摘要
基于水稻两个亚种indica与japonica已被证实的高度共线性,在本中心1997年构建的第一代水稻基因组物理图的基础上,结合指纹法、分子标记锚标定位STC-PCR等技术,构建了构建了水稻籼稻广陆矮4号(GLA4)与粳稻日本晴(Nipponbare )4号染色体的物理图,并据此最终完成了Nipponbare 4号染色体的精确测序工作。完成序列(finished sequence)全长34.6 Mb,覆盖染色体的97.3%。使用基因中心自主开发的基因组注解软件包对Nipponbare 4号染色体长臂中段74.5～78.2 cM、全长1946125 bp的区段进行了基因预测和人工修正,确认预测基因297个,其中包括8个known基因,52个novel基因,134个putative基因及103个hypothetical基因。基因的平均长度2764 bp,平均外显子数目5.4个。根据上述基因注解结果并结合同源数据搜索,我们发现并实验验证了水稻中的两组表达基因——frr和trs。其中,frr具有两个同源基因——位于4号染色体上的OsfrrA和7号染色体上的OsfrrB。frr编码的核糖体再循环因子(Ribosome Recycling Factor,RRF)在原核生物的蛋白质合成系统中起着不可或缺的作用:帮助翻译后复合体的解聚,并且避免翻译错误,因此是除了古细菌外所有基因组已被研究的原核生物中均有的一个高度保守的基因。OsfrrA和OsfrrB在基因组中都是单拷贝的,分别编码260个氨基酸残基的OsRRFA和242个氨基酸残基的OsRRFB。根据OsRRFA与OsRRFB不同的N端特性、在不同组织和时期的转录谱及与其它35个原核和真核RRF的同源比较,我们推断,OsRRFA和OsRRFB虽然由核基因编码,但将被分别转运并定位于线粒体和叶绿体中,并在其蛋白质翻译系统中发挥与其在原核生物中相似的功能。鉴于水稻线粒体与叶绿体基因组中并没有发现这两个基因的同源序列,因此有理由相信它们是在进化的过程中从细胞器基因组转移到核基因组中的。对从GeneBank中搜集到的37个涵盖了各类已进行了基因组研究的原核生物及有限的几个真核生物的RRF的进化分析结果还揭示了RRF在分子进化研究中作为标尺的潜在价值。而水稻中的trs-like基因则与酵母转运蛋白颗粒(Transport Protein Particle,TRAPP)具有保守结构域的6种亚基的编码基因高度同源。TRAPP是小泡牵引复合体(vesicle tethering complex)的一种,可能在内
质网-高尔基体转运后期发挥作用——通过激活膜表面的一个Rab-GTPase(Ypt1)触发SNARE桥的形成,两种细胞器膜融合,从而达成相关大分子物质从内质网到高尔基体的靶向性转运。我们发现水稻中至少有10个具有广泛转录活性的trs-like基因。这10个基因的编码产物与酵母TRAPP蛋白复合体中已知10个亚基中的6个—Bet3p、Bet5p、Trs20p、Trs23p、Trs31p和Trs33p分别同源。其中4对基因是双拷贝的,另2个(Os1bet5和Os1trs31)则是单拷贝的(基于已知的水稻基因组序列)。它们同时存在于水稻的两个亚种籼稻和粳稻中,且序列和结构上几乎完全相同。与其它真核生物中的同源基因在基因和蛋白质序列、结构及进化各层次上的比较说明,这是一组高度保守的基因,尤其是其中的bet3,其基因结构的保守性甚至跨越了动物与植物的界限。其中双拷贝的4对基因,其两个拷贝均位于不同的染色体上,且在同种组织中的转录强度往往有很大的差异,因此不排除它们还具有其它功能的可能性,正如人的trs20的两个拷贝SEDL基因和MIP-2A基因那样。
The two cultivated rice sspecies of` Oryza sativa—indica and japonica—are proved highly syntenic at their genomic sequence level. On the basis of the first physical map of indica Guangluai4 constructed by NCGR, CAS in 1997, the physical maps of GLA4 and Nipponbare chromosome 4 are updated with the strategy which combined finger-printing, BAC-end sequencing, marker location, cytogenetic approach and STC-PCR techniques together. According to this updated physical map of Nipponbare chromosome 4, a tiling path consisting of 287 BACs and 2 PACs is constructed and sequenced. The finished sequence of Nipponbare chromosome 4 spans 34.6 Mb and represents 97.3% of the chromosome. The sequence accuracy is as high as 99.99%. Using the automatic annotation pipeline developed by NCGR, the 74.5～78.2 cM region of Nipponbare chromosome 4 which includes 19 overlapped BAC clones with a total length of 1946125 bp is analyzed and modified with manual check. 297 candidate genes are discovered in this region including 8 known, 52 novel, 134 putative and 103 hypothetical genes. The average ORF length is 2764bp, while the average exon number is 5.4. Two sets of rice genes--frr homologues and trs-like families—are identified and characterized through sequence analysis and experimental studies. There are two frr homologues in rice genome--frrosA on chromosome 4 and frrosB on chromosome 7. The frr gene was firstly identified and cloned in Escherichia coli. Its encoding protein ribosome recycling factor (RRF) was characterized to be playing an indispensable role in prokaryotic protein synthesis at two aspects. First, RRF is responsible for disassembling the post-translation complex into mRNA, tRNA and ribosome. Second, it prevents the ribosome from reading through the stop codon and initiating another unscheduled translation in all frames without depending on any initiation signal. Thus the transltion errors are reduced. So it is not surprising that every prokaryote
whose genome has been sequenced so far has frr homologue except for Archae. Both OsfrrA and OsfrrB are unique in rice genome, encoding a 260 residues’ OsRRFA and a 242 residues’ OsRRFB. The different N-termial transit peptide characters and transcription profiles suggest that OsRRFA should be located in rice mitochondrion while OsRRFB in chloroplast, and playing similar role in the protein synthesis system of those semi-autonomous organelles. The homologous comparison among 37 species spanning both prokaryotes and eukaryotes demonstrates the potential value of RRF in molecular evolution research, considering its solid conservation in terms of both residue identity and length, and its prevailing existence in organisms. The trs-like genes in rice are highly homologous with the genes encoding 6 of the 10 TRAPP subunits in yeast. TRAPP is a kind of vesicle tethering complexes. It activates a RAB-GTPase (Ypt1) to trigger the combination between v-SNARE and t-SNARE. The combined SNARE complex then induces the membrane fusion between the vesicles from ER and their Golgi targets. So the specific membrane transport from ER to Golgi accomplishes. There are at least ten transcriptional trs-like genes in rice. Fours pairs of them are duplicates and the other two are unique according to the known rice genomic sequences. Their sequences and structures are almost identical in indica and japonica. It is a conserved gene family according to the homologous comparison results with other eukaryotic trs-like homologues at gene structure and protein sequence levels. And bet3 is the most conservative one among them. The transcription profile between two copies of the duplicates is quite different, which demonstrates the existence of the functional difference between them, just like the human trs20 duplicates—SEDL and MIP-2A.
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