转录组测序在高等植物中的研究进展

随着高通量测序技术的发展,在高等植物中应用转录组测序的研究越来越多。转录组测序针对物种转录本直接进行测序,能够有效的获得基因编码序列,主要研究物种在不同时期,不同环境条件,不同处理方式等各个不同条件下的基因表达差异及调控模式差异。本文主要介绍了转录组测序的测序平台,实验流程,生物信息分析内容以及目前在高等植物中的广泛应用,讨论了转录组测序在高等植物中研究的问题和新的研究内容。     

Deletions in the genomes of fifteen inbred mouse lines and their possible implications for fat accumulation

Copy number variants (CNVs) are pieces of genomic DNA of 1000 base pairs or longer which occur in a given genome at a different frequency than in a reference genome. Their importance as a source for phenotypic variability has been recognized only in the last couple of years. Chromosomal deletions can be seen as a special case of CNVs where stretches of DNA are missing in certain lines when compared to the reference genome of the mouse line C57BL/6, for example. Based upon more than 8 million single nucleotide polymorphisms (SNPs) in the fifteen inbred mouse lines which were determined in a whole genome chip based resequencing project by Perlegen Sciences, we detected 20166 such long chromosomal deletions. They cover altogether between 4.4 million and 8.8 million base pairs, depending on the mouse line. Thus, their extent is comparable to that of SNPs. The chromosomal deletions were found by searching for clusters of missing values in the genotyping data by applying bioinformatics and biostatistical methods. In contrast to isolated missing values, clusters are likely the consequence of missing DNA probe rather than of a failed hybridization or deficient oligos. We analyzed these deletion sites in various ways. Twenty-two percent of these deletion sites overlap with exons; they could therefore affect a gene's functioning. The corresponding genes seem to exist in alternative forms, a phenomenon that reminds of the alternative forms of mRNA generated during gene splicing. We furthermore detected statistically significant association between hundreds of deletion sites and fat weight at the age of eight weeks.     

How to Understand the Gene in the Twenty-First Century?

It is widely acknowledged in the literature on philosophy of biology and, more recently, among biologists themselves that the gene concept is currently in crisis. This crisis concerns the so-called “classical molecular concept”, according to which a gene is a DNA segment encoding one functional product, which can be either a RNA molecule or a polypeptide. In this paper, we first describe three categories of anomalies that challenge this way of understanding genes. Then, we discuss proposals for revising the gene concept so as to accommodate the increasingly known complexity of genomic architecture and dynamics. Our intention is to provide an informative overview of recent proposals concerning how we should conceive of genes, which are probably not very familiar to many science educators and teachers, but can bring relevant contributions to genetics teaching, in particular, to a more critical treatment of genes and their role in living systems.     

基因组重排的反转排序的近似算法

本文主要研究基因无方向的基因组重排的反转排序问题.本文算法基于断点图的概念,给出一个时间复杂性为O(maxb3(π),nb(π)),空间复杂性为O(n)的求解近似最优解的算法,其中n为基因组中基因个数,π=(π1,π2,...πn)表示n个基因的一种排列,b(π)表示排列π中的断点数.数据试验的结果表明,该近似算法可以求得较好的结果.     

一种由piggyBac转座子介导高效构建稳定细胞株的策略

构建稳定细胞株的关键是如何提高外源目的基因插入基因组的效率.piggyBac转座子(简称PB)通过"切离-粘贴"的机制实现在基因组上的转座,其本质是在基因组的不同位点间进行插入.利用PB转座子高效插入基因组的特性,并通过改造该转座系统,即分别在PB转座酶的3'端和5'端加入核定位信号肽,然后在人源胚胎肾293细胞系中评...     

可移动的内含子

已发现部分Ⅰ型和Ⅱ型内含子能在基因间移动．这种移动现象可分为内含子回归和内含子转座．移动是通DNA位点专一重组或内含子的反向剪接实现的．     

西文蜜蜂LOC724521基因座的cDNA序列克隆及TSS的分析

5’LongSAGE标签得自于全长mRNA分子的5’末端的前19 nt.该研究利用定位在西方蜜蜂基因组中的一个预测基因座LOC724521的2条5’LongSAGE标签序列作为5’引物,通过RT-PCR克隆了该预测基因座的2条长335 bp和337 bp的cDNA序列（GenBank登录号：GU358205,GU358204）.此cDNA编码一条长88个氨基酸残基的多肽.用所克隆的cDNA序列对基因座LOC724521进行功能注释发现,该基因含有3个外显子和2个＂GT-AG＂型内含子.5’LongSAGE标签序列的基因组定位结果显示：基因座LOC724521在雄蜂的头部中表达丰度很高,RNA PolⅡ可从5个转录起始位点（TSS）上以不同效率起始转录,该基因的59%和31%的转录是从2个优势TSS上起始.有趣的是,有一条5’LongSAGE标签序列被定位在内含子区,暗示该基因存在外显子的可变性选择现象.该研究结果不仅在转录水平上证实了软件预测的基因座LOC724521确实存在,同时揭示了该基因存在可变性转录起始位点和可变性外显子选择等转录调控机制.     

卵菌基因组学研究进展

卵菌是一类真核生物,包括许多植物病原菌,引起许多作物、花卉等发生灾难性病害.卵菌具有独特分类地位的群体,由于表现出丝状等特性传统上被划分到真菌界中.近年来,随着基因组学理论和技术的发展,大量的卵菌基因组相关的数据库被建立.本文综述了现有的卵菌基因组资源以及卵菌分子遗传学的研究进展.重点论述了卵菌基因组的大小、组成和不稳定性以及卵菌的基因结构与功能基因组等五个方面,并分析了卵菌基因组的特点.     

昆虫杆状病毒在生物技术研究中的应用

随着昆虫杆状病毒分子生物学研究的不断深入，昆虫杆状病毒在生物技术研究中也得到了应用。利用杆状病毒作为载体，在昆虫细胞和虫体内表达外源基因，形成了昆虫杆状病毒载体表达系统，利用杆状病毒携带外源基因，通过同源重组将外源基因导入到家蚕的基因组构建了转基因家蚕；通过向杆状病毒基因组中插入毒素、激素、酶抑制剂等的基因或从杆状病毒基因组中删除某个基因，或通过不同杆状病毒间的杂交，使子代病毒的宿主域扩大，从而提高了杆状病毒作为生物杀虫剂的杀虫效果。本文对杆状病毒在上述几个方面的应用进行了简要综述。     

题目：遗传改造细菌基因组的策略：基因定点突变、基因失活和基因过表达

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.     

Maximal sequence length of exact match between members from a gene family during early evolution

INTRODUCTION Gene duplication provides a main resource of new genes in genomes (Ohno, 1970; Brown, 1999). Sequences of two paralogous genes from a duplication event will become different from each other along with evolutionary processes. And the difference in sequences caused by substitution, deletion, insertion of nucleotide acid will cause maximal sequence lengths of exact match (MALE) between paralogous members from a gene family to become shorter during evolution. As for example, o…     

Salinity tolerance in barley during germination—homologs and potential genes

Salinity affects more than 6% of the world’s total land area, causing massive losses in crop yield. Salinity inhibits plant growth and development through osmotic and ionic stresses; however, some plants exhibit adaptations through osmotic regulation, exclusion, and translocation of accumulated Na⁺ or Cl^-. Currently, there are no practical, economically viable methods for managing salinity, so the best practice is to grow crops with improved tolerance. Germination is the stage in a plant’s life cycle most adversely affected by salinity. Barley, the fourth most important cereal crop in the world, has outstanding salinity tolerance, relative to other cereal crops. Here, we review the genetics of salinity tolerance in barley during germination by summarizing reported quantitative trait loci (QTLs) and functional genes. The homologs of candidate genes for salinity tolerance in Arabidopsis, soybean, maize, wheat, and rice have been blasted and mapped on the barley reference genome. The genetic diversity of three reported functional gene families for salt tolerance during barley germination, namely dehydration-responsive element-binding (DREB) protein, somatic embryogenesis receptor-like kinase and aquaporin genes, is discussed. While all three gene families show great diversity in most plant species, the DREB gene family is more diverse in barley than in wheat and rice. Further to this review, a convenient method for screening for salinity tolerance at germination is needed, and the mechanisms of action of the genes involved in salt tolerance need to be identified, validated, and transferred to commercial cultivars for field production in saline soil.

     

几个宫颈癌相关抑癌基因的研究进展

目前,人们研究肿瘤的热点已经从癌基因转到抑癌基因,在已发现的数个抑癌基因中,p53、Fas、p16、Fhit、nm23、BLCAP与宫颈癌的关系密切,也是宫颈癌研究的热点.p53和p16基因通过调控细胞周期来抑制细胞增殖;Fas通过与其配体FasL或其单克隆抗体结合后,诱导Fas所在细胞凋亡;Fhit通过诱导细胞凋亡、细胞周期阻滞发挥着抑制肿瘤细胞增殖的作用;nm23则是通过改变nm23基因的编码产物二磷酸核苷酸激酶(NDPK)从而引起NTP产生不足,参与浸润、转移过程;BLCAP是一个新近发现的宫颈癌相关抑癌基因,其功能及性质尚不大清楚.     

A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.     

甜樱桃S等位基因与育种

樱桃为蔷薇科李属植物,属配子体自交不亲和系统.该属植物其S基因在雌蕊中特异表达的蛋白产物S-RNase,能够降解同一品种花粉管中的RNA,抑制自体花粉的受精作用.S等位基因的结构在基因水平上都具有3个外显子和2个内含子,在蛋白水平上有5个高度保守区.S等位基因在雌蕊和花粉中的表达具有时间和组织特异性,并且受植物生长发育时期的调控,这种特异性的自交不亲和反应由多态的S位点单模型决定.分子系统发育树显示甜樱桃S等位基因聚为三类.S等位基因在樱桃各品种间配置授粉树和亲本育种的选配方面起重要作用.     

用5＇ LongSAGE标签分析蜜蜂Yps基因转录起始位点的多样性

使用5＇ LongSAGE标签序列确定了一个新的西方蜜蜂Ypsilon Schachtel（Yps）基因的转录起始位点,并进而预测了该基因的启动子序列．5＇ LongSAGE标签的蜜蜂基因组定位结果表明：在成年雄蜂的头部中,蜜蜂Yps基因存在23个转录起始位点,其中优势转录起始位点有4个,其起始频率分别为27．9％、23．1％、13．5％和11．5％．Yps基因TSS的碱基组成分析发现,此23个TSS第一个碱基为A、G、T、C的概率分别为52％、39％、4%、4％．这些结果暗示RNA聚合酶和调控因子在Yps基因启动子的一个较宽范围内的互作控制着不同转录本的起始效率．