水稻蜡质基因及其利用研究进展

简要介绍了水稻蜡质基因的等位基因位点及其表达特性。并从蜡质基因第1内含子核苷酸和蜡质基因剪切方式以及蜡质基因相关的顺式作用元件与反式作用因子等方面阐述了水稻蜡质基因表达调控,还概述了水稻蜡质基因的分子标记辅助选育方面的研究。从近年来的报道显示,利用反义RNA技术抑制水稻内源蜡质蛋白的表达是目前蜡质基因应用研究的主要方向。关于蜡质基因的研究,在其它作物中也都有报道。     

水稻蜡质基因5‘非翻译区一个与调控有关的内含子

从发育的水稻种子中分离出ＲＮＡ,经ＲＴ－ＰＣＲ反应并结合顺序测定,在籼稻２３２蜡质基因编码区５‘上游非翻译区中证明确实存在一个长度为１１２６ｂｐ的内含子,其Ａ＋Ｔ碱基的含量高达６７．４％,它的边界符合真核基因内含子的ＧＴ－ＡＧ规则。表明该内含子与蜡质基因的表达调控有一定的关系。     

糯性水稻中蜡质基因第1内含子的剪接活性

以前的研究结果表明 ,非糯性的稻米中直链淀粉的含量与各品种中蜡质基因 (Wx)第 1内含子被剪接的效率有关 ,即剪接效率高的品种直链淀粉含量也高。为弄清糯米中不含直链淀粉是否也与此内含子剪接效率有关系 ,构建了蜡质基因启动子 (来自籼稻品种2 32 )、蜡质基因第 1内含子 [来自籼稻品种 2 32 (高直链淀粉含量的品种 )或粳稻品种寒丰 6 36 6 (中、低直链淀粉含量的品种 ) ]与GUS报告基因组成的两种嵌合基因 ,将含有这两种嵌合基因的质粒分别转化进粳性糯稻品种奉糯 5 93中 ,同时还分别转化进非糯性的籼稻品种特青和粳稻品种中花 11中作为对照 ,测定它们的抗性愈伤组织与转基因植株未成熟种子胚乳中的GUS酶活性。结果表明与在非糯性的籼稻和粳稻中一样 ,这两种嵌合质粒在不合成直链淀粉的糯稻中也有相当水平的表达。从此结果可以推测 ,糯稻品种有从嵌合基因的转录本中剪接蜡质基因第 1内含子的正常能力 ,而在糯稻中缺乏直链淀粉可能是糯稻蜡质基因第1内含子中的其它突变所造成的     

糯性水稻中蜡质基因第1内含子的剪接活性

以前的研究结果表明,非糯性的稻米中直链淀粉的含量与各品种中蜡质基因（Ｗｘ）第１内含子被剪接的效率有关,即剪接效率高的品种直链淀粉含量也高。为弄清糯米中不含直链淀粉是否也与此内含子剪接效率有关系,构建了蜡质基因启动子,（来自灿稻品种２３２）、蜡质基因第１内含子「来自籼稻品种２３２（高直链淀粉含量的品种）或粳稻品种寒丰６３６６（中、低直链淀粉含量的品种）或粳稻品种寒丰６３６６（中、低直链淀粉含量的品种     

水稻蜡质基因第一内含子碱基突变引起其RNA二级结构的可能变化

通过体外转录得到籼稻品种232蜡质基因第一内含子5’端430 bp的ssRNA分子,以及在此区域发生了自然突变的粳稻品种寒丰蜡质基因第一内含子5’端同样长度的ssRNA分子。部分变性胶电泳结果表明两种ssRNA分子的迁移速率不同。将两种ssRNA分子的核酸序列用计算机分析,表明此两种ssRNA分子能形成不同的茎-环结构,自由能值也有差异。对突变引起的这些不同与两种水稻品种蜡质基因转录本剪接的差异进行了讨论。     

水稻蜡质基因第一内含子中g片段上核蛋白因子结合位点的确定

水稻蜡质基因５’非翻译区中的第一内含子具有增强基因表达的作用,本实验室曾检测到该内含子中的一段１７１ｂｐ长的ｇ的片段与水稻未成熟种子核蛋白存在特异性结合。本文进一步用凝胶滞后实验和足印实验确定了该核蛋白在ｇ片段上的结合位点位于蜡质基因转录起始点下游７８３－８１８ｂｐ处,该结合位点富含ＡＴ碱基;Ｓｏｕｔｈｗｅｓｔｅｍ ｂｌｏｔ实验测出该蛋白的分子量约为２０ｋＤ。用硫酸铵分步沉淀和肝素－Ｓｅｐｈａｒｏ     

水稻蜡质基因第一内含子中g片段上核蛋白因子结合位点的确定

水稻蜡质基因 5’非翻译区中的第一内含子具有增强基因表达的作用 ,本实验室曾检测到该内含子中的一段 171bp长的g片段与水稻未成熟种子核蛋白存在特异性结合。本文进一步用凝胶滞后实验和足印实验确定了该核蛋白在g片段上的结合位点位于蜡质基因转录起始点下游 783～ 818bp处 ,该结合位点富含AT碱基 ;Southwesternblot实验测出该蛋白的分子量约为 2 0kD。用硫酸铵分步沉淀和肝素 Sepharose柱层析的方法对这一蛋白进行了初步纯化。还初步检测了此蛋白DNA结合活性对温度的耐受性。以上特征提示它可能属于HMG类DNA结合蛋白     

水稻蜡质基因第一内含子碱基突变引起其RNA二级结构的可能变化

通过体外转录得到籼稻品种２３２蜡质基因第一内含于５’端４３０ｂｐ的ｓｓＲＮＡ分子,以及在此区域发生了自然突变的粳稻品种寒丰蜡质基因经一内含子５’端同样长度的ｓｓＲＮＡ分子。部分变性胶电泳结果表明两种ｓｓＲＮＡ分子的迁移速率不同。将两种ｓｓＲＮＡ分子的核酸序列用计算机分析,表明此两种ｓｓＲＮＡ分子能形成不同的茎－环结构,自由能值也有差异。对突变引起的这些不同与两种水稻品种蜡质基因转录本剪接的差异进行     

用于筛选直链淀粉含量为中等的籼稻品种的分子标记

用PCR AccⅠ分子标记检测方法 ,检测了来自不同地区的 6 3个栽培水稻品种 (系 )蜡质基因第 1内含子剪接供体 1位碱基是G或是T。另外 ,还测定了这些水稻成熟种子的直链淀粉含量。结果显示该位置是G碱基的水稻品系成熟种子中直链淀粉含量均高于 2 0 % ,该位置是T的均低于 18%。在杂交育种过程中 ,这一分子标记可用于预测水稻植株种子的直链淀粉含量。对高直链淀粉含量的水稻亲本与中等直链淀粉含量的水稻亲本之间 5个籼型杂交组合F2 群体的分析表明 ,蜡质基因第 1内含子 1位碱基是G或是T与水稻种子中直链淀粉含量的高或低是紧密连锁 ,共同分离的。这些结果表明PCR AccⅠ分子标记检测方法可用于选育中等直链淀粉含量的籼稻新品系     

小麦Wx基因及其在农业生产中的潜在应用

围绕影响小麦品质的蜡质基因,介绍了小麦蜡质基因突变体筛选、蜡质基因对淀粉合成的影响、蜡质基因的分子遗传和蜡质小麦培育的研究近况,并对Wx基因的结构特征和分子标记辅助选择进行了分析,还就Wx基因的研究前景和问题作了分析和展望.     

Increment of hFIX expression with endogenous intron 1 in Vitro

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence.hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA,and plasmid vector pKG5i‘IX,retroviral vector G1NaCi‘IX were constructed.These vectors were transduced into target cells of PA317,C2C12,primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF).The expression level of mixed colonies are PA317/pKG5i‘IX,151 ng/10^6 cells/24h;PA317/G1NaCi‘IX,308 ng/10^6 cells/24 h;C2C12/G1NaCi‘IX,186 ng/10^6 cells/24 h;RSF/G1NaCi‘IX,1929 ng/10^6 cells/24 h;HSF/G1NaCi‘IX,1646 ng/10^6 cells/24 h.These results indicated that hFIX minigene with intron l is able to increase the expression level to about 3 times of that of hFIX cDNA.Meanwhile,in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production,a retroviral vector G1NaCi‘IXR with reversely inserted hFIX minigene expression cassette was constructed.The expression level of reverse constructor in PA317 cells was 390 ng/10^6 cells/24 h with 79% of bioactivity.PCR detection of HT/G1NaCi‘IXR cells infected with PA317/G1NaCi‘IXR supernatant confirmed the existence of intron 1 sequence.These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene transfer,but when using the retroviral-mediated gene transfer system,reversely-inserted intronl-carrying hFIX expression cassette should be considered.     

外显子和内含子的序列复杂性

引入了两个新的关于序列复杂性的测度,并以此为指标分析比较了结构基因序列中的外显子和内含子的复杂性差异。     

A new method for precipitating bacterial exopolysaccharides

Summary Several methods for exopolysaccharide precipitation are compared and a new method is proposed which uses nitron (3,5,6-triphenyl-2,3,5,6-tetraaza[2.1.1]bicyclo-1-hexene) as the precipitating agent and gives recoveries of about 99%, using only 0.1 vol.. The highest recovery obtained with the usual methods was around 75%, using 3 vol. propanol, but this makes the final recovery more difficult.     

转基因动物乳腺暂时性表达系统的建立

以乳清酸蛋白(WAP)基因5′区为调控序列,人基因组G-CSF基因为目的片段,同时将WAP基因第一内含子插入G-CSF基因5′端,构建成转基因动物乳腺表达载体。将其直接注射到小鼠乳腺,在泌乳期表达出人G-CSF。表明乳腺直接注射质粒的方法可以做为暂时性的一种表达系统,同时也表明内含子对表达有一定的作用。     

人癌细胞内质网分子伴侣Grp94基因近3′端的初步分析

采用PCR及RT－PCR方法结合核酸分子杂交技术,首次确定了与Grp94cDNA2231～2500碱基片段对应的Grp94基因序列上有内含子．含有内含子的扩增片段长约708bP．对三种癌细胞系进行了PCR和RT－PCR反应,电泳结果显示存在差异．研究结果为进一步探讨Grp94基因3'端精细结构及其在正常细胞和肿瘤细胞中表达的改变打下了基础．     

Characterization of 5‘—proximal sequence of mouse GABA transporter gene （GAT—1）

The cDNA molecule encoding the mouse GABA transporter gene(GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library.A positive clone,harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G,was fished out from the library,the 5‘ proximal region and intron 1 were sequenced and analysed,and low homology was found in the above region between GAT-1 genes from mouse and human except some short conserved sequences.The DNA-protein interactions between DNA fragments containing the conserved sequences in the 5‘ proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shift assay,and Southern-Western blot.The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.     

A comparative spin trapping study of hypobromous and hypochlorous acids interaction with tert-butyl hydroperoxide

We have demonstrated that hypochlorite (HOCI/OCl-) and hypobromite (HOBr/OBr-) can react with tert-butyl hydroperoxide with close rate constants (k(HOCl) = 10,8 M(-1) x s(1); k(HOBr) = 8,9 M(-1) x (s(-1)). By means of the spin trap 4-pyridyl-1-oxide-N-tert-butyl nitron we have found that both reactions proceed through decomposition of tert-butyl hydroperoxide and generation of tert-butyl peroxyl (OOC(CH3)3) and tert-butoxyl (OC(CH3)3) radicals, the ratio of their the concentrations being dependent on the concentration of tert-butyl hydroperoxide. Thus, hypobromite, similar to hypochlorite, is a precursor of free radicals produced in the reaction with organic hydroperoxides. This reaction can be of great importance in the intensification of free radical processes, namely, in lipid peroxidation at the stage of chain branching.     

Differential growth rates of Candida utilis mother and daughter cells under phased cultivation.

The yeast Candida utilis was continuously synchronized by the phased method of cultivation with the nitrogen source as the growth-limiting nutrient. The doubling time (phasing period) of cells was 6 h. Both cell number and deoxyribonucleic acid synthesis showed a characteristic stepwise increase during the phased growth. The time of bud emergence coincided with the time of initiation of deoxyribonucleic acid synthesis. Size distribution studies combined with microscopic analysis showed that the cells expanded only during the unbudded phase of growth. Usually the cells stopped increasing in size about 30 min before bud emergence, and the arrest of the increase in cell volume coincided with the exhaustion of nitron from the medium. There was no net change in the volume of cells during the bud expansion phase of growth, suggesting that as the bud expanded, the volume of the mother portion of the cell decreased. After division the cells expanded slightly. The postdivision expansion of cells, unlike the growth before bud initiation, occurred in the absence of the growth-limiting nutrient. The newly formed daughter cells were smaller than the mother cells and expanded at a faster rate, so that both types of cells reached maximum size at the same time. Possible reasons for the different rates of expansion of mother and daughter cells are discussed.     

Immunochemical detection of hemoglobin-derived radicals formed by reaction with hydrogen peroxide: involvement of a protein-tyrosyl radical

To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/H2O2 system, we have used a new approach called "immuno-spin trapping," which combines the specificity and sensitivity of both spin trapping and antigen:antibody interactions. Previously, a novel rabbit polyclonal anti-DMPO nitrone adduct antiserum, which specifically recognizes protein radical-derived nitrone adducts, was developed and validated in our laboratory. In the present study, the formation of nitrone adducts on hemoglobin was shown to depend on the oxidation state of the iron heme, the concentrations of H2O2 and DMPO, and time as determined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The presence of reduced glutathione or L-ascorbate significantly decreased the level of nitrone adducts on metHb in a dose-dependent manner. To confirm the ELISA results, Western blotting analysis showed that only the complete system (oxy- or metHb/DMPO/H2O2) generates epitopes recognized by the antiserum. The specific modification of tyrosine residues on metHb by iodination nearly abolished antibody binding, while the thiylation of cysteine residues caused a small but reproducible decrease in the amount of nitrone adducts. These findings strongly suggest that tyrosine residues are the site of formation of the immunochemically detectable hemoglobin radical-derived nitrone adducts. In addition, we were able to demonstrate the presence of hemoglobin radical-derived nitrone adducts inside red blood cells exposed to H2O2 and DMPO. In conclusion, our new approach showed several advantages over EPR spin trapping with the anti-DMPO nitrone adduct antiserum by demonstrating the formation of tyrosyl radical-derived nitrone adduct(s) in human oxyHb/metHb at much lower concentrations than was possible with EPR and detecting radicals inside RBC exposed to H2O2.