利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。     

利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。     

Cloning and characterization of chloroplast ribosomal protein-encoding genes, rpl16 and rps3, of the marine macro-algae, Gracilaria tenuistipitata

In Gracilaria tenuistipitata, a highly differentiated multicellular member of the marine red algae, Rhodophyta, chloroplast (cp) DNA can be separated as a satellite band from the nuclear DNA in a CsCl gradient. Using a heterologous probe from Chlamydomonas, the ribosomal protein-encoding gene, rpl16, was located on a 4.5-kb EcoRI fragment of cp DNA. The fragment was cloned and a 1365-bp region around rpl16 was sequenced. The gene order around rpl16, 5′ rpl22-rps3-rpl16, is identical to that detected in the chloroplast DNA of liverwort, tobacco and maize. Both the nucleotide sequence and the amino-acid sequence of rpl16 are more conserved than that of rps3. The rpl16 gene contains no intron, a feature which shows more similarity to the unicellular green algae, Chlamydomonas, than the other land plants. Sequences that may form a stable stem-loop structure were detected within the coding sequence of rpl16.     

过表达钾转运蛋白基因trkH提高玉米的钾营养

钾是植物生长发育必须的营养元素,参与多种生理生化过程。土壤缺钾严重影响了玉米的产量和品质,通过遗传改良的方式提高玉米的钾营养是解决这个问题的有效途径。本实验室前期从海洋微生物宏基因组DNA中克隆得到细菌钾转运蛋白基因trkH,在酵母和烟草中验证了功能。为进一步分析trkH基因在玉米中的功能,以Hi-Ⅱ基因型玉米为转化受体,采用农杆菌介导法将trkH基因转入玉米,获得具有Baster抗性的独立转化苗21株。PCR检测结果表明trkH基因已成功转入到玉米基因组中。Bar试纸条检测结果表明Bar基因在蛋白水平上成功表达。将部分T₀代转基因植株与玉米骨干自交系PH6WC杂交,获得8个T₁代株系,半定量RT-PCR检测结果表明转基因植株在转录水平上均能表达。三叶一心期喷洒除草剂进行筛选,选择比例接近1：1的L3、L5和L7转基因株系进行PCR检测,统计检测结果并进行χ²测验,结果表明符合孟德尔分离定律。L3、L5和L7转基因株系玉米苗期的钾耗竭实验结果表明过表达trkH基因能够提高转基因玉米的K⁺吸收,为培育钾营养高效玉米新品种奠定基础。     

Sequence of histidyl tRNA,present as a chloroplast insert in mtDNA ofZea mays

Unfractionated tRNA, isolated from maize mitochondria, has been specifically labeled at the -CCA end and used to recover a tRNA gene-bearing fragment from a clone bank of maize mitochondrial DNA. This gene has been mapped, sequenced and found to carry the anticodon for histidine. The sequence of the gene and that of bases in its near vicinity are identical to maize chloroplast tRNA^His, although sequences more distant on the fragment are not homologous with cpDNA. The junction of the cpDNA insert has been sequenced.     

Characterization of a rice sucrose-phosphate synthase-encoding gene

A rice genomic clone (spsl) coding for sucrose phosphate synthase (SPS) was isolated and sequenced. Rice spsl contains 13 exons and 12 introns, an unusually long 366-bp leader region with a highly organized primary structure and a promoter region with no obvious homology with eukaryotic promoter consensus sequences. Southern blot analysis showed that SPS is encoded by a single-copy gene in the rice genome. Comparison of the rice, maize, potato and spinach SPS deduced amino acid (aa) sequences showed that these enzymes have a well conserved region comprising their first 700 aa, and a variable C-terminal region. Analysis of rice spsl expression showed that mRNA levels change during leaf development. SPS activity and mRNA were undetectable in roots.     

Convergent loss of an anciently duplicated, functionally divergent RH2 opsin gene in the fugu and Tetraodon pufferfish lineages

We describe the complete opsin gene families from the sequenced fugu and Tetraodon pufferfish genomes. We report the convergent loss of function of an anciently duplicated, functionally divergent RH2 or “green-sensitive” opsin gene in both pufferfish lineages, designated RH2-2. In fugu, RH2-2 apparently ceased to function very recently following a transposon-induced deletion that truncated the N-terminal 115 amino acids from the translated protein. Although a lack of frameshift or nonsense mutations in the fugu RH2-2 pseudogene suggests that the gene was lost very recently in this lineage, we were unable to detect any evidence of a selective sweep associated with the fixation of the truncated allele from population data. Interspecific comparison of the remaining fugu RH2-2 coding sequence paradoxically indicates that the gene was under strong purifying selection until the truncation occurred.     

Structure, organization and expression of the eukaryotic translation initiation factor 5, eIF-5, gene in Zea mays

The maize genomic DNA sequence encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from genomic library of maize seedlings and the exon–intron structure determined (accession number AJ132240). The length of genomic DNA sequenced was about 7 kb and contained two exons with the translation start site in exon 2. The only intron is located in the non-coding 5′ region and it is 1298 bp long with the splice acceptor and donor sites conforming to the AG/GT rules. Repetitive sequence fragments are located in the 5′ and 3′ intergenic region. The accumulation of eIF-5 mRNA was studied by RNA blot and in situ hybridization. The observed distribution of mRNA may correlate with the function of the protein, as it appears to be highly abundant in tissues where the proportion of cells actively dividing is very high, such as meristematic regions.     

是金子无论在何处都发光: 玉米和水稻驯化中的趋同选择

野生植物的驯化为人类定居与文明起源奠定了重要基础。在世界范围内不同地区生活的古人类分别对当地不同的野生植物进行了驯化, 而经过驯化的作物常常表现出相似的驯化综合性状。在基因组层面上对趋同选择规律的解析, 可为作物育种提供重要信息与遗传资源。近日, 中国农业大学杨小红/李建生和华中农业大学严建兵领衔的团队从单基因和全基因组2个层次系统解析了玉米(Zea mays)和水稻(Oryza sativa)趋同选择的遗传基础, 发现玉米KRN2与水稻OsKRN2受到了趋同选择, 并通过相似的途径调控玉米与水稻的粒数与产量。他们还发现玉米与水稻在全基因组范围内存在大量趋同选择同源基因对(gene pair), 这些基因在淀粉代谢、糖及辅酶合成等途径特异富集。该研究不仅克隆了在玉米与水稻中均具有重要育种价值的趋同选择同源基因对KRN2/OsKRN2, 而且在全基因组水平上揭示了玉米与水稻趋同选择的规律, 为进一步解析驯化综合性状形成的分子机理及其在育种中的应用奠定了重要理论基础。     

A novel gene encoding a ras-like GTP-binding protein from Trypanosoma brucei: an evolutionary ancestor of the ras and rap genes of higher eukaryotes?

The ras superfamily of GTP binding proteins encompasses a wide range of family members, related by conserved amino-acid motifs, and act as molecular binary switches that play key roles in cellular processes. Gene duplication and divergence has been postulated as the mechanism by which such family members have evolved their specific functions. We have cloned and sequenced a ras-like gene, tbrlp, from the primitive eukaryote Trypanosoma brucei. The gene encodes a protein of 227 amino acids and contains the six conserved subdomains that designate it as a ras/rap subfamily member. However, the presence of key diagnostic residues characteristic of both the ras and rap families of GTP confuse the familial classification of this gene. Phylogenetic analysis of the GTP binding domain places its origins at the divergence point of the ras/rap families and suggests that tbrlp is an ancestral gene to the ras/rap genes of higher eukaryotes.     

The tyrosyl-tRNA synthetase from Escherichia coli: Complete nucleotide sequence of the structural gene

The structural component of the tyrS gene of Escherichia coli, comprising 1269 base pairs, has been fully sequenced by the combined M13/dideoxychain termination approach. The gene has a codon usage pattern which is typical of highly expressed proteins and similar to other Escherichia coli aminoacyl-tRNA synthetase genes. Peptide purification and sequencing has been used to locate the N-terminus and to provide confirmation of 95% of the translated protein sequence. This latter yields on M_r of 47 403 for the Escherichia coli tyrosyl-tRNA synthetase, and reveals considerable homology with the primary structure of the analogous enzyme isolated from Bacillus staerothermophilus.     

Nucleotide sequence and organization of full length human U4 RNA pseudogenes

Two loci encoding human U4 RNA, designated U4/7 and U4/14, have been isolated and sequenced. Both are pseudogenes in that their sequences do not match any identified human U4 RNA species perfectly. The U4/7 locus harbours a full-length pseudogene of 144 bp with eight base substitutions in the structural region. This pseudogene might be derived from a hitherto unidentified human U4 RNA gene. The second locus, U4/14, has a complex structure; the structural sequence of a U4 gene has apparently been integrated into an Alu sequence.     

转基因玉米HGK60在不同遗传背景下抗虫性鉴定及农艺性状分析

为研究转基因玉米HGK60在不同遗传背景下遗传稳定性和抗虫效果,利用转Bt cry1Ah基因的转基因玉米HGK60为供体,通过回交转育的方式将cry1Ah基因分别导入玉米自交系郑58、昌7-2、lx05-4、lx03-2,获得转基因玉米自交系HGK60-郑58、HGK60-昌7-2、HGK60-lx03-2、HGK60-lx05-4,并杂交获得HGK60-郑单958（HGK60-郑58 × HGK60-昌7-2）和HGK60-鲁单9066（HGK60-lx05-4 × HGK60-lx03-2）,转化体特异性PCR证明cry1Ah基因已转入不同遗传背景玉米中,ELISA检测不同遗传背景转基因玉米叶片中Cry1Ah蛋白表达情况,结果表明在不同遗传背景玉米自交系和杂交种中Cry1Ah蛋白表达没有显著差异;田间人工接虫和室内玉米螟抗虫性鉴定结果表明,不同遗传背景的转基因玉米高抗玉米螟,室内接虫后4 d幼虫死亡率达到100%;对不同遗传背景转基因玉米HGK60进行农艺性状分析,结果显示与受体对照玉米相比,两者之间农艺性状没有显著差异,转基因玉米HGK60可用于抗虫玉米品种的选育。     

The Tub alpha 3 gene from Zea mays: structure and expression in dividing plant tissues.

A gene (Tub alpha 3) coding for an alpha-Tub, expressed in dividing tissues, has been cloned from Zea mays. The deduced amino acid (aa) sequence, 450 aa long, is very similar to the other plant alpha-Tub (85-89% homology) so far reported, and in particular to the other two aa sequences (alpha 1-Tub and alpha 2-Tub) already published from the same species (93% homology). The genomic structure is also very similar, having three introns located at the same positions as in the Tub alpha 1 and Tub alpha 2 genes, one of them placed at the same position in the homologous genes from Arabidopsis thaliana. Nevertheless, the noncoding sequences are very different from the two other maize genomic sequences. In particular, no homology has been found either in the 5' upstream or in the 3'-untranslated sequences. Using specific 3' probes, it has been possible to detect the mRNA coded by this gene in many of the plant organs measured, but its highest abundance is observed in the organs rich in dividing cells, a pattern correlated with that of the histone H4-encoding gene. A cDNA clone has been identified in maize coleoptiles and sequenced, confirming the expression of the Tub alpha 3 in this organ. No preferential accumulation in any organ of the plant was found, in contrast with what was observed in the Tub alpha 1 and Tub alpha 2 genes already described. The Tub alpha gene family seems to consist in maize by at least two groups of homologous sequences, each one including a maximum of two or three coding units.