根瘤菌在植物内的迁移运动及其与植物相互作用的蛋白质组学研究

根瘤菌不但可以在豆科植物的根部形成共生固氮的根瘤，而且还可以在自然条件下与重要的谷类作物的根形成内生的联合作用。尽管内生菌在植物中广泛存在，但是关于根瘤菌在植物根内的定殖方式还有许多未知。根瘤菌作为内生菌与水稻相互作用的分子机制目前还不清楚。本研究应用显微镜观察、分子生物学和蛋白质组学的研究技术，对内生根瘤菌与植物相互作用，并促进生长的机制进行了探索。 我们用带有gfp标记的根瘤菌分别接种非豆科植物水稻、烟草和豆科植物，应用激光共聚焦显微镜和平板分离，检测其在健康植物组织内的侵染、定殖和分布过程及其对植物生长生理的影响。结果表明： 1．根瘤菌对水稻的侵染是一个动态的过程，它开始于根际表面的定殖，然后从根的裂隙进入根内，向上迁移到叶鞘和叶部分，并且发展为较高的群体密度。水稻接种不同种类的根瘤菌，显著增加了根和地上部分的生物量，提高了光合速率、气孔导度、蒸腾速率、水分利用效率和旗叶的叶面积，并且在植物体内积累了更高浓度的植物激素（生长素和赤霉素）。 2．根瘤菌同样可以在烟草体内由根部向植物地上部分的茎、叶迁移，并从叶的气孔溢出到叶的表面，具有附生－内生－附生生活方式的转换。同时，根瘤菌还可以沿植物的表面从根到地上部分迁移。在植物的生殖生长阶段，内生根瘤菌仍然保持活动性，可以进入烟草子房的子房壁、胎座和胚珠内，暗示根瘤菌通过种子向子代垂直传播的可能性。 3．根瘤菌与豆科植物形成共生固氮根瘤的同时，还可以以内生菌的生态方式定殖于豆科植物中，同样有类似于水稻、烟草的方式在体内由根向地上部分迁移。这种定殖和迁移与根瘤菌胞外多糖和鞭毛的有和无没有关系。 内生根瘤菌促进植物生长的原因是人们一直关心的问题。将根瘤菌固氮正调控基因nifA的启动子与gfp基因构建成融合质粒，设计其他nif相关基因的引物，对有内生根瘤菌的水稻和豆科植物的RNA，进行RT-PCR，表明，虽然定殖于豆科植物体内的内生根瘤菌nifA基因有表达，但是其他的nif基因不表达，因而内生根瘤菌对植物的促生作用不是固氮作用的结果。 我们还用蛋白质组学的方法，分析了Sinorhizobium meliloti 1021和Azorhizobium caulinodans ORS 571接种水稻根部后的植物根、叶鞘和叶组织的蛋白质表达的差异变化。结果表明Sinorhizobium meliloti 1021接种水稻引起的差异蛋白在根内有21个，叶鞘内有19个，叶内有12个;Azorhizobium caulinodans ORS 571接种水稻引起的差异蛋白在根内有7个，叶鞘内有 8个，叶内有8个。蛋白功能的归类中有防卫反应、光合作用、植物生长素、碳和能量代谢及氮代谢相关蛋白的变化。特别是光合作用、植物生长素等相关蛋白的表达，与生理测定光合作用和生长素有提高是一致的，为内生根瘤菌促进水稻生长提供了一个方面的分子证据。 综上所述，表明内生根瘤菌和植物的联合作用比以前所认识的更为复杂，更具有侵染力和动态性。因此，本研究提高了人们对根瘤菌的新认识，不仅与豆科植物根部结瘤，进行共生固氮，而且以内生菌与水稻等植物联合，提高光合作用和生长素含量，促进生长，从另一个方面补充了根瘤菌对植物的有益作用，为根瘤菌作为广谱生物肥料的发展策略奠定分子基础，对可持续农业有重要意义。

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.

Detecting lineage-specific adaptive evolution of brain-expressed genes in human using rhesus macaque as outgroup

Comparative genetic analysis between human and chimpanzee may detect genetic divergences responsible for human-specific characteristics. Previous studies have identified a series of genes that potentially underwent Darwinian positive selection during huma

No accelerated evolution of 3 ' UTR region in human for brain-expressed genes

The difference in cognitive skills between humans and nonhuman primates is one of the major characters that define our own species. It was previously hypothesized that this divergence might be attributable to genetic differences at gene expression level,

Detecting positive darwinian selection in brain-expressed genes during human evolution

To understand the genetic basis that underlies the phenotypic divergence between human and non-human primates, we screened a total of 7176 protein-coding genes expressed in the human brain and compared them with the chimpanzee orthologs to identity genes

Phylogenetic study of complete cytochrome b genes in musk deer (genus Moschus) using museum samples

As an endangered animal group, musk deer (genus Moschus) are not only a great concern of wildlife conservation, but also of special interest to evolutionary studies due to long-standing arguments on the taxonomic and phylogenetic associations in this group. Using museum samples, we sequenced complete mitochondrial cytochrome b genes (1140 bp) of all suggested species of musk deer in order to reconstruct their phylogenetic history through molecular information. Our results showed that the cytochrome b gene tree is rather robust and concurred for all the algorithms employed (parsimony, maximum likelihood, and distance methods). Further, the relative rate test indicated a constant sequence substitution rate among all the species, permitting the dating of divergence events by molecular clock. According to the molecular topology, M. moschiferus branched off the earliest from a common ancestor of musk deer (about 700,000 years ago); then followed the bifurcation forming the M. berezouskii lineage and the lineage clustering M. fuscus, M. chrysogaster, and M. leucogaster (around 370,000 years before present), interestingly the most recent speciation event in musk deer happened rather recently (140,000 years ago), which might have resulted from the diversified habitats and geographic barriers in southwest China caused by gigantic movements of the Qinghai-Tibetan Plateau in history. Combining the data of current distributions, fossil records, and molecular data of this study, we suggest that the historical dispersion of musk deer might be from north to south in China. Additionally, in our further analyses involving other pecora species, musk deer was strongly supported as a monophyletic group and a valid family in Artiodactyla, closely related to Cervidae. (C) 1999 Academic Press.

Functional consequences of new exon acquisition in mammalian chromodomain Y-like (CDYL) genes

The origin of new exons is an important mechanism for proteome diversity. Here, we report the recurrent origination of new exons in mammalian chromodomain Y-like (CDYL) genes and the functional consequences associated with the acquisition of the new exons

Cloning and developmental expression of the Xenopus Nkx6 genes

The evolutionarily conserved Nkx6 family transcription factors play important roles in the patterning of the central nervous system (CNS) and pancreas in vertebrates. In this study, we describe the cloning and expression patterns of the three Nkx6 family

Differential expression of the Brunol/CELF family genes during Xenopus laevis early development

The BRUNOL/CELF family of RNA-binding proteins plays important roles in post-transcriptional regulation and has been implicated in several developmental processes. In this study, we describe the cloning and expression patterns of five Brunol genes in Xenopus laevis. Among them, only Brunol2 is maternally expressed and the zygotic expression of the other four Brunol genes starts at different developmental stages. During Xenopus development, Brunol1, 4-5 are exclusively expressed in the nervous system including domains in the brain, spinal cord, optic and otic vesicles. Brunol2 and 3 are expressed in both the somatic mesoderm and the nervous system. Brunol2 is also extensively expressed in the lens. In transfected Hela cells, BRUNOL1, 2 and 3 proteins are localized in both the cytoplasm and the nucleus, while BRUNOL4 and 5 are only present in the cytoplasm, indicating their different functions.

Methylation of tumor associated genes in tissue and plasma samples from liver disease patients

To investigate whether aberrant hypermethylation in plasma DNA could be used as diagnosis makers for hepatocellular carcinoma (HCC), we performed methylation-specific PCR (MSP) to check the methylation status of five tumor associated genes in 36 cases of

Sox and Zfx genes in giant panda

By using PCR cloning techniques, the DNA sequences of the HMG box regions of six Sox genes (pSox) and the zinc finger domains of two Zfx genes (pZfx) in the giant panda were identified. The giant panda Sox genes fell into two subfamilies, SOX-S1 and SOX-S2. The pSox and pZfx genes of the giant panda were highly homologous to the corresponding genes in mammals and revealed close substitution rates to those in the primates.

Molecular evolution of GH in primates: characterisation of the GH genes from slow loris and marmoset defines an episode of rapid evolutionary change

Pituitary growth hormone (GH), like several other protein hormones, shows an unusual episodic pattern of molecular evolution in which sustained bursts of rapid change are imposed on long periods of very slow evolution (near-stasis). A marked period of rap

Adaptive diversification of bitter taste receptor genes in mammalian evolution

The diversity and evolution of bitter taste perception in mammals is not well understood. Recent discoveries of bitter taste receptor (T2R) genes provide an opportunity for a genetic approach to this question. We here report the identification of 10 and 30 putative T2R genes from the draft human and mouse genome sequences, respectively, in addition to the 23 and 6 previously known T2R genes from the two species. A phylogenetic analysis of the T2R genes suggests that they can be classified into three main groups, which are designated A, B, and C. Interestingly, while the one-to-one gene orthology between the human and mouse is common to group B and C genes, group A genes show a pattern of species- or lineage-specific duplication. It is possible that group B and C genes are necessary for detecting bitter tastants common to both humans and mice, whereas group A genes are used for species-specific bitter tastants. The analysis also reveals that phylogenetically closely related T2R genes are close in their chromosomal locations, demonstrating tandem gene duplication as the primary source of new T2Rs. For closely related paralogous genes, a rate of nonsynonymous nucleotide substitution significantly higher than the rate of synonymous substitution was observed in the extracellular regions of T2Rs, which are presumably involved in tastant-binding. This suggests the role of positive selection in the diversification of newly duplicated T2R genes. Because many natural poisonous substances are bitter, we conjecture that the mammalian T2R genes are under diversifying selection for the ability to recognize a diverse array of poisons that the organisms may encounter in exploring new habitats and diets.