚乳表达特异性,与内源基因定量表达分析的结果一致;而1.1 kb的ISA1启动子则不具备该表达特性,它在胚乳、茎、茎节及谷壳中都有很高的表达。这可能是因为 2.1 kb启动子中含有抑制目的基因在茎等组织或器官中表达的顺式作用元件。     

Specific expression of a sesame storage protein in transgenic rice bran

Rice oil bodies enclosed by unique structural proteins, oleosins, are found in the embryo and the aleurone layer, but not the starchy endosperm where starch and storage proteins are accumulated. To examine oleosin promoter specificity, a sesame storage protein, 2S albumin, was expressed in transgenic rice seeds under the control of a rice oleosin promoter. In all transgenic rice seeds, the sesame 2S albumin was found exclusively in the bran fraction after milling. Immunological staining revealed that the sesame 2S albumin was also located in the embryo and the outermost cells of the starchy endosperm. Furthermore, immunogold labeling showed that the transgenic 2S albumin was deposited in both type-I and type-II protein bodies of the outermost cells of the endosperm as well as in the type-II protein bodies of the embryo. The methionine and cysteine contents in the bran from four homozygous transgenic lines were elevated by 24–38 and 50–62%, respectively, compared with those of wild-type plants. The results suggest that the rice oleosin promoter is bran-specific and could be used to add value to rice bran, an abundant by-product of rice polishing, by genetic engineering.     

额种子蛋白体靶向表达口服重组胰岛素原载体构建及转化水稻

应用水稻种子生物反应器开发口服重组胰岛素原具有重要应用前景。通过分子设计保证重组胰岛素原在人体肠道内的自主加工成熟,根据水稻密码子偏爱性人工合成了霍乱毒素β亚基和人胰岛素原的融合基因(cholera toxin B subunit fused with human proinsulin,CTBIN),并在C末端添加内质网滞留信号KDEL。通过PCR技术从粳稻品种日本晴全基因组中克隆谷蛋白启动子及其信号肽序列pGluB1sig(GluB1 promoter and its signal peptide)用于驱动融合基因CTBIN的表达,插入载体pCAMBIA1302,构建了水稻种子蛋白体靶向表达口服重组胰岛素原的载体pCAMBIA1302-pGluB1sig-CTBIN-Nos。采用农杆菌介导法转化日本晴,获得了46株转基因水稻植株,Western杂交检测到CTB-人胰岛素原融合蛋白在水稻种子中表达。     

Effects of prolamin on the textural and pasting properties of rice flour and starch

Prolamin is a major class of rice proteins but its influence on the physicochemical properties of rice is not clear. Rapid Visco Analyser (RVA) and TA-XT2 TPA textural analyses were performed on rice starch with the addition of prolamin extracted from three rice cultivars (Hitomebore, M103 and Amaroo), and on rice flour with the prolamin removed by propan-2-ol extraction. Addition of prolamin to rice starch was found to cause a significant (P<0.05) increase in RVA breakdown viscosity but significant (P<0.05) decreases in hardness, adhesiveness and gumminess of the starch gel. Similarly, when prolamin was removed from rice flour, exactly the opposite effect was observed. Addition of prolamin to rice starch also caused it to absorb water faster during cooking but the gelatinised starch absorbed less water compared with control samples without prolamin.     

New angiotensin-converting enzyme inhibitory peptide from Coix prolamin and its influence on the gene expression of renin-angiotensin system in vein endothelial cells

Coix seed, which is a traditional Chinese medicine, has been used to treat hypertension for thousands of years. It has been shown that Coix prolamin peptides display high levels of angiotensin I converting enzyme (ACE) inhibitory activity. Hence, we purified the ACE inhibitory peptides from Coix prolamin hydrolysates and evaluated the influence of the most potent peptide on the renin-angiotensin system (RAS) genes expression in human umbilical vein endothelial cells (HUVECs). In this study, Coix prolamin peptides were sequentially separated by ultrafiltration, ion exchange chromatography, gel filtration chromatography and RP-HPLC, while the peptide structure was analyzed by mass spectrometry. Next, in silico proteolysis, pharmacophore and molecular docking were further applied to screen and optimize the structure of peptides. Finally, a novel ACE inhibitory peptide VDMF was obtained, in which its influence on the gene expression of RAS signaling pathway in AngⅡ-injury HUVECs was evaluated by quantitative real-time PCR. VDMF significantly down-regulated ACE, AngII type 1 receptor (AT1R) and ACE2 mRNA expression in comparation with model group, while up-regulating Mas gene expression. Hence, we obtained a novel antihypertensive candidate that was derived from the Coix peptides, which could involve a multi-modulation mechanism that regulates blood pressure.     

反义TrxS基因在小麦中的表达及对小麦种子蛋白酶活性和蛋白组分的影响

为了揭示反义硫氧还蛋白基因(anti-TrxS)在抗穗发芽小麦中的作用机制,探讨转基因小麦中蛋白酶活性和蛋白组分的变化规律,以转反义TrxS基因小麦株系为材料,用荧光定量RT-PCR、SDS-PAGE和生理指标测定的方法,对转基因株系和对照种子萌发过程中反义TrxS基因表达、蛋白酶活性和蛋白组分进行了检测。结果表明,反义TrxS基因在RNA水平上能够表达;转基因小麦籽粒蛋白酶活性明显下降,种子吸涨12、24、48、729、6和120 h后,转基因小麦种子蛋白酶活性比对照分别降低27.3%、30.7%、19.1%、19.4%、23.7%和13.5%;转基因小麦籽粒谷蛋白、醇溶蛋白的SDS-PAGE分析表明,反义TrxS基因的导入抑制了转基因小麦籽粒谷蛋白和醇溶蛋白大分子亚基二硫键(S-S)向巯基(-SH)的转化,从而使蛋白质的降解延缓。     

水稻种子特异性谷蛋白GluB1启动子在水稻愈伤组织中驱动外源基因表达

【目的】水稻谷蛋白启动子GluB1(GluB1promoter,pGluB1)常用于外源基因在种子中特异性高效表达的研究,也是研究种子储藏蛋白基因表达调控机制的模型。前人研究表明,pGluB1只在水稻胚乳中表达,而在根、茎、叶片、叶鞘、颖壳等组织中均无表达活性。研究的目的是为了克服种子特异表达启动子筛选周期长的缺点。【方法】将由pGluB1驱动的霍乱毒素B亚单位和重组胰岛素原组成的融合基因(a fusion gene of the cholera toxin B subunit and human proinsulin, CTBIN)表达载体pCAMBIA1302-pGluB1sig-CTBIN-NOS经农杆菌介导法转化水稻成熟胚愈伤组织。通过RT-PCR和蛋白质印迹杂交试验检测融合基因CTBIN在水稻愈伤组织中的转录和翻译表达。【结果】获得的7个转基因愈伤克隆中,有6个克隆的融合基因CTBIN在转录水平上表达。选取其中4个克隆进一步进行蛋白质印迹杂交试验检测证实融合基因CTBIN均在翻译水平上表达,而且从分子量大小推断融合蛋白包含的谷蛋白GluB1的N-端信号肽序列(24个氨基酸残基)在所测的愈伤组织细胞中均被成功切除。【结论】水稻种子特异性启动子pGluB1在愈伤组织中具有驱动外源基因表达活性,种子蛋白体亚细胞定位信号肽序列可在愈伤组织细胞中被切除。这为在愈伤组织细胞中快速检测种子特异表达启动子活性和探索愈伤组织中蛋白质的亚细胞分拣机制奠定了基础。     

拟南芥AtLURP1基因启动子在转基因烟草和水稻中的功能分析

克隆拟南芥LURP1基因上游1515 bp的启动子调控序列并命名为AtLURP1p,将其与GUS报告基因融合,构建成植物表达载体,分别遗传转化烟草和水稻,获得LURP1p::GUS的烟草和水稻转基因植株及其相应的T2代株系,分别研究LURP1p对水稻稻瘟病、辣椒青枯病侵染及SA、MeJA、ABA等几种重要的植物激素信号分子处理的应答.结果表明:(1)转基因烟草和水稻在几种激素,包括SA、MeJA、ABA的诱导处理下,GUS基因均可以被诱导表达;(2)转基因烟草在细菌性病菌青枯病的侵染下,GUS可被诱导表达并表现出持续表达的趋势,转基因水稻在稻瘟病的侵染下,其GUS基因也被诱导表达,并表现出后期持续上调的趋势.这些研究结果表明,拟南芥LURP1的应答逆境信号通路也存在于烟草和水稻等植物,该启动子可用作诱导型启动子广泛地应用于不同植物抗病基因工程.     

Analysis of Storage Protein Distribution in Rice Grain of Seed-Protein Mutant Cultivars by Immunofluorescence Microscopy

Abstract

Localization of storage proteins in rice grains of seed-protein mutant cultivars, low-glutelin cultivars LGC-1, LGCsoft and a 26-kDa-globulin-deficient low-glutelin cultivar LGC-Katsu, was examined by immunofluorescence microscopy using fluorescence-labeled antibodies of 13 kDa prolamin and 23 kDa glutelin. Abundant 13 kDa prolamin and 23 kDa glutelin was observed in the outer regions of rice grains. Image analysis revealed that peaks of fluorescence intensity of both proteins were located at 2–7% of the width or longitudinal length of brown rice distant from the outer surface of brown rice (RDOS) on the dorsal, ventral, and apical sides of brown rice. In these seed-protein mutant cultivars and a normal-type cultivar Nihonmasari, the foot of the distribution peaks of both proteins were located at 20–30% RDOS on the ventral and apical side. In contrast, on the dorsal side of rice grain, varietal differences of RDOS at the foot of the distribution peak of both proteins varied with the cultivar. The RDOS was 20 –40%; and Nihonmasari >LGC-1≒LGCsoft >LGC-Katsu. Although the quantity of 13 kDa prolamin and 23 kDa glutelin greatly varied with the cultivar in the outer region of rice grain, both proteins were distributed uniformly at low levels in the inner region in all cultivars. Furthermore, SDS-PAGE analysis showed that a larger quantity of 13 kDa prolamin localized on the ventral than the dorsal side of rice grains in seed-protein mutant cultivars, especially in LGC-Katsu.     

水稻种胚LOX3基因在逆境胁迫中的作用

LOX3是主要的水稻种胚脂氧合酶同工酶。为了研究水稻LOX3基因在逆境胁迫中的作用,构建了LOX3基因的反义植物表达载体,用农杆菌介导法转化水稻品种武运粳7号和Kasalath,获得了转基因植株。PCR和Southern鉴定证实基因已经导入水稻基因组中,种胚LOX3缺失鉴定和半定量RT PCR分析证实反义RNA抑制了LOX3基因的表达。对T2代转基因植株进行了水分胁迫处理及稻瘟病和白叶枯病的接种鉴定。结果显示,与非转基因对照相比,反义LOX3转基因植株对水分胁迫、稻瘟病和白叶枯病都表现敏感,说明水稻种胚LOX3基因在逆境胁迫反应中发挥一定的作用。