The series Variantia Ching et S.H.Wu mainly occur in China and its members are highly variable in morphology. The denomination on this group of Asplenium is very confused in the herbaria. We hope by means of a biosystematic study to find out their genetic relationships in the reticulate evolution, and to raise a suggestion on their taxonomic treatment. Evidence from cytology, allozyme, morphology, and palynology shows that three ancestor diploids have formed Asplenium sarelii complex comprising 13 members. A. sarelii Hook. Should be typified as a diploid. The so_called tetraploid “A. sarelii" before is an allotetraploid that comes from the doubled hybrid between diploid A. sarelii and A. tenuicaule Hayata, which should be treated as a new species A. wudangense Z. R. Wang et X. Hou. A. pekinense Hance is an autotetraploid that comes from the doubled diploid ancestor A. sarelii. A. lushanense C. C hr., a diploid species and the only ancestor of A. yunnanense group, should not been sunk as a synonym of tetraploid A. yunnanense Franch. Most probably, A. varians Wall. E x Hook. Et Grev. Is an autotetraploid of A. tenuicaule Hayata. Three new natural tetraploid hybrids and their origins have been found out: they are A.×longmenense (= A. pekinense × varians), A. × jingyunense (= A. pekinense × yunnanense) and A. × kidoi (=A. pekinense × wudangense). Three other new natural triploid hybrids have been found and their origins have been inferred: they are A. × huawuense (=A. sarelii × wudangense), A. × luyunense (=A. Lushanense × yunnanense) and A. × tenuivaians (=A. tenuicaule × varians). The method of allozyme comparion combined with cytological observation is employed to reveal the complicated relationships among the members of Asplenium sarelii complex in reticulate evolution and proved to be a highly effective tool to investigate the origin of polyploid and hybrid.
This study reports the anatomical structures of a kind of lepidodendralean stem in coal balls from the early Early Permian Taiyuan Formation in Yanzhou Mining District, southwestern Shandong Province, North China. The leaf cushion is slightly rhomboid in outline with a height of 9-10 mm and a width of 7.5-9.0 mm and its lower sides are slightly longer than the upper ones. The top and basic angles of the leaf cushion are truncate and the basic angle is slightly elongate. The upper part of the leaf cushion is strongly high_rising. The leaf scar is large and lenticular in shape. The leaf trace is wide and V_shaped in the leaf scar, and horizontally elongate within the leaf cushion. The leaf trace and lateral parichnos strand extend at a nearly horizontal course outward within the leaf cushion. The ligular pit is deep and extends outward at an oblique course and its aperture is located near the top angle of the leaf scar. No infrafoliar parichnos strands are present. The stem is probably siphonostelic and its pith is probably parenchymatous. The primary xylem is exarch with a nearly smooth outer margin. Only the outer cortex is present and it consists of alternately_arranged radial cell bands and gaps within which the arc_shaped or V_shaped leaf traces can be seen. The concave side of the leaf trace is toward the center of the stem. No bundle sheath is developed. Periderm is well_developed and consists of phelloderm and phellem in nearly equal thickness. Compared with the lepidodendralean stems of the Cathaysian and Euramerican Floras, the present specimens are most close to an impression_compression species Lepidodendron pulchrum Zhang in morphology of the leaf cushion and they are put into this species temporarily. Whether the present specimens or the type specimens of L. pulchrum are very different from Lepidodendron Sternburg sensu DiMichele, thus the correct nomenclature and classification of L. pulchrum needs to be reconsidered based on the study of better_ and anatomically_preserved stems and fertile organs in the future. Because “L”. Pulchrum possesses the mixed features of several genera of Euramerican lepidodendralean stems, it bears significance to study the origin and evolution of the Cathaysian lepidodendralean lycopods. Key words: Cathaysian Flora; Early permian; coal balls; “Lepidodendron” pulchrum; stems; anatomy
陈贵仁1 王士俊2* 田宝霖1
（1. 中国矿业大学，北京 100083；2. 中国科学院植物研究所，北京 100093）
摘要： 描述了山东兖州矿区太原组（早二叠世早期）煤核中一种具叶座的鳞木类茎的解剖构造。叶座呈略不对称的菱形，下侧边略长于上侧边，顶、底角均平截，底角并略呈拖延状；叶座上部突起较强烈，叶痕双凸镜形，叶迹呈宽V 字形，叶迹与侧通气束可能都以近水平状在叶座内向外延伸；叶舌穴深，自叶座最内处向外斜伸，开口位于叶痕顶角附近。叶座底部表面具横皱纹。茎可能具管状中柱和薄壁组织的髓。初生木质部外始式，外缘较平滑。仅见有外部皮层，由径向伸长的壁较厚的细胞条带和夹于其间的空腔交替排列构成，空腔内可见弧形或宽b 字形叶迹，其凹面朝向内方。周皮较发育，由近等量发育的木栓层和栓内层组成。经比较，当前标本与华夏植物区的印痕———压型化石种美丽鳞木在叶座的形态上非常相似，暂归入该种。由于无论是当前标本还是美丽鳞木的模式标本与狭义的鳞木属都有很大区别，因此美丽“鳞木”的确切归属还有待于今后进一步对保存更好的具解剖构造的茎和生殖器官的研究。美丽“鳞木”与欧美植物区几个乔木状鳞木类植物属的茎都不完全相同，具有它们的混合特征，很可能代表了一个新属。美丽“鳞木”是目前华夏植物区研究得最详细的一种具解剖构造的鳞木类的茎，对于研究华夏植物区鳞木类植物的起源和演化具有一定的意义。
NAD(P)H oxidases were detected in suspension cultured cells of ginseng (Panax ginseng C.A. Meyer). The activities of these enzymes were induced by an elicitor (Cle) extracted from cell walls of Colletotrichum lagerarium. In addition, Cle induced an oxidative burst and enhanced the synthesis of saponin, activity of phenylalanine ammonialyase (PAL), accumulation of chalcone synthase (CHS) and the transcription of a hydroxyproline_rich glycoprotein gene (hrgp). Pre_treatments with DPI and quinacrine (two inhibitors of mammalian neutrophil plasma membrane NADPH oxidase) for 30 min prior to Cle addition blocked the NAD(P)H oxidase activity induced by Cle. These inhibitors also inhibited the release of H2O2, the synthesis of saponin, PAL activity and CHS accumulation. Our data revealed homology between plasma membrane NAD(P)H oxidases of mammalian neutrophil cells and ginseng suspension cells. They also indicated that Cle_activated NAD(P)H oxidases catalysed the release of H2O2 and that H2O2 was functioning as a second messenger stimulating PAL activity, saponin synthesis and hrgp transcription. Elevations of Ca2+ and protein phosphorylation/dephosphorylation were required for this defense process. We propose that NAD(P)H oxidases mediate the processes of Cle_induced defense responses in ginseng suspensions, and postulate the existence of a signalling cascade including extracellular Cle stimulation, activation of plasma membrane NAD(P)H oxidases, release of H2O2, and the intracellular responses of metabolism and gene transcription in ginseng suspension cells.
质膜NAD（P）H 氧化酶参予金瓜炭疽（Colletotrichum lagerarium）细胞壁激发子诱导人参细胞产生激发反应
胡向阳1 Steven J. Neill2 方建颖1 蔡伟明1* 汤章城1
（1. 中国科学院上海生命科学研究院植物生理生态研究所，上海 200032；
2. Centra for Research in Plant Science, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, United Kindom）
摘要： 在人参（Panax ginseng C. A. Meyer）悬浮细胞质膜上测出了NAD（P）H 氧化酶活性。这类NAD（P）H 氧化酶活性可以被金瓜炭疽细胞壁激发子（Cle）诱导。Cle 处理还能诱导人参悬浮细胞的氧迸发、促进人参悬浮细胞的皂苷合成、提高苯丙氨酸解氨酶（PAL）的活力、以及诱导查尔式酮酶（CHS）的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp（Hydroxyprolin-rich glycoproteins）的表达。当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二亚苯基碘（Diphenylene iodonium, DPI）与奎吖因（quinacrine）预处理人参悬浮细胞30 min 后，Cle诱导的H2O2 释放与Cle 激活的质膜NAD(P)H 氧化酶活性被抑制，同时Cle诱导的PAL活性及CHS 的积累下降，皂苷合成与hrgp的表达被抑制。由此推测：人参细胞质膜NAD(P)H 氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性。在Cle激发人参悬浮细胞产生氧迸发的过程中，NAD(P)H 氧化酶活性被诱导从而导致H2O2 的产生，H2O2 作为第二信使，激活苯丙氨酸途径，诱发人参皂苷的合成及hrgp防御基因的表达。这一过程中还涉及到Ca2+ 内流，胞内Ca2+浓度的升高，蛋白磷酸化与去磷酸化。人参细胞质膜NAD(P)H氧化酶在人参细胞对Cle的反应过程中起一种介导作用。因此可能存在由Cle刺激, NAD(P)H 氧化酶被诱导，H2O2释放，到人参细胞产生激发反应这样一个由外及内的级联反应。
关键词： NAD(P)H 氧化酶；人参；金瓜炭疽；激发子；信号转导
As a possible peptide primary messenger, extracellular calmodulin (CaM) may regulate processes such as cell proliferation, pollen germination and expression of some genes. Stomata open or close quickly in response to environmental stimuli. CaM was found to be extracellular both in guard cells of broad bean leaves and in epidermal cells by immuno_electron microscopy and immuno_fluorescence microscopy techniques. Exogenous purified CaM enhanced stomatal closure and inhibited stomatal opening with an optimal concentration of 10-8 mol/L; CaM antagonist W7_agarose and anti_CaM serum, which were membrane_impermeable macromolecules, inhibited stomatal closure and promoted stomatal opening. All these data showed that endogenous extracellular CaM of guard cells did promote stomatal closure and inhibit stomatal opening, and could be active only outside the cells. Therefore under natural conditions, the endogenous extracellular CaM of guard cells might regulate stomatal movements as a primary messenger together with other signal molecules, and might be an important linkage between environmental stimuli and cell responses.
陈玉玲 张学琴 陈珈 王学臣*
The self_incompatibility (S) loci from the Solanaceae, Rosaceae and Scrophulariaceae encode a class of ribonucleases, known as S Rnases, which have been shown to control the pistil expression of self_incompatible reaction. In the former two families, the S loci have been shown to be located near centromere. However, the chromosomal location of the S locus in Antirrhinum, a species of the Scrophulariaceae, is not known. To determine its chromosomal location and genomic organization, an S2 Rnase gene and its corresponding 63 kb BAC clone were separately used for fluorescence in situ hybridization (FISH) of mitotic metaphase chromosomes of a self_incompatible Antirrhinum line of S2S5. The results showed that the S2 Rnase detected a doublet signal near the centromere of the smallest chromosome (2n=16). Two separate doublet signals of the tested BAC sequence were shown on both sides of the centromeres of all eight pairs of the chromosomes, suggesting that the Antirrhinum S locus is located in a pericentromeric region. Furthermore, a retrotransposon, named RIS1 (retrotransposon in the S locus), which has not been identified yet in Antirrhinum, was found next to S2 Rnase. Taken together, the centromeric location of the S locus from the three S_Rnase_based self_incompatible families provides a further support on a common origin of their evolution as well as suppressed recombination.
马闻师 周君莉 赖钊 张燕生 薛勇彪
摘要： 在蔷薇科、茄科和玄参科配子体自交不亲和中，编码花柱的S RNase控制花柱的自交不亲和性。在前两科植物中，自交不亲和（s）位点定位于着丝粒的附近，但在玄参科植物金鱼草（Antirrhinum）中自交不亲和位点至今未知。为了确定它在染色体上的位置和基因组结构，以基因型S2S5金鱼草根尖为材料，进行染色体的制备观察，利用地高辛标记的S2RNase和含有其全长的&JE 克隆（S2BAC）为探针进行荧光染色体原位杂交（FISH），发现S2RNase 杂交信号位于染色体的着丝粒附近，而S2BAC的杂交信号位于每条染色体的着丝粒的周边区，呈对称的4个，表明金鱼草! 位点位于着丝粒的周边区。对S2BAC 预测基因的分析表明，发现一个金鱼草新的反转座子（RLS1）。结果显示，金鱼草S位点位于染色体着丝粒的周边区，富含转座子和反转座子，和其他两类配子体自交不亲和的位置类似，预示它们的共同起源和具有抑制重组的能。
Programmed cell death (PCD) of the nucellar cells at the micropylar end is involved in pollen chamber morphogenesis in Ginkgo biloba L. A development_course observation of the morphological changes in the nucellar cells undergoing PCD to form pollen chamber was performed. During the PCD, the nucellar cells degraded their cellular components through an orderly progression. Through the vacuolation, the cytosol was engulfed by the enlarging vacuole, leaving out various organelles, which remained morphologically integrated. As the vacuolation continued, the vacuole collapsed with the breakage of the tonoplast and the cytosol disappeared completely. Organelles were subsequently destroyed. Ultimately, nucellar cells digested away all of their cytoplasm, leaving with cell walls. They became collapsed as the nucellus developed. Intracellular membranes were strikingly changed, playing a role in leading to cell death. Some of these noticeable changes were the appearance of multivesicular body, multicycle_like membranes, membrane_bounded bodies containing some organelles, tonoplast rupture and numerous vesicles. The dehiscence of the apical epidermis, resulting in the opening, appeared to have followed two different pathways with one involving a specific epidermal cell autolysis and the other by detachment from middle lamella of two neighboring epidermal cells without cell autolysis. The specific epidermal cells had been dead prior to the dehiscence of the apical epidermis, which marked the sites of the dehiscence followed. In view of the changes in the cellular morphology, a process of nucellar cell PCD in the course of the pollen chamber formation was demonstrated.
李大辉 杨雄 崔克明 李正理
摘要： 银杏（Ginkgo biloba L.）贮粉室的发生涉及位于珠孔端的珠心细胞的程序性死亡（PCD）。本研究观察了贮粉室发生过程中发生PCD的珠心细胞的形态学变化。这些珠心细胞在PCD过程中形态变化显著，细胞组分有序地降解，液泡在此起关键作用。在液泡化过程中，细胞质基质和一些细胞器被液泡所吞噬，此时的细胞器结构完整。当液泡膜破裂、细胞质基质消失之后，细胞器才逐步解体。最终，这些珠心细胞仅具有残留的细胞壁。随着胚珠的生长，细胞壁也被破坏。在整个PCD过程中，内膜系统发生明显改变：细胞质膜出泡，产生多泡体；形成多环膜结构；出现由膜包围的小体，其中含有细胞质基质和一些细胞器；液泡膜破裂；细胞器解体；细胞中出现大量的小膜泡。珠孔端的珠心表皮开裂形成贮粉室的开口有两种方式：一种为专一细胞的自溶，而另一种是在两个邻接细胞的中胶层处分离，没有发生细胞的自溶破裂。贮粉室开口位置的特定表皮细胞在开裂发生前就死亡，从而提前标示出表皮开裂的发生位置。这些细胞形态的变化反映出银杏珠心细胞的死亡是受发育调控的J?Q 过程。
A regulated gene expression system would offer the unique opportunity to study the gene physiological functions at different developmental stages. For realizing gene special expression in plant anther at given time, we constructed a new system that combined tetracycline_inducible elements with TA29 promoter, a tapetum_specific promoter of tobacco. The system was tested in transient GUS assay system by electroporation (gene gun) transformation of tobacco (Nicotiana tabacum L. cv. Winsconsin 38) anther. In the absence of tetracycline as the inducer, no GUS activity was detected. However, strong GUS expression was observed in tapetum tissue upon tetracycline induction, and little GUS activity was found outside the tapetum. Our results suggested that gene expression can be restricted to a specific tissue at the given time under the control of this new system, and this system would be a very useful tool for both basic plant biology research and biotechnological applications.
唐孙勇 余波澜 牛恒尧 张利明 孙勇如 李文彬
摘要： 如果获得一种可以特异调控基因表达的系统，对于研究某一基因在各个不同发育时期的功能会有很大的帮助。为了控制外源基因在特定的时间和组织内在转基因植物中表达，将四环素诱导元件和花药绒毡层特异表达启动子TA29结合，构建了一个新的系统。通过基因枪将该系统转入烟草（Nicotiana tabacum L.cv.Wisconsin 38）组织，GUS瞬时表达结果表明，四环素诱导前，没有任何烟草组织染上蓝色，表明该系统不表达；而四环素诱导仅30min 后，花药就染上明显蓝色，而且集中在花药绒毡层。其对照子房在四环素诱导前和诱导后均未染上蓝色。这些结果表明，该系统既是四环素诱导又是花药绒毡层特异表达的。
关键词： 四环素诱导系统；TA29; 植物基因工程
Previously the partial sequence of an ethylene receptor gene NTHK2 was isolated from tobacco (Nicotiana tabacumL. Var. Xanthi) plants and it was wound and drought inducible. In the present study full_length cDNA of NTHK2was cloned by 5′_RACE method.NTHK2 gene has 3 216 bp, with 509 bp of 5′_non_coding region and 427 bp of 3′_non_coding region, and encodes an ethylene_receptor homolog of 760 amino acids. NTHK2 protein has a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. In the putative histidine kinase domain, the histidine at the phosphorylation site was replaced by an asparagine. To study the biochemical property of NTHK2, its kinase domain was expressed as a fusion protein with glutathione S_transferase (GST) using yeast Schizosaccharomyces pombe as an expression system. In vitro kinase assay showed that NTHK2 kinase domain can autophosphorylate in the presence of Mg2+, indicating that NTHK2 may function as a kinase. Further studies will elucidate the function of NTHK2 in plant.
张志刚 巩燕. 何新建 王玉军 孙仲序 张劲松 陈受宜
摘要： 应用5'-RACE方法克隆到烟草NTHK2的全长cDNA。其全长cDNA共有3216bp，其中5'非编码区为509bp,3'非编码区为427bp,编码区为2280bp,编码产物为760个氨基酸。NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性，具有推测的组氨酸激酶结构域和接受域；但是，在激酶结构域中没有保守的组氨酸，而是被一个天冬氨酸残基所替代。为了研究其生化特性，在酵母中以融合蛋白的形式表达了激酶结构域。体外激酶分析表明，当有Mg2+存在的情况下NTHK2 能够自我磷酸化。进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体，参与乙烯的信号传导过程。
The inheritance of chloroplast DNA (cpDNA) in sweet potato (Ipomoea batatas Lam.) was analyzed using DNA restriction fingerprinting. The cpDNA fingerprints of hybrids from reciprocal crosses between Xushu18 and AB78_1 were found to be identical to those of their female parents, which reveals that cpDNA of sweet potato is maternally inherited in this intervarietal crossing. This maternal cpDNA transmission pattern does not accord with the putative one based on former cytological studies. The plastid inheritance in Convolvulaceae has been briefly reviewed in this study, and the utility of DNA restriction fingerprinting analysis in the study of plastid inheritance is also discussed.
By mRNA differential display, eight induced cDNAs were obtained from rice leaves infected with an incompatible race 131 of Magnaporthe grisea, and one of these cDNAs was highly similar to salt_induced mannose_binding lectin gene. Using this fragment as a probe, a full length cDNA was isolated from a rice cDNA library, which was constructed using mRNA from the incompatible race_infected leaves. Sequence analysis indicates that the cDNA encodes a protein of 15 kD with145 amino acids and shares 96% identity at nucleotide level with MRL and salT, but is identical to MRL at amino acid level. Genomic Southern blotting shows that there are two mannose_binding lectin genes in rice genome. Northern blotting analysis indicates that the gene was strongly and specifically induced in rice leaves infected with the incompatible race, suggesting that the lectin induction be involved in the defense of rice to M. grisea.
秦庆明 张全 赵文生 王云月 彭友良
（1. 中国农业大学植物病理系，北京100094; 2. 云南农业大学植物病理系，昆明 650201）
摘要: 利用mRNA差异显示技术(DDRT-PCR),从非亲和性稻瘟菌生理小种131侵染的水稻品种爱知旭(Oryza smti-va L.cv Aichi-asahi)叶片中分离了8个诱导差异表达的cDNA片段。对这8个差示片段进行了回收、重扩增和克隆,以其中一个长度为321碱基并与甘露糖结合水稻凝集素和水稻盐诱导蛋白基因高度同源的差示片段为探针,筛选水稻非亲和性cDNA文库,获得12个阳性克隆。序列测定和数据库查询表明该基因的cDNA与水稻凝集素基因的cDNA及盐诱导蛋白基因的cDNA核苷酸同源性高达96％,推定的氨基酸序列与甘露糖结合水稻凝集素的氨基酸序列一致,与水稻盐诱导蛋白仅相差2个氨基酸。Southern杂交显示该基因在水稻基因组中有两个同源拷贝数，Northern杂交表明非亲和性稻瘟菌侵染可强烈诱导该基因表达。因此推测该基因参与了水稻对稻瘟菌侵染的防御反应。
* 通讯作者。E-mail: email@example.com。
EREBP/AP2_type proteins are members of a large DNA binding protein (DBP) family found in plants. Some members like APETALA2 and AtDREB/CBF can regulate flower development and response to environmental stresses, respectively. To characterize transcription factors involved in plant responses to salt stress, we constructed cDNA library from salt_treated halophyte (Atriplex hortensis) and isolated a novel gene encoding EREBP/AP2_type protein from this library. This cDNA contained an ORF of 723 bp and a long 3′_Untranslated_Region (UTR) of 655 bp. The deduced amino acid sequence showed one conserved DNA binding domain of EREBP/AP2, thus the corresponding gene was named AhDREB1 with a calculated molecular mass of 26.1 kD. AhDREB1 under the control of CaMV 35S promoter was then transformed into tobacco and nine independent transgenic lines were obtained and subjected to long term salt stress. The results suggested that overexpression of AhDREB1 improved the salt tolerance in transgenic tobacco through functioning as a regulatory molecule in response to salt stress. Analysis of Arabidopsis genome in database resulted in dozens of EREBP/AP2_type homologous proteins, of which seven members showed high similarity to AhDREB1. Secondary structure analysis predicted similar arrangement of α_helix in their DNA binding domains.
（1. 中国科学院遗传和发育生物学研究所，北京 100101；2. 西北农林科技大学生命科学学院，陕西杨凌 712100；3. 清华大学生物科学和生物技术系，北京 100084）
摘要: EREBP/AP2类蛋白是一个仅存在于植物中的DNA结合蛋白(DBP)大家族.其中一些成员如APETALA2和tDREB/CBF分别调控花的发育和植物对环境胁迫的反应.为了阐明在植物响应盐胁迫过程中所涉及的转录因子的特点,用高盐浓度处理盐生植物山菠菜,构建了cDNA文库,并从此文库中分离得到了一个编码EREBP/AP2类蛋白的基因.此cDNA包含一个723bp的开放读码框和一个长达655bp的3＇端非编码区,推导的氨基酸序列显示其有一个保守的EREBP/AP2的DNA结合域,此基因被命名为AhDREB1.将AhDREB1置于CaMV 35S启动子下转入烟草,获得9个独立的转基因株系,对其进行了长期的盐胁迫实验.结果显示,AhDREB1的组成性表达显著提高了转基因烟草的耐盐能力,这可能是由于其激活了一些具有抗盐效应的下游基因.通过与拟南芥的全基因组进行同源性比较得到了具有较高相似性的7个EREBP/AP2家族成员,二级结构预测显示了它们在DNA结合区段的α螺旋结构上具有相似性.
关键词: 山菠菜; EREBP/AP2类DNA结合蛋白; 转基因烟草
** 通讯作者。E-mail: firstname.lastname@example.org; email@example.com。
The ast (anthocyanin spotted testa) mutant, which was induced by carbon ion radiation, was a single recessive gene mutant of Arabidopsis thaliana (L.) Heynh. With spotted pigment in seed coats, and involved in the anthocyanin biosynthesis. To clone the AST gene by map_based cloning strategy, a series of molecular markers were designed according to the SNPs (single nucleotide polymophisms) and insertion/deletion polymophisms in the Arabidopsis database. With these molecular markers, the fine_structure mapping of the AST gene was finished, the AST locus was located in BAC clone T13M11. It was suggested that the AST candidate gene was T13M11.8 in the T13M11. This gene was 1 432 bp long with 6 exons and 5 introns. The putative protein of T13M11.8 gene was similar to dihydroflavonol 4_reductase (DFR), which was an important enzyme in the anthocyanin biosynthesis pathway.
毛爱军 王台 宋艳茹
摘要： 拟南芥(Arabidopsis thaliana （L.）Heynh.）ast（anthocyanin spotted testa）突变体是由碳离子辐射诱导产生的与花青苷生物合成有关的基因突变体，受单隐性核基因控制。根据拟南芥数据库中的SNPs(single nucleotide polymophisms)序列和插入/缺失多态性（insertion/deletion polymorphisms）序列，设计了一系列分子标记。采用图位克隆策略，应用这些分子标记完成了对拟南芥AST基因的精细作图，成功地将AST基因定位到BAC克隆T13M11 上，初步认为该BAC 克隆中的基因T13M11.8 可能是AST 基因。该基因的DNA 序列长1432hp，含有6 个外显子和5 个内含子，编码的蛋白与花青苷生物合成途径中的二氢黄酮醇-4-还原酶有较高的同源性。将进一步通过功能互补实验验证图位克隆的结果。
关键词： ast 突变AST 基因；分子标记；精细作图；图位克隆；拟南芥
* 通讯作者。E-mail: firstname.lastname@example.org.
Two different cDNA clones (Sscat1 and Sscat2) encoding catalase, the primary important H2O2_scavenging enzyme, were isolated from a λZap_cDNA library constructed from a 400 mmol/L NaCl_treated library of Suaeda salsa (L.) Pall aerial tissue. Sscat1 (1.7 kb) contains a full open reading frame of 492 amino acids and Sscat2 (1.1 kb) is a partial clone. BLAST analysis indicates that the two clones share 71.9% identity in nucleotide sequence and 75% identity in deduced amino acid sequence within the last 287 amino acid residues of Sscat1. Southern blotting analysis showed that Sscat1 is multicopy in S. salsa genome, while Sscat2 is a single copy gene. Northern blotting analysis showed a rapid increase in the steady_level of both genes in roots after 48 h salt treatment, but only Sscat1 was induced in salinity treated leaves. Time_course analysis carried out in leaves confirmed that Sscat1 was induced by salt stress, in contrast to Sscat2. These implied that the expression of Sscat1 and Sscat2 genes are differentially regulated in S. salsa. The activity of total catalase is dramatically increased in response to salt stress.
To produce mouse metallothionein_Ⅰ (mMT_Ⅰ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia coli_cyanobacterium shuttle fusion expression vector, pKG_MT, was constructed. Via this vector, mMT_Ⅰ cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione_S_transferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS_polyacrylamid gel electrophoresis (SDS_PAGE) showed that the fusion protein GST_MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio_β_D_galactoside (IPTG). Glutatione_S_transferase metallothionein (GST_MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT_Ⅰ was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G_50. SDS_PAGE demonstrated that the purified mMT_Ⅰ was the desired protein. The result of ELISA for the purified mMT_Ⅰ showed that the recovery of mMT_Ⅰ from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal_binding activity of the purified mMT_Ⅰ was almost the same as that of wild type MT.
The Agrobacterium mediated transgenic rice (Oryza sativa L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T_DNA were analyzed with inverse PCR. Results showed that 65.4% of the T_DNA was integrated in different locations of rice genome, and some T_DNA flanking sequences were located on certain chromosomes. A number of T_DNA was found to have inserted into protein coding regions. In order to induce transposition of the inserted Ds elements, 354 crosses of Ac×Ds and Ds×Ac were constructed. The excision frequency of Ds element trans_activated by Ac transposase was 22.7% in the F2 populations, and the transposition was confirmed with analyses of DNA sequences flanking the Ds elements. In addition to the transposition due to “cut_paste" mechanism, Ds can replicate itself and integrate into a new locus, and inaccurate excisions were also found. A proportion of DNA segments flanking the Ds elements showed no homologies to sequences published in GenBank, of which two were registered under the accession numbers AF355153 and AF355770. The strategy of using transposon tagging for rice genomics study was discussed.
!" : ;- 转座系统在水稻转化群体中的转座活性及
朱正歌N，W 付亚萍N 肖晗N 胡国成N 斯华敏N 于永红N 孙宗修N!
摘要： 利用本实验室构建的转D)（D)<B*!$）及-! （-&!!")&*+&"(）的水稻（C%-(+ .+18A+ H9）转化群体，配置了D) g -! 的杂交组合>a^ 个。检测了转基因植株的<1-ED 插入位点右侧旁邻序列，研究了D) ‘ -! 转座系统在水稻转化群体中的转座活性。结果表明，有些转化植株<1-ED 插入位点相同或相距很近，插入位点互不相同的占?a9 ^h。检测到<1-ED 可插入到编码蛋白的基因中。在D) g -! 的:W
代中，-! 因子的转座频率为WW9 Xh。对D) g -! 杂交子代中-! 因子旁侧序列的分析，进一步表明了-! 因子在水稻基因组中的转座活性，除了从原插入位点解离并转座到新的位点之外，还有复制———转座和不完全切离等现象。获得的旁侧序列中，有些序列与F$(M*(4 中的数据没有同源性，目前有W 个-ED 片段在F$(M*(4 登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。
关键词： 转座子D) ‘ -!；转座活性；旁侧序列；水稻
!通讯作者。K1#*&.：j !/(];k#*&.9 ,]9 ]l9 )( m 。
A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum_retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API_B genes was constructed. These two insect_resistant genes were transferred into two cotton (Gossypium hirsutum L.) varieties (or lines) via Agrobacterium_mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0%-99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Acand API_B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect_resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API_B with that expressing Cry1Ac only revealed that the insect_resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect_resistant genes reported here is reasonable.
郭洪年1 吴家和2 陈晓英1 罗晓丽2 卢 睿1 石跃进2 秦红敏1 肖娟丽2 田颖川1*
（1. 中国科学院微生物研究所，北京 100080；2. 山西省农业科学院棉花研究所，运城 044000)
摘要： 根据植物基因的结构特征，合成了Cry1Ac 活性杀虫蛋白的编码序列并与内质网定位肽编码序列组成嵌合杀虫蛋白基因Bt29K。构建了含Bt29K基因及慈菇蛋白酶抑制剂[（ 8<)B$）基因表达框的双抗虫基因植物表达载
体。通过根癌土壤杆菌（830.@1-%+0"92 %92+I1-"+#6（>W+2. /2 \A1,0/,H）-A,, =[‘bbOb）介导转化了棉花（J.66?’"92 5"069B
%92 =*）的两个生产品种（系）。根据抗棉铃虫（*+,".%5"6 102"3+01）试验及农艺性状的观察调查结果，经N 代筛选，获得
了抗棉铃虫FO*Og h FF*)g且农艺性状优良的F 个双价抗虫棉纯合品系。分子生物学分析结果表明，两个抗虫基
因在棉花基因组中的插入拷贝数为’ 个或$ 个。活性-8J’‘9 和‘?G<[ 蛋白在转基因抗虫棉株系中的表达量分别约
关键词： 合成的嵌合&0?C8- 基因；慈菇蛋白酶抑制剂基因；抗虫转基因棉花
中图分类号：UFb&*$ 文献标识码：‘ 文章编号：OV))<)bFN（$OO&）O’<O’OX<ON
!通讯作者。\/5：O’O<N$Nb$V))；3<W6+5：i 2+6,J9j0Q,* +W* 69* 9, k 。
Fermentation of the succulent bamboo shoots of Bambusa balcooa Roxb. Resulted in an enrichment of phytosterols from 0.12% to 0.62% dry weight as compared to that of the fresh unfermented samples. The bacterial strains responsible for higher accumulation of phytosterols during fermentation of the bamboo shoots have been isolated and further extraction and purification of the crude phytosterols (isolated from the fermented samples) were done by TLC, UV, NMR, IR and Mass spectral analysis. The isolated phytosterols (β_sitosterols) were then subjected to microbial transformation which yielded a considerable amount of androsta_1, 4_diene_3,17_dione (ADD) in the incubation mixture in presence of metabolic inhibitors (α, α′_dipyridyl and sodium arsenate).
Two novel hydroperoxylated Lycopodium alkaloids, 11α_hydroperoxyphlegmariurine B (1) and 7_hydroperoxyphlegmariurine B 2), along with a known compound, phlegmariurine B (3), were isolated from the total alkaloid fraction of the Chinese medicinal herb Huperzia serrata (Thunb.) Trev. Their structures and relative configurations were elucidated on the basis of spectroscopic analyses.
Two new triterpenoid saponins, monepaloside K (1) and monepaloside L (2), together with a known saponin, mazusaponin I (3), were isolated from the water_soluble part of the whole plant of Morina nepalensis var. alba Hand._Mazz. The structures of monepalosides K and L were determined to be 3_O_α_L_arabinopyranosyl_(1→3)_β_D_xylopyranosyl siaresinolic acid (1) and 3_O_β_D_glucopyranosyl_(1→3)_α_L_arabinopyranosyl siaresinolic acid 28_O_β_D_glucopyranosyl_(1→6)_β_D_glucopyranoside (2) respectively, on the basis of chemical and spectroscopic evidence.
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