一种水稻酰基辅酶A结合蛋白cDNA的鉴定

对一水稻ｃＤＮＡ克隆（Ｒ１９０８）的分析表明,其可能编码水稻酰基辅酶Ａ结合蛋白（ａｃｙｌ－ＣｏＡ－ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ,ＡＣＢＰ）。Ｓｏｕｔｈ－ｅｒｎ杂交显示水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ）基因组中仅有一个该基因的拷贝。Ｎｏｒｔｈｅｒｎ分析表明水稻的ＡＣＢＰ基因在水稻的根、茎、叶、叶鞘、黄化苗和幼穗中皆表达,而以黄化苗的绿苗叶鞘中的表达高于绿苗叶片。     

大鼠酰胺化酶的信号肽引导的昆虫细胞表达外泌系统

将大鼠酰胺化酶的信号肽及前导肽编码序列引入昆虫核多角体病毒转移表达载体,构建ＰＡＢＣｈＧＲＦ（Ｇｌｙ）、ＰＡＢＣＩＧＦＩ融合基因的昆虫细胞分泌表达质粒ｐＢａｃＰＡＧ２、ｐＢａｃＰＡＩ,并与经修饰的银纹夜蛾核多角体病毒ＢａｃＰＡＫ６线性化ＤＮＡ共转染秋粘虫细胞Ｓｆ２１,通过同源重组、筛选和鉴定,得到它们的重组病毒ＢａｃＰＡＧ、ＢａｃＰＡＩ。将重组病毒感染Ｓｆ２１细胞,ＰＡＢＣｈＧＲＦ（Ｇｌｙ）和ＰＡＢＣＩＧＦＩ均得到有效外泌表达,表达产物通过ＩｇＧＳｅｐｈａｒｏｓｅ柱可获得快速纯化。     

青蒿转杜松烯合成酶基因发根系的培养

将已克隆的棉花杜松烯合成酶的ｃＤＮＡ（ｃａｄＣ１４）插入到植物表达载体ｐＢＩ１２１中,构建含ＣａＭＶ３５Ｓ启动子驱动下的杜松烯合成酶基因的植物表达载体ｐＢＩＣ１４。用含ｐＢＩＣ１４质粒的发根农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ）１５８３４感染青蒿（ＡｒｔｅｍｉｓｉａａｎｎｕａＬ．）叶片并诱导发根,共建立１２１个生长迅速的发根系。经浓度为２０ｍｇ／Ｌ的Ｋａｎ筛选,获得１２个抗Ｋａｎ阳性根系。ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ分析表明,外源杜松烯合成酶基因已整合到青蒿基因组中,其转基因频率为３％。ＲＴＰＣＲ分析表明,外源杜松烯合成酶基因在Ｃ３７根系中,在转录水平上已有表达。     

蛋白质N端豆蔻酰化修饰的基因工程表达偶联加工系统

对酵母ＮＭＴ基因在大肠杆菌中表达进行较详细的研究,进而构建了复制子为ｐ１５Ａ并含卡那霉素抗性基因的相容性表达质粒ｐＫＺＭＴ,将其与表达质粒ｐＣＺｍＣα１共转化进大肠杆菌ＢＬ２１（ＤＥ３）Ｆ′,进行双质粒表达偶联加工修饰研究,其中ｐＣＺｍＣα１表达底物蛋白小鼠ｃＡＭＰ依赖的蛋白激酶催化亚基α（ＰＫＡ-ｍＣα）。ＳＤＳＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ分析表明,双质粒表达系统中,ＰＫＡ-ｍＣα都得到了稳定的高表达,尤其在２３℃低温诱导表达时,表达产物的可溶性部分明显增多;而酵母ＮＭＴ被控制在有利于活性功能的可溶性低水平表达。［^３Ｈ］ｍｙｒｉｓｔｉｃａｃｉｄ标记测定及放射自显影的结果显示,在大肠杆菌中表达的重组ＰＫＡ-ｍＣα被豆蔻酰化修饰。     

抗癌胚抗原单链抗体基因在家蚕细胞和幼虫中的表达

将抗癌胚抗原（ＣＥＡ）单链抗体基因插入家蚕杆状病毒转移载体ｐＢａｃＰＡＫＨｉｓ, 与修饰的家蚕核型多角体病毒ＢｍＢａｃＰＡＫＤＮＡ共转染家蚕细胞, 经同源重组得到含有在多角体蛋白基因启动子控制下的抗ＣＥＡＳｃＦｖ 基因的重组病毒ＢｍＢａｃＳｃＦｖ。用重组病毒分别感染家蚕细胞和幼虫, 在两者中均得到了高效表达, 产物分子量为２８ｋＤ, 前者占细胞总蛋白的６ ％ , 后者为０ ．３ ｍｇ／ 蚕。目的基因在家蚕细胞和幼虫中表达产物经Ｎｉ２＋ＩＤＡＳｅｐｈａｒｏｓｅ６Ｂ亲和柱纯化, 前者纯度可达９０％ 以上, 后者纯度较低; 纯化后的融合蛋白具有ＣＥＡ 结合活力, 其亲和常数分别为５ ．４×１０８／ｍｏｌ·Ｌ－ １ 和２．３ ×１０８／ｍｏｌ·Ｌ－１ , 略低于其亲本单抗Ｅ７Ｂ１０ ２．７ ×１０９／ｍｏｌ·Ｌ－ １ 。     

蚕豆叶片下表皮ABA结合蛋白提取及分离条件的选择

以蚕豆（ＶｉｃｉａｆａｂａＬ．）叶片下表皮为材料,比较ＴｒｉｔｏｎＸ１００、冷丙酮和（ＮＨ４）２ＳＯ４对ＡＢＡ结合蛋白（简称ＡＢＡＢＰ）的提取效果。结果表明：０．５％（Ｗ／Ｖ）ＴｒｉｔｏｎＸ１００去垢剂提取的ＡＢＡＢＰ与ＡＢＡ特异结合活性较高（０．４８７ｎｍｏｌ／ｇｐｒｏｔｅｉｎ）,维持结合活性的时间较长（４℃下反应４０ｈ保持最大结合的６０％）;而冷丙酮法提取的ＡＢＡＢＰ特异结合活性只有０．３２５ｎｍｏｌ／ｇｐｒｏｔｅｉｎ,且容易失活,１０ｈ仅保持最大结合的３０％左右。实验比较了各种盐离子对ＡＢＡＢＰ的影响,高盐（＞３００ｍｍｏｌ／ＬＮａＣｌ）不利于ＡＢＡＢＰ的结合反应,低浓度ＫＣｌ对ＡＢＡＢＰ活性略有促进。ＡＢＡＢＰ的结合活性需要介质中有一定量的Ｃａ２＋和Ｍｇ２＋,用ＥＤＴＡ螯合介质中Ｍｇ２＋、Ｃａ２＋后,ＡＢＡＢＰ活性大大降低,分别为最大结合的７５％和６０％。ＡＢＡＢＰ与ＡＢＡ反应的最适ｐＨ在６．５,这些条件为亲和层析纯化ＡＢＡＢＰ提供了依据。     

高产稳产聚羟基烷酸的重组大肠杆菌的构建

重组大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＨＭＳ１７４（ｐＴＺ１８ＵＰＨＢ） 含有携带聚羟基烷酸（ＰＨＡ） 合成基因（ ｐｈａＣＡＢ）＊＊ 的质粒ｐＴＺ１８ＵＰＨＢ,是很有潜力的ＰＨＡ 生产菌,但存在着质粒不稳定和不能合成３羟基丁酸（３ＨＢ） 与３羟基戊酸（３ＨＶ） 共聚物［Ｐ（３ＨＢｃｏ３ＨＶ）］ 的缺陷。将ＲＫ２ 质粒上的ｐａｒ ＤＥ 基因引入ｐＴＺ１８ＵＰＨＢ 构成质粒ｐＪＭＣ２ ,该质粒可以在宿主Ｅ．ＣｏｌｉＨＭＳ１７４ 中稳定遗传。将培养基中的磷酸盐浓度降至１８ ｍ ｍｏｌ／Ｌ,发现Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） 能够以丙酸为前体合成Ｐ（３ＨＢｃｏ３ＨＶ） ,其中３ＨＶ 在共聚物中的含量为５ ％ ～８ ％ 。在５Ｌ自动发酵罐中分批补料培养Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） ,培养基初始磷酸盐浓度为１５ ｍ ｍｏｌ／Ｌ,３０ ｈ 后每升培养液中干菌体可达４２－５ ｇ,Ｐ（３ＨＢｃｏ３ＨＶ） 占干重的７０ ％ ,其中３ＨＶ 在共聚物中的含量为４－９ ％ 。     

编码Synechococcus sp. PCC 7002 FNR 中FNR区的基因克隆及其在大肠杆菌中的表达

用ＰＣＲ的方法克隆出了编码蓝细菌Ｓｙｎｅｃｈｏｃｏｃｃｕｓｓｐ．ＰＣＣ７００２ＦＮＲ中ＦＮＲ区的基因ｐｅｔＨＬ,克隆到达载体ｐＥＴ３ａ上,转化大肠杆菌ＢＬ２１（ＤＥ３）后实现了大量表达。重组ＦＮＲ区（ｒＦＮＲＤ）经ＤＥＡＥＳｅｐｈｄｅｘＡ５０离子交换层析及ＳｅｐｈａｄｅｘＧ１００凝胶层析得到大量的电泳均一的ｒＦＮＲＤ。Ｎ末端氨基酸序列分析表明,表达产物确为ｐｅｔＨＬ所编码。且起始Ｍｅｔ翻译后未被除去。ｒＦＮＲＤ与ｒＦＮＲ的吸收光谱相同,其黄递酶活性的最适ｐＨ和最适温度也相同。ｒＦＮＲＤ能在体外催化电子从Ｐ７００到ＮＡＤＰ＋的传递     

植物表达载体pKC—3的构建及大分子DNA连接策略

以根癌农杆菌双元载体ｐＣＡＭＢＬＡ１３００为基础,先后连接含有马铃薯蛋白酶抑制剂－Ⅱ基因（ＰⅡ）、苏云金杆菌毒蛋白基因（Ｂ．ｔ ｃｒｙＩ（Ａ））及雪花莲外源凝集素基因（ＧＮＡ）的完整表达片段,构建了抗多种稻田害虫的表达载体ｐＫＣ－３,将其导入根癌农杆菌ＬＢＡ４４０４,可进一步用于水稻抗虫基因转化的研究。载体构建进程采用多次的大分子ＤＮＡ片段连接,总结出了一套适合大片段连接转化的可行策略。     

Ⅰ-A~k基因克隆转导及对肿瘤细胞成瘤的影响

采用ＲＴＰＣＲ方法合成小鼠ＭＨＣⅡ类分子ⅠＡｋ基因α和β链ｃＤＮＡ,插入逆转录病毒载体ｐＬＳＸＮ,构建ⅠＡｋα和ⅠＡｋβ表达载体,采用脂质体介导的重组质粒转移方法将ⅠＡｋα和ⅠＡｋβ基因导入ＥＬ４小鼠淋巴瘤细胞和Ｐ８１５小鼠肥大细胞瘤细胞,经流式细胞仪检测在细胞表面有ⅠＡｋ表达．将以上两种细胞注射到同源小鼠Ｃ５７ＢＬ／６（Ｈ２ｄ）皮下,观察到肿瘤产生后又消退,证明在肿瘤细胞中单独导入同种异型ＭＨＣⅡ类分子基因也能激活肿瘤的细胞免疫,为进一步开展肿瘤的基因治疗奠定了基础．     

Proteome approach to characterize the methylmalonate-semialdehyde dehydrogenase that is regulated by gibberellin

Proteins regulated by gibberellin (GA) in rice were determined by proteome analysis. Proteins extracted from suspension culture cells of slr1, a constitutive GA response mutant of rice, were separated by two-dimensional polyacrylamide gel electrophoresis, and three proteins were greatly accumulated in the mutant. The most up-regulated protein was methylmalonate-semialdehyde dehydrogenase (MMSDH), and the amount of protein was 7-fold that of wild type. In this study, the function of MMSDH in rice was analyzed. MMSDH gene expression in suspension culture cells, roots, and leaf sheaths ofslr1 was higher than that in its wild-type. MMSDH expression in wild-type roots was increased by exogenous GA(3). Analyzed by in situ hybridization, MMSDH mRNA was expressed in root primordia of slr1, where cells are undergoing growth. MMSDH gene expression in the root zone of tissue differentiation was higher than in the elongation zone or meristem. Transgenic rice expressing antisense MMSDH showed that its seminal roots were thinner than that of control, and that the leaf sheath elongation was slightly inhibited compared to control. Concentrations of TCA cycle metabolites were decreased in the antisense plants as compared with the control plants, suggesting that acetyl-CoA was reduced in the antisense plants. These results suggest that one of the regulations by GA signal transduction including SLR1 is the expression of MMSDH, and that MMSDH may play a role in root development and leaf sheath elongation in rice.     

Interaction of N-acetylglutamate kinase with a PII-like protein in rice

PII protein in bacteria is a sensor for 2-oxoglutarate and a transmitter for glutamine signaling. We identified an OsGlnB gene that encoded a bacterial PII-like protein in rice. Yeast two-hybrid analysis showed that an OsGlnB gene product interacted with N-acetylglutamate kinase 1 (OsNAGK1) and PII-like protein (OsGlnB) itself in rice. In cyanobacteria, NAGK is a key enzyme in arginine biosynthesis. Transient expression of OsGlnB cDNA or OsNAGK1 cDNA fused with sGFP in rice leaf blades strongly suggested that the PII-like protein as well as OsNAGK1 protein is located in chloroplasts. Both OsGlnB and OsNAGK1 genes were expressed in roots, leaf blades, leaf sheaths and spikelets of rice, and these two genes were coordinately expressed in leaf blades during the life span. Thus, PII-like protein in rice plants is potentially able to interact with OsNAGK1 protein in vivo. This finding will provide a clue to the precise physiological function of PII-like protein in rice.     

Infection Process of Burkholderia glumae Before Booting Stage of Rice

Burkholderia glumae is a well‐known pathogen for causing bacterial panicle blight of rice. In this study, the infection process of B. glumae in rice plants at different growing stages was tracked by means of real‐time fluorescence quantitative PCR. Burkholderia glumae tended to colonize at the growing point of rice plants, and the biomass of population was 10⁴ to 10⁸ CFU/g. The most intensive colonization was detected in the upmost leaf in the two‐leaf period. However, after the two‐leaf period, the population of pathogens decreased significantly, and they successfully recovered in the booting stage and broke out in panicles. We also illustrated the incubation location of B. glumae by presenting the infection pattern in the seedling and tillering stage of rice. Under fluorescent microscopy, the gfp‐labelled pathogens were first found in the vascular bundle of lateral roots, taproots and injured cells, then they were observed in the root hairs, epidermal cells and main root cap. The pathogens in the vascular bundle laterally dispersed towards the epidermal cells. By spray application of a bacterial suspension, the pathogens landed on the leaf sheaths and leaves, colonized in the epidermal hairs and leaf hairs, or invaded into the cells through the stomas. At the same time, the pathogens from the vascular bundle of the roots spread into the vascular bundle of leaf sheaths and leaves, which caused the leaves to curl and wilt, beginning from the tip.     

Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis

We have isolated a new recessive dwarf mutant of rice (Oryza sativa L. cv Nipponbare). Under normal growth conditions, the mutant has very short leaf sheaths; has short, curled, and frizzled leaf blades; has few tillers; and is sterile. Longitudinal sections of the leaf sheaths revealed that the cell length along the longitudinal axis is reduced, which explains the short leaf sheaths. Transverse sections of the leaf blades revealed enlargement of the motor cells along the dorsal-ventral axis, which explains the curled and frizzled leaf blades. In addition, the number of crown roots was smaller and the growth of branch roots was weaker than those in the wild-type plant. Because exogenously supplied brassinolide considerably restored the normal phenotypes, we designated the mutant brassinosteroid-dependent 1 (brd1). Further, under darkness, brd1 showed constitutive photomorphogenesis. Quantitative analyses of endogenous sterols and brassinosteroids (BRs) indicated that BR-6-oxidase, a BR biosynthesis enzyme, would be defective. In fact, a 0.2-kb deletion was detected in the genomic region of OsBR6ox (a rice BR-6-oxidase gene) in the brd1 mutant. These results indicate that BRs are involved in many morphological and physiological processes in rice, including the elongation and unrolling of leaves, development of tillers, skotomorphogenesis, root differentiation, and reproductive growth, and that the defect of BR-6-oxidase caused the brd1 phenotype.     

Fate of fructose supplied to leaf sheaths after defoliation of Lolium perenne L.: assessment by 13C-fructose labelling

The role of fructans from leaf sheaths for the refoliation of Lolium perenne after severe defoliation was assessed by following the fate of (13)C-fructose supplied to leaf sheaths at the time of defoliation. At the end of the 4 h labelling period on defoliated plants, 77% of the (13)C incorporated was still located in leaf sheaths. Only 4% and 0.9% were, respectively, allocated to stem and roots, while 18% was imported by the growing leaves where (13)C was allocated first to the proximal part of the leaf growth zone (0-10 mm). In all tissues, the most highly (13)C-labelled carbohydrates was not fructose but sucrose. In leaf sheaths, (13)C-loliose was produced. In the leaf growth zone (0-20 mm), fructans were simultanously synthesized from (13)C entering the leaves and degraded. The export of (13)C from leaf sheaths continued during the first day of regrowth but stopped afterwards. There was no net loss of C from (13)C-fructose over the first 2 d of regrowth. The role of fructans and loliose is discussed as well as the physiological mechanisms contributing to defoliation tolerance in L. perenne.     

Overexpression of rice OSH genes induces ectopic shoots on leaf sheaths of transgenic rice plants

Five rice homeobox (OSH) genes were overexpressed under the control of the cauliflower mosaic virus 35S promoter or the rice actin gene promoter in transgenic rice plants. Almost all of the transgenic plants showed abnormal phenotypes, which could be classified into three types according to their severity. Plants with the most severe phenotype formed only green organs, with many shoot apices on their adaxial sides. Plants with an intermediate phenotype formed bladeless leaves with normally developed leaf sheaths. Plants with a mild phenotype formed normal leaf sheaths and blades, but lacked ligules and showed diffusion of the blade-sheath boundary. The leaf structure of this phenotype was similar to that of dominant maize mutants, such as Kn1, Rs1, Lg3, and Lg4. Based on these phenotypes, we suggest that ectopic expression of the rice OSH genes interferes with the development of leaf blades and maintains leaves in less differentiated states. These results are discussed in relation to the leaf maturation schedule hypothesis (M. Freeling et al., 1992, BioEssays 14, 227-236).     

Isolation of a 1 195 bp 5′-Flanking Region of Rice Cytosolic Fructose-1,6-bisphosphatase and Analysis of Its Expression in Transgenic Rice

A genomic DNA fragment containing the 5′-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 bp 5′-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding β-glucuronidase (GUS) and stably transferred to rice via particle bombardment. Strong GUS activity was detected in leaves and leaf sheaths of transgenic rice, but not in culms and roots. Histochemical localization revealed that GUS expression was exclusively restricted to mesophyll cells in transgenic rice. Our results indicate that the 1 195 bp fragment contains all the cis-elements required for directing mesophyll-specific expression pattern in rice. Key words: rice (Oryza sativa); promoter;　cytosolic fructose-1,6-bisphosphatase gene; mesophyll-specific expression