水稻谷蛋白基因GluC启动子的克隆及表达分析

水稻谷蛋白仅在水稻种子胚乳中表达,其启动子是分离胚乳特异性表达启动子的理想材料。本研究克隆了GluC基因启动子pGluC,生物信息学分析表明pGluC内部含有胚乳特异性表达所需要的Skn-1 motif和ACGT-box元件。将pGluC启动子和7个5'端缺失启动子片段构建到pGPTV-GUS载体上,转化水稻愈伤组织,进行组织化学染色和GUS酶活分析。结果表明：全长及截短的-1 911、-1 611、-1 311 bp启动子均能驱动GUS基因在水稻种子胚乳中高效稳定表达。-999、-451、-203、-102 bp启动子失去了胚乳表达特异性,在根、茎或者叶中也检测到GUS表达。该结果为实现外源目的基因在水稻胚乳中特异高效表达提供了理论依据。     

水稻胚乳特异性启动子的克隆及序列分析

目的:克隆获得水稻胚乳特异表达启动子pGluB-1。方法:采用PCR方法从水稻"台粳9号"基因组DNA中扩增出pGluB-1启动子序列,并克隆至pMD20-T载体上,酶切鉴定后进行测序并对测序结果进行生物信息学分析。结果:获得大小为1 353bp的pGluB-1启动子序列,该序列与已报道序列的同源性为97%。启动子功能预测及顺式作用元件分析表晨所克隆的启动子序列含有ACGT基序、AACA基序、GCN4基序和醇溶蛋白框等胚乳特异表达必需的调控元件,其序列差异可能是由于不同品种个体差异及多态性的影响。结论:成功克隆出pGluB-1启动子,为实现外源目的基因在水稻胚乳中特异性表达奠定了实验基础。     

水稻胚乳特异表达基因的挖掘及顺式元件分析

本研究旨在利用计算机方法对水稻胚乳特异性表达基因进行挖掘和功能分析,及特异性顺式调控元件的预测。我们将基因在不同组织中的表达信号谱看作多维空间内的向量,利用向量夹角余弦法计算其与理想状态下该基因在某一组织特异表达向量的相似度,以此来判断组织特异性表达基因。本文通过对水稻芯片数据的大规模分析,共挖掘出了127个在水稻胚乳中特异表达基因。并对其启动子进行顺式元件预测,发现两个与胚乳特异表达相关的顺式调控元件,其保守序列分别为CATGCATSCM和GATCGATCGR。与已知功能的顺式元件比较显示,前者为种子特异基因表达相关的RY repeat元件,而后者则与元件RNFG1相似,但其具体功能尚不明确。     

小鼠DEAF1调控基因的生物信息学分析

Deeaf1在小鼠体内可调控约600个基因的表达。用Clover程序对受Deaf1调控的基因的启动子区进行分析,结果发现受DEAF1调控的基因中约半数(263/590)含有能被DEAF1结合的保守序列TTTC(C/G)G。联合组织特异性基因表达数据库(TiGER)和小鼠基因组信息学(MGI)数据库对这些基因的组织特异表达谱进行分析,发现这些基因大部分为多个组织表达,也有在单个组织或少数几个组织中表达。有228个基因在胰腺特异表达,其中131个基因既在胰腺表达,又在淋巴结中表达,在胰腺及淋巴结中特异表达的这些基因大部分启动子区含DEAF1特异结合的保守序列。这些结果为下一步外周组织抗原在淋巴结中表达调控机制的研究及外周免疫耐受建立和维持机制的研究奠定了基础。     

栽培草莓潜在印记基因FaTRG-31的克隆与特征分析北大核心CSCD

在前期通过筛选获得草莓候选印记基因FaTRG-31(turgor-responsive protein 31)的基础上,以八倍体栽培草莓‘红颜’(Fragaria×ananassa Duch.‘Benihoppe’)为材料,对FaTRG-31基因的编码序列进行克隆、生物信息学、组织表达、启动子和印记特性分析,以揭示该基因的作用机理,为草莓印记基因表达调控及生物学功能研究奠定基础。结果显示:(1)草莓FaTRG-31基因开放阅读框(ORF)全长999 bp,编码332个氨基酸,含有典型的Asn-Pro-Ala(NPA)基序和6个跨膜结构,定位于细胞质膜,属于典型的AQP蛋白(aquaporin)家族;系统进化树深入分析表明FaTRG-31为PIPs(plasma intrinsic proteins)亚家族1型蛋白。(2)草莓FaTRG-31在不同组织中均有表达,胚乳中的表达量最高,且在FaTRG-31基因上游克隆出的1989 bp启动子序列中发现含有与胚乳特异表达相关的作用元件,此外还有多种非生物胁迫响应元件和激素响应元件等。(3)草莓印记特性分析结果显示,FaTRG-31在胚乳中的SNP(single nucleotide polymorphism)位点处测序峰为单峰且是母本表达,即该基因在栽培草莓胚乳中为母本表达印记基因。(4)非生物胁迫处理后分析发现,草莓FaTRG-31基因能在不同程度上响应非生物胁迫。     

小麦高分子量麦谷蛋白亚基1Dx5启动子驱动外源基因的表达

将小麦高分子量麦谷蛋白亚基(HMW-GS)基因的胚乳组织特异性表达启动子驱动的外源突变型1Dx5基因和gus基因导入小麦中.对其转基因植株连续3代的跟踪研究表明,突变型1Dx5基因的重复序列导致其表达蛋白分子量增大,并影响其它1Bx17 1By18亚基基因的表达.组织化学分析观察到gus基因在1Dx5基因启动子驱动下的表达表现出胚乳组织特异性,在开花2周后开始表达,表达量呈持续上升,至腊熟期达到最高,其次为籽粒成熟期.     

水稻种子醇溶蛋白4a基因的表达受串联复合启动子调控

从水稻中花8号中克隆了醇溶蛋白4a的基因5＇侧翼区并首次报道该基因5＇端-680～-18区段具有胚乳特异性表达调控功能.本研究通过 5’缺失方法对该基因5＇侧翼区的结构和功能进行了进一步分析,结果表明:(1)醇溶蛋白4a基因启动子Ⅰ能够特定地在转基因烟草开花发育 22 d后的种子胚乳中激活下游 GUS融合基因表达,是该基因有功能的启动子;(2)prolamin box Ⅱ对该基因的表达具有增强功能;(3)启动子Ⅰ的转录起始点是位于该基因起始ATG上游63 bp处的C碱基.     

普通野生稻绿色组织特异表达启动子的克隆与鉴定

绿色组织特异表达启动子可调控外源基因只在受体作物的绿色组织中定点、高效地表达。以普通野生稻为实验材料,克隆了绿色组织特异表达启动子Or GSP,构建Or GSP和GUS基因融合的表达载体,转入拟南芥中鉴定功能。启动子Or GSP长度为825 bp,含有基本的转录起始元件TATA-box和CAAT-box,以及光响应元件TCCC-motif、Sp1、G-box、I-box、GA-motif和as-2-box等。转基因拟南芥GUS组织化学染色结果表明,启动子Or GSP调控GUS基因只在绿色组织中特异表达。GUS活性测定结果显示,叶和茎中的GUS活性比根中明显提高。普通野生稻中克隆的启动子Or GSP为绿色组织特异表达启动子,可为作物分子育种提供新的调控元件。     

油葵种子特异启动子Ha ds10 G1的克隆及功能鉴定

从油葵中克隆得到LEA蛋白基因家族Ha ds10 G1基因的启动子序列,并对其进行功能分析。利用PCR技术从油葵品种"矮大头"基因组DNA中分离Ha ds10 G1基因上游的调控序列,将其与GUS基因融合,构建种子特异性表达载体p BI121-PHa ds10,通过根癌农杆菌介导法转化烟草(Nicotiana tabacum)NC89,对再生植株进行PCR、RT-PCR和GUS组织化学分析,以检测GUS基因在转基因烟草中的表达情况。结果表明,油葵Ha ds10 G1基因启动子长度为1 417 bp,与已报道的向日葵Ha ds10G1基因启动子序列同源性为89.42%。作用元件分析发现该区域除了具有启动子核心调控序列外,还含有多个与组织特异性、激素、逆境等表达相关的顺式作用元件,如RY重复元件、ABRE元件、TC-rich元件等。转基因植株的PCR结果显示,成功地获得了转基因阳性植株;GUS活性检测表明,该启动子序列仅能够驱动GUS基因在烟草种子表达,而在根、茎、叶等组织中均未检测到GUS基因表达。因此,油葵LEA蛋白基因家族Ha ds10 G1基因上游1 417 bp片段具有种子特异性启动子功能。研究结果为油葵等油料作物的油脂遗传改良提供组织特异性启动子。     

水稻ADP-葡萄糖焦磷酸化酶小亚基Ⅰ基因的启动子分析

水稻(Oryaz sativa L.)基因组中的ADP-葡萄糖焦磷酸化酶小亚基(ADP-Glucose pyrophosphorylase smallsubunit,OsAgpS)由两个基因编码,即OsAgpS1和OsAgpS2.其中OsAgpS1基因产生两个转录本OsAgpS1a和OsAgpS1b,区别在第一个外显子的位置不同.通过RT-PCR方法分析了两个转录本在水稻组织和胚乳不同发育时期的表达特性;同时通过报告基因GUS检测了两个转录本上游转录调节区DNA片段的转录启动特性.结果表明,两个启动了与其下游转录本的表达模式完全一致,即OsAgpS1a转录本和OsAgpS1a 上游启动子控制的GUS基因主要在胚乳中高水平表达,在叶片中有很低水平的表达;而OsAgpS1b转录本和OsAgpS1b上游启动子控制的GUS基因主要在叶片和胚乳发育早期低水平表达.这说明OsAgpS1基因产生的两个转录本是由不同的启动子控制转录的,OsAgpS1a上游启动了可以作为胚乳表达用启动子.     

Scanning Electron Microscope Observation on Endosperm Starch Grain Cnaracters in Orvza sativa subsp. Hsien

Scanning electron microscope observation on the characters of endosperm starch grains of wild rice anti seventeen hsien rice varieties belonging to first and second season cropping indicated that endosperm starch grains of hsien rice generally were polyhedron in sharp and edgy angle indistinct of polyhedron, individual starch grain nearly subrounded in the parts called white belly and white centre. General diameters are in an extent of 3.3–9.3 mm. The shapes of the endosperm starch grains between wild rice and cultivars are similar and sizes leas than hsien rice varieties, but the sizes of the endosperm starch grains of the first eropp4ng axe large than those of the second cropping. The shapes of endosperm starch grains of the varieties to have good exterior quality are polyhedron and structures are arranged closely, Hance, it can be primarily distinguished the good quality of grains from the poor by observation on the endosperm starch grains of varieties.     

The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.     

污染作物籽实中As的分布及其结合形态初探

对水稻和小麦籽实中Ａｓ的分布,结合形态及加工去除进行了初步研究,结果表明,Ａｓ在谷物各结构部位中的分布不均,在水稻籽实中浓度分布顺序为胚〉种皮〉颖壳〉胚乳,小麦中Ａｓ的浓度分布顺序为胚〉种皮〉胚乳;Ａｓ在各形态部位的累积量不同,主要存留在胚乳中;Ａｓ主要与蛋白质相结合的形态存在;在稻谷去颖壳、粗米糠和精米糠的过程中,可分别去除Ａｓ１６．５１％、１２．４１％和１０．２６％,小麦也随着加工深度的升级,     

不同倍性水稻胚乳蛋白的差异表达研究

以4份同源四倍体水稻和4份相应的二倍体水稻为研究材料, 对其种子内清、球蛋白(清蛋白和球蛋白)、醇溶蛋白和谷蛋白的含量进行了测定, 利用聚丙烯酰胺凝胶电泳(SDS-PAGE)技术分析了其特异性。结果表明: 同源四倍体水稻胚乳蛋白各组分含量与相应的二倍体相比, 大部分呈增加趋势; 同源四倍体水稻胚乳蛋白的亚基类型与相应的二倍体水稻基本一致, 仅在全蛋白电泳和清、球蛋白电泳中各发现1条差异条带。研究结果认为: 二倍体水稻经过染色体组加倍之后, 同源四倍体水稻重复基因组在蛋白质水平上的表达结果趋于“二倍化”, 即与二倍体水稻表现出相似的特点, 但在蛋白质表达量上前者往往高于后者。基因组多倍化对同源四倍体水稻重复基因组进化最主要的影响并不是体现在蛋白质结构上的差异, 而是体现在蛋白质表达量上的差异。     

The trafficking pathway of a wheat storage protein in transgenic rice endosperm

Background and Aims

The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these.

Methods

The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies.

Key results

The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells.

Conclusions

The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.     

Storage-protein Deposition in the Developing Rice Caryopsis in Relation to the Transport Tissues

The developing caryopsis of rice (Oryza sativa L.) was examinedhistologically at successive stages of grain-filling in orderto identify the factors which determine the distribution ofstorage protein in the endosperm, and which terminate the depositionof endosperm protein. The storage protein was deposited at theperiphery of the endosperm, and this distribution was apparentlycaused by the radial pattern of cell development in the endosperm,and by the proximity of the peripheral endosperm cells to thenucellar epidermis. The nucellar epidermis directly surroundsthe endosperm and functions as the pathway for amino acid transportto the endosperm. During the later stages of caryopsis developmentthe nucellar epidermis became compressed by being ‘sandwiched’between the expanding endosperm and the rigid hull (the tightlylocked palea and lemma) which encloses the caryopsis. It isproposed that this compression of the nucellar epidermis blocksthe supply of amino acids to the endosperm and thereby terminatesthe deposition of storage protein in the rice grain. Oryza sativa, rice, caryopsis (development), endosperm, grain filling, nucellar epidermis, storage protein     

Purification and some properties of starch branching enzyme (Q-enzyme) from tuberous root of sweet potato

The pattern of isoforms of starch branching enzyme II or Q-enzyme II in the tuberous root of sweet potato was distinct from those of other organs; altogether 7 isoforms of QEII were contained in the sweet potato plant. The QEIIf isoform, one of the two major QEII isoforms in the tuberous root, was purified to homogeneity by using a variety of HPLC columns. The purified QEIIf was a monomeric protein with a molecular mass of about 85 kDa. Western blot analysis showed that the polyclonal antibodies raised against the purified QEIIf was significantly reactive to the rice endosperm QEI, but not to the rice endosperm QEIIa. Furthermore, the sweet potato QEIIf reacted with the antiserum raised against the rice endosperm QEI, but not with that against the rice endosperm QEIIa. The results suggest that the sweet potato QEIIf is more similar to the rice endosperm QEI than to the rice endosperm QEIIa.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds II. Scutellum as the Site of Sucrose Synthesis

In a close parallel to the developmental pattern of α-amylase activity, a rapid increase of maltase activity occurred in the endosperm tissue of germinating rice seeds after about 4 days of the seed imbibition. The overall pattern of the 2 hydrolytic enzyme activities strongly suggest that amylolytic breakdown is the major metabolic route of starch utilization in the germinating rice seeds. Results of the chemical analyses of sugar constituents as well as the measurements of sucrose synthetase activity show that the scutellum is the site of sucrose synthesis in the germinating rice seeds. It is thus supported that glucose derived from the reserve starch in endosperm is transported to scutellum, where it is converted to sucrose. Sucrose is further mobilized to the growing tissues, shoots and roots.     

Thin Frozen Film Method for Visualization of Storage Proteins in Mature Rice Grains

There are technical difficulties in obtaining intact sections of cereal grains in which mature cells and their subcellular structures are well preserved. Here we describe a simple method for sectioning hard mature rice grains. It makes possible accurate localization of storage proteins in high-quality histological sections of rice endosperm.