芒果子叶切段不定根形成的影响因素分析

分别用不同成熟时间、不同取材部位、不同品种、不同大小的芒果子叶切段为外植体进行不定根的诱导,以探讨影响芒果子叶切段不定根形成能力的原因.结果表明,芒果子叶切段的生根能力随着芒果成熟度的增加而逐渐提高,花后50和60 d的2.0 cm长子叶切段都无不定根形成,从花后70 d开始有不定根形成,此时生根率为28.6%,之后其生根能力迅速提高,在花后90 d生根率达到76.7%,之后生根率稳定保持直到110 d果实成熟.成熟芒果的子叶切段长度(2.0、1.0、0.5和0.2 cm)对不定根的形成有显著影响,0.2 cm的子叶切段上无不定根形成,另外3个长度的切段都有不定根形成,且生根能力随着长度的增大而逐渐提高.取材位置(靠近或远离胚轴)则对生根影响不大,且紫花芒、红芒、青皮芒等几种常见芒果品种的不定根形成能力基本相同.     

与芒果子叶切段不定根形成有关的活性物质初探

在芒果子叶维管渗出物中检测到多种物质,其中生物碱和甾体两种物质为维管渗出物所特有.芒果子叶的甲醇提取液可提高黄瓜子叶不定根形成的百分率、生根数和根鲜重, 促进小麦胚芽鞘的伸长.     

应用代表性差异分析法筛选人癌候选抑癌基因

运用cDNA代表性差异分析法(cDNA representational difference analysis,cDNA RDA),以正常人鼻咽上皮细胞及鼻咽癌HNE1细胞作为比较的样品来源,分离了四个在鼻咽癌中缺失的cDNA片段.以此四个片段作探针,分别进行DNA杂交、RNA杂交,结果显示,这些差异性的cDNA序列确实来自正常人鼻咽上皮且只在其中表达和/或在鼻咽癌HNE1中表达降低,并在鼻咽癌病人中存在不同程度的缺失.序列分析结果表明这些差异性表达的基因为具有相当抑癌基因功能的已知基因和可能与鼻咽癌相关的抑癌基因的新基因.从而说明cDNA RDA是一种高效、敏感、假阳性低的克隆抑癌基因的有效方法.     

表达序列标签及基因芯片技术在植物抗性基因研究中的应用

表达序列标签和基因芯片技术是基因组学研究的重要手段。表达序列标签是cDNA的3’或5’端的一段序列,通过表达序列标签可以寻找在某种胁迫条件下特异表达的基因并推测其可能的功能。基因芯片技术是指将大量基因探针分子固定于载体上并与标记的样品分子进行杂交,通过检测每个探针分子的杂交信号强度获取样品分子数量和序列信息,通过基因芯片技术,可以研究基因在不同的条件下的表达量,进而研究植物抗性机理。     

甜瓜子叶离体培养直接再生不定芽的形态学和解剖学观察

对甜瓜品种“西莫洛托”子叶离体培养直接再生不定芽过程进行了形态学和解剖学观察,结果表明：以子叶远轴面接触培养基培养能直接再生不定芽,以子叶近轴面接触培养基培养只能得到无序组织块;不定芽再生过程中,培养4d时,子叶变绿,但还不见有细胞分裂,约6d时,在子叶外植体的形态学下端切口处的近轴面到有局部的表皮细胞和亚表皮细胞分裂活跃,初步形成了拟分生组织,7－9d时,这些拟分生组织形成了肉眼可见的小突起,10－14d时,这些突起变得狭长,发育成幼叶或叶状体,它们在外植体上成族存在,但此时还没有典型的“芽”结构出现,15－20d时,成族突起形成幼叶丛,一些幼叶和叶状体的近轴面的基部出现叶腋分生组织,这些将外植体转入伸长培养基,3－5d后,幼叶充分展开,成丛叶状,多个叶腋分生组织同时发育成芽,再过2－7d,相继有不定芽从丛叶外植体上伸长,将伸长的不定芽切下,可促使外 植体上的其它不定芽伸长。     

小麦NBS类抗病基因同源cDNA序列的克隆与特征分析

根据已克隆植物抗病(R)基因NBS保守结构域设计简并引物,采用RT-PCR和cDNA末端快速扩增技术(RACE),在小麦抗叶锈病近等基因系材料TcLr19中进行抗病同源基因cDNA全长的扩增。获得了1个通读的NBS类抗病同源基因S11A11cDNA序列,该序列全长2923bp,编码878个氨基酸序列。生物信息学分析结果表明,该片段含有NB-ARC保守结构域和多个LRR结构域。聚类分析表明,S11A11编码的蛋白与小麦抗叶锈病基因Lr1编码的蛋白亲缘关系较近,而与Lr10亲缘关系较远。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr19小麦中成功获得了抗病基因同源序列,为最终克隆小麦抗叶锈病目的基因奠定了基础。     

以绵羊MHC区段BAC克隆酶切片段为探针杂交筛选绵羊混合组织cDNA文库

在已知中国美利奴羊MHC(Major histocompatibility complex)区段BAC(Bacterial artificial chromosome)克隆序列信息和预测的基因注释前提下,用位于中国美利奴羊基因组BAC文库MHC区段的6个BAC克隆酶切片段为探针,以噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA文库(库库杂交),对分离到的cDNA阳性克隆进行全序列测定,并与相应的已知序列信息和基因注释的BAC克隆比对以及在NCBI Blastn数据库中序列相似性检索,旨在验证基因注释结果的准确性和对基因(序列)功能的初步分析。实验中,经过两轮杂交共筛选出27个cDNA阳性克隆(序列),并发现这些序列均可定位到相应的BAC克隆上,且25条序列处在注释基因的外显子部分;在NCBI数据库中经Blastn序列相似性检索发现,23条序列与牛基因的序列相似性最高,且与免疫功能密切相关。     

无核荔枝果实形成差异表达基因cDNA的克隆

采用抑制差减杂交技术(Suppression Subtractive Hybridization,SSH)分离与海南无核荔枝果实形成相关的差异表达基因的cDNA片段,为克隆相关基因提供研究基础。分别以无核荔枝的有核幼果为driver;无核幼果为tester,建立差减cDNA文库。经Reverse Northern Dot-Blot筛选该文库,共获得61个阳性克隆,随机选取17个克隆进行测序,共获得10条非重复序列,对其中较长的7个序列进行同源分析,结果表明:有6个序列在荔枝中为首次报道。     

生长素和模拟微重力效应对大白菜不定根形态发生的影响

在生长素诱导下,大白菜(Brassicacampestris．Spp．Pekinensis)的下胚轴切段显示了一定的发根能力,其中,0．4—1．0mg／LIAA显著地促进大白菜不定根的发生。在生长素诱导24h后,可借助显微切片观察到下胚轴切面明显的解剖学变化首先是中柱鞘内靠近韧皮部的薄壁细胞的细胞质与细胞核变浓,染色加深,部分细胞分裂;随后是分裂的细胞团增大,逐渐形成根原基并分化出根冠。当下胚轴切段培养5天后,大量不定根穿破皮层,达到肉眼可见的程度。同一外植体中不定根的发育是不同步的,下胚轴不同部位的切段具有不同的发根能力;当下胚轴切段在培养基上反插时,提高外源IAA可修饰根发生的极性,提高蔗糖浓度能增强IAA的修饰作用;在模拟微重力效应条件下,不定根发生的极性没有明显变化,但是,增加了外植体对IAA诱导发根的敏感性。本结果为进一步研究不定根发生的分子机制建立了试验系统。     

一个东亚钳蝎蝎毒素BmTXKβ基因的克隆及其基因表达调控

从东亚钳蝎 (ButhusmartensiiKarsch ,BmK)毒腺组织cDNA文库中分离的长链钾通道毒素BmTXKβcDNA序列 ,克隆了BmTXKβ基因组序列 .BmTXKβ基因含有一个长度为 886bp的内含子 ,定位于BmTXKβ成熟肽中 ,与其它蝎毒素基因内含子定位于信号肽的基因结构不同 .并且 ,BmTXKβ基因的内含子特征也与其它蝎毒素基因不同 .研究结果从基因水平上证实了BmTXKβ是一个新的蝎毒素样肽 .以BmTXKβcDNA序列为探针与蝎基因组DNASouthern杂交出现 2条特异性杂交带 .杂交结果为蝎毒素基因可能通过DNA重排、多拷贝或多基因家族来调控基因表达提供了证据 .     

芒果生长素反应因子类蛋白的cDNA克隆和表达

通过SSH法获得了一个与不定根形成相关的差异表达的cDNA片段,其推导的氨基酸序列与拟南芥的生长素反应因子(ARF)类蛋白具有较大的同源性,因此将它命名为MiARF。用所设计的基因特异引物进行3′RACE扩增获得包含完整读码框架(ORF)的MiARF1（GenBank登录号为AY255705）和MiARF2（GenBank登录号为AY300808）。MiARF1全长为3272bp,其中,ORF含2523bp,5′非翻译区（5′UTR）含285bp, 3′非翻译区(3′UTR)含464bp。由该序列所推导的氨基酸序列与拟南芥ARF2（BAB10162）的ID值为64%, E值为0,在DNA结合区域（DBD）、III和IV区域的同源性更高,ID值均大于80%。MiARF2cDNA全长为1474bp,其中ORF含981bp,5′非翻译区含285bp, 3′非翻译区含208bp,由该序列所推导的氨基酸序列与拟南芥ARF2（BAB10162）的ID值为84%,E值为e-151。 MiARF2仅具有DBD保守区并与MiARF1的基本相同,但缺乏III和IV区域。Virtural Northern 杂交表明：MiARF2在生根的组织中表达水平高, 而在非生根的组织中未见表达;MiARF1在生根及非生根的组织中均有表达。     

Characteristics of adventitious root formation in cotyledon segments of mango (Mangifera indica L. cv. Zihua): two induction patterns, histological origins and the relationship with polar auxin transport

Cotyledon segments derived from zygote embryos of mango (Mangifera indica L. cv. Zihua) were cultured on agar medium for 28 days. Depending on different pre-treatments with plant growth regulators, two distinct patterns of adventitious roots were observed. A first pattern of adventitious roots was seen at the proximal cut surface, whereas no roots were formed on the opposite, distal cut surface. The rooting ability depended on the segment length and was significantly promoted by pre-treatment of embryos with indol-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for 1 h. A pre-treatment with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) completely inhibited adventitious root formation on proximal cut surfaces. A second pattern of roots was observed on abaxial surfaces of cotyledon segments when embryos were pre-treated with 2,700 μM 1-naphthalenacetic acid (NAA) for 1 h. Histological observations indicated that both patterns of adventitious roots originated from parenchymal cells, but developmental directions of the root primordia were different. A polar auxin transport assay was used to demonstrate transport of [³H] indole-3-acetic acid (IAA) in cotyledon segments from the distal to the proximal cut surface. In conclusion, we suggest that polar auxin transport plays a role in adventitious root formation at the proximal cut surface, whereas NAA levels (influx by diffusion; carrier mediated efflux) seem to control development of adventitious roots on the abaxial surface of cotyledon segments.     

用抑制差减杂交法分离和克隆梭梭幼苗受渗透胁迫诱导相关基因的cDNA片段

以Hoagland溶液培养的梭梭幼苗(H)为对照群体,甘露醇处理的梭梭幼苗(M)为目标群体,进行抑制差减杂交.用经过H cDNA差减的M cDNA构建了一个含有大约400个独立克隆的差减文库;采用差减前的H cDNA和M cDNA以及正向/反向差减杂交后的cDNA为模板标记探针,对随机挑取的100个重组质粒进行差示筛选,获得了21个阳性候选克隆.从这些阳性候选克隆中随机挑取了8个进行Northern blot分析,证实其中3个候选克隆代表了在M中特异表达或表达增强的基因,序列分析和同源性比较表明它们与逆境胁迫有关;而另外5个候选克隆无Northem杂交信号,推测它们为低丰度转录本.     

EXPERIMENTS WITH ROOT CUTTINGS OF BRUSSELS SPROUT

A technique for the propagation of Brussels sprout by means of root cuttings is described. Adventitious shoots arise exogenously on callus tissue which develops around the base of side roots. Cuttings sometimes rot without forming adventitious shoots, and cuttings which remain sound do not all produce shoots. Rotting may largely be prevented by planting cuttings with the proximal end exposed above the surface of the medium, and by allowing the root portions to dry before planting. Surface sterilization with mercuric chloride controls rotting but reduces bud formation. Individual plants differ in their capacity to form buds on root cuttings, and this difference is carried by the clones derived from them. Portions of root form more buds if cut into several pieces than if planted intact.     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

黄瓜芽黄突变体抑制消减杂交文库的构建及初步分析

利用抑制消减杂交技术(suppression subtractive hybridization,SSH)分离了黄瓜芽黄突变体及其野生型之间差异表达的cDNA片段.以突变体和野生型分别作检测子和驱赶子,建立正向和反向两个消减杂交cDNA文库;经阳性克隆鉴定,在正向文库中获得特异表达的阳性克隆有133个,在反向文库中得到的阳性克隆有73个.测序后将所得到的159条非重复且非黄瓜的ESTs(登录号:GH270133～GH270291)进行序列同源性比对分析,发现这些ESTs分别与叶绿素合成、光合系统、信号转导、转录因子、氨基酸代谢、糖类代谢、脂类代谢等相关酶及蛋白基因高度同源.     

Human chromosome 21-encoded cDNA clones

We have employed two strategies to isolate random cDNA clones encoded by chromosome 21. In the first approach, a cDNA library representing expressed genes of WA17, a mouse-human somatic cell hybrid carrying chromosome 21 as its sole human chromosome, was screened with total human DNA to identify human chromosome 21-specific cDNAs. The second approach utilized previously characterized single-copy genomic fragments from chromosome 21 as probes to retrieve homologous coding sequences from a human fetal brain cDNA library. Six cDNA clones on chromosome 21 were obtained in this manner. Two were localized to the proximal long arm of chromosome 21, two to the distal portion of the long arm, and one to the region of 21q22 implicated in the pathology of Down syndrome.