低温对龙井43种胚基因表达效应的初步研究

为了探讨低温对茶树种子特定基因表达的效应,以便为茶树种子长期低温储存提供分子生物学方面的参考依据,采用cDNA-AFLP方法对茶树无性系龙井43种子经超低温（-70 ℃）储存1年后种胚的基因转录活性进行了初步分析,结果显示：64对引物组合共产生了132条PCR产物.对其中的15条进行克隆测序,最终获得8条差异片段.在GenBank进行序列分析,发现有6条与已知功能基因相关,其中包括参与植物转运、转录、能量产生及老化相关的蛋白,2条在GenBank数据库中未找到相似序列.     

陆地棉体细胞胚胎发生过程的cDNA-AFLP分析

以陆地棉标准系TM-1未经诱导的下胚轴、下胚轴经诱导40 d的愈伤组织和胚性愈伤组织为材料,利用cDNA-AFLP差异显示技术对陆地棉体细胞胚胎发生过程中的cDNA差异表达进行了初步分析.经反转录获得3个不同时期的cDNA,利用180对引物组合进行AFLP分析,结果表明,在总共显示的约3 000条谱带中,其中38条为胚性愈伤组织中所特有的差异谱带.对这些差异片段进行克隆、序列测定和同源性分析,其中12个差异片段同已知陆地棉、雷蒙德氏棉和亚洲棉不同发育时期器官的EST序列高度同源,其相似性达到90%以上.结果说明,棉属在形态建成早期（胚胎发育阶段）,其特定器官的相关基因已经表达.     

授粉后黄瓜果实膨大相关基因的鉴别

利用cDNA—AFLP技术分析了授粉前后黄瓜幼果的基因表达差异。有64条转录本片段（TDF）出现在授粉后6-72h的黄瓜幼果中,其中5条经反向Northern斑点杂交证实主要在授粉后的幼果组织中表达。序列分析确定,有一条TDF属于编码黄瓜扩张蛋白的新基因,命名为Cs．Expansin10（CsExplO）,可能与授粉后黄瓜果实膨大生长相关;另一条与谷胱甘肽还原酶基因高度同源;其余3务在基因库中未找到相似序列。     

甘蓝型油菜Ogura 雄性不育×白菜的回交杂种后代与亲本之间蕾期基因表达差异比较研究*

以甘蓝型油菜（Brassica napus L, AACC,2n＝38）Ogura细胞质雄性不育材料为母本,以不同白菜（B campestris ssp. chinensis Makino, AA, 2n＝20） 自交系‘新选一号’和‘矮脚黄’为父本进行杂交,获得了杂种F1、BC1、BC2代。利用cDNA-AFLP技术对两种材料的不同回交世代BC1、BC2代与其亲本在蕾期的基因表达进行分析。结果表明,两种白菜回交世代与其亲本的基因表达有明显差异,在质和量上都存在差异。基因表达模式有5类共7种：（1）单亲沉默型（2种）,（2）单亲一致型（2种）,（3）双亲共沉默型,（4）杂种特异型,（5）表达一致型。随着回交世代的增加,回交杂种和亲本的基因表达在单亲沉默型、双亲共沉默型呈增加趋势。而在母本一致型、父本一致型、杂交种特异型、表达一致型呈下降的趋势。两种白菜在F1、BC1、BC2 3个世代与回交亲本花蕾间的基因差异表达有15种类型,其中以在轮回亲本、F1、BC1、BC2中共同出现表达的带的比例最高。Abstract: Crosses between female parent of Ogura male sterility Brassica napus L. and male parents of B. campestris ssp. chinensis Makino were made and F1, BC1 and BC2 generations produced. Gene expression of two Chinese cabbage backcross hybrid BC1, BC2 and their parents at bud stage was analyzed by means of cDNA-AFLP technique. The results indicated that the patterns of gene expression differ significantly between BC1 and BC2 generations and their parents. There were many patterns of gene expression, including gene overexpression and gene silencing. Five patterns (seven kinds) of gene expression were observed, which include: (1) bands occurring in only one parent (two kinds); (2) bands observed in hybrids and one parent (two kinds); (3) bands occurring in only parents (one kind); (4) bands visualized in only hybrids (one kind); (5) bands observed in parents and hybrids (one kind). In accompany with the addition of backcross, the increase trend in backcross hybrids and their parents were described in the aspects of differential gene expression, bands expressed only in one parent and bands expressed only in both parents. The declined trend in backcross hybrids and their parents were observed in the aspects of bands expressed in both hybrids and one parent (two kinds), bands visualized in only hybrids and bands observed in parents and hybrid. Fifteen patterns of gene expression were observed in F1、BC1、BC2 and backcross parents. The percent of bands expressed in F1、BC1、BC2 and backcross was highest.     

Cloning of differential expression fragments in cauliflower after Xanthomonas campestris inoculation

A near isogenic line (NIL) of Brassica oleracea var. botrytis with resistant and susceptible lines C712 and C731, was used in this study. More than 100 differentially expressed cDNA fragments were obtained from black rot resistant cauliflower plants obtained using cDNA-amplified fragment length polymorphism (AFLP) after infection with the pathogen. Thirteen of these fragments were cloned and subjected to reverse Northern blot analysis using both infected and control cDNA pools. Two positive clones, M2 and M6, were isolated. Northern dot blot and Northern blot analyses showed that M2 was constitutively expressed, whereas M6 contained a gene that was differentially expressed during pathogen infection. Moreover, M6 cDNA fragment was also highly expressed 16–24 h after H₂O₂ treatment. Southern blots showed that M6 is a single copy gene in the cauliflower genome, and encodes a protein with 84 % homology to gene on Arabidopsis chromosome 1. The deduced M6 protein has 91 % positive homology with the Arabidopsis 2A6 protein, which regulates ethylene synthesis; 76 % homology with a 1-aminocyclopropane-1-carboxylate oxidase (ACO), the last enzyme in ethylene synthesis; and 70 % homology with an ethylene induced DNA binding factor. These results suggest that M6 gene fragment is a new H₂O₂ downstream defense related gene fragment and can be induced by Xanthomonas campestris pv. campestris and H₂O₂.     

Collateral gene expression changes induced by distinct plant viruses during the hypersensitive resistance reaction in Chenopodium amaranticolor

Hypersensitive reactions to plant diseases are typically mediated by R genes. Many R genes that have been cloned only confer resistance to a particular pathogen. However, Chenopodium spp. have multivirus hypersensitive resistance, thus making the understanding of this broad-spectrum resistance mechanism attractive. Using tobacco mosaic virus (TMV) tagged with green fluorescent protein to follow infection over time, cDNA-AFLP to find genes up-regulated during virus infection in C. amaranticolor and quantitative RT-PCR to accurately measure gene expression at different time points, the first dissection of this significant defense response pathway is presented. The detected disease-expressed sequences in C. amaranticolor (DESCA) are similar to those that encode p450 monooxegenases, hypersensitivity-related genes, cellulases, ABC transporters, receptor-like kinases, serine/threonine kinases, phosphoribosylanthranilate transferases and hypothetical R genes, many of which are associated with pathogen defense in other plants. The expressions of these DESCA genes are also induced by infection with the taxonomically distinct tobacco rattle virus (TRV) in C. amaranticolor. In particular, DESCA1, one of the gene fragments from C. amaranticolor that lacks similarity to any other sequence in the GenBank database, is induced at least 200 fold 4 d after infection (dai) by both TMV and TRV. The potential role of DESCA genes in a C. amaranticolor multivirus defense response with regard to their levels and time of gene expression is discussed.     

The response of Spartium junceum roots to slope: anchorage and gene factors

BACKGROUND AND AIMS: Plant anchorage is governed by complex, finely regulated mechanisms that occur at a morphological, architectural and anatomical level. Spanish broom (Spartium junceum) is a woody plant frequently found on slopes--a condition that affects plant anchorage. This plant grows throughout the Mediterranean area where it plays an important role in preventing landslides. Spanish broom seedlings respond promptly to slope by altering stem and root morphology. The aim of this study was to investigate the mechanisms whereby the root system of Spanish broom seedlings adapts to ensure anchorage to the ground. METHODS: Seedlings were grown in tilted and untilted pots under controlled conditions. The root apparatus was removed at different times of growth and subjected to morphological, biomechanical and molecular analyses. KEY RESULTS: In slope-grown seedlings, changes in root system morphology, pulling strength and chemical lignin content, all features related to plant anchorage in the soil, were related to seedling age. cDNA-AFLP analysis revealed changes in the expression of several genes in root systems of slope-grown plants. BLAST analysis showed that some differentially expressed genes are homologues of genes induced by environmental stresses in other plant species, and/or are involved in the production of strengthening materials. CONCLUSION: Plants use various mechanisms/strategies to respond to slope depending on their developmental stage.