piggyBac转座子及其在转基因昆虫中的应用

piggyBac是一种从粉纹夜蛾Trichoplusiani.中分离到的、具有TTAA插入位点特异性的DNA转座子。piggyBac可在昆虫基因组中准确切离,转化频率较高,并且不受宿主因子的限制,是目前转基因昆虫研究中应用最广的转座子载体。近年来的研究发现,piggyBac类转座子广泛分布于昆虫和其他生物基因组中。文章从piggyBac的结构、转座特性、在转基因昆虫中的应用以及piggyBac类转座子的分布等几个方面综述了piggyBac的研究进展。     

Mobilization of the active MITE transposons mPing and Pong in rice by introgression from wild rice (Zizania latifolia Griseb.)

Hybridization between different species plays an important role in plant genome evolution, as well as is a widely used approach for crop improvement. McClintock has predicted that plant wide hybridization constitutes a "genomic shock" whereby cryptic transposable elements may be activated. However, direct experimental evidence showing a causal relationship between plant wide hybridization and transposon mobilization has not yet been reported. The miniature-Ping (mPing) is a recently isolated active miniature inverted-repeat transposable element transposon from rice, which is mobilized by tissue culture and gamma-ray irradiation. We show herein that mPing, together with its putative transposase-encoding partner, Pong, is mobilized in three homologous recombinant inbred lines (RILs), derived from hybridization between rice (cultivar Matsumae) and wild rice (Zizania latifolia Griseb.), harboring introgressed genomic DNA from wild rice. In contrast, both elements remain immobile in two lines sharing the same parentage to the RILs but possessing no introgressed DNA. Thus, we have presented direct evidence that is consistent with McClintock's insight by demonstrating a causal link between wide hybridization and transposon mobilization in rice. In addition, we report an atypical behavior of mPing/Pong mobilization in these lines, i.e., the exclusive absence of footprints after excision.     

Transposition and target preferences of an active nonautonomous DNA transposon nDart1 and its relatives belonging to the hAT superfamily in rice

The nonautonomous nDart1 element in the hAT superfamily is one of a few active DNA transposons in rice. Its transposition can be induced by crossing with a line containing an active autonomous element, aDart1, and stabilized by segregating aDart1. No somaclonal variation should occur in nDart1-promoted gene tagging because no tissue culture is involved in nDart1 activation. By transposon display analysis, we examined the activities of nDart1-related elements in the selfed progeny of a mutable virescent pyl-v plant containing aDart1. Although various nDart1-related elements are present in the rice genome, only nDart1-3 subgroup elements, nDart1-0 and nDart1-3 in particular, were found to be transposed frequently and integrated into various sites almost all over the genome, and a fraction of the transposed elements were found to be transmitted to the next generation. More than half of the newly integrated elements were identified as nDart1-0. Analysis of the newly inserted sites revealed that the nDart1-3 subgroup elements were predominantly integrated into single-copy regions. More than 60% of the transposed elements were inserted into the genic regions that comprise putative coding regions and their 0.5-kb flanking segments, and approximately two-thirds of them were within the 0.5-kb area in front of the putative initiation codons, i.e., promoter-proximal genic regions. These characteristic features of nDart1-3 subgroup elements seem to be suitable for developing an efficient and somaclonal variation-free gene tagging system for rice functional genomics.     

Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis

Pairwise comparison of whole plastid and draft nuclear genomic sequences of Arabidopsis thaliana and Oryza sativa L. ssp. indica shows that rice nuclear genomic sequences contain homologs of plastid DNA covering about 94 kb (83%) of plastid genome and including one or more full-length intact (without mutations resulting in premature stop codons) homologues of 26 known protein-coding (KPC) plastid genes. By contrast, only about 20 kb (16%) of chloroplast DNA, including a single intact plastid-derived KPC gene, is presented in the nucleus of A. thaliana. Sixteen rice plastid genes have at least one nuclear copy without any mutation or with only synonymous substitutions. Nuclear copies for other ten plastid genes contain both synonymous and non-synonymous substitutions. Multiple ESTs for 25 out of 26 KPC genes were also found, as well as putative promoters for some of them. The study of substitutions pattern shows that some of nuclear homologues of plastid genes may be functional and/or are under the pressure of the positive natural selection. The similar comparative analysis performed on rice chromosome 1 revealed 27 contigs containing plastid-derived sequences, totalling about 84 kb and covering two thirds of chloroplast DNA, with the intact nuclear copies of 26 different KPC genes. One of these contigs, AP003280, includes almost 57 kb (45%) of chloroplast genome with the intact copies of 22 KPC genes. At the same time, we observed that relative locations of homologues in plastid DNA and the nuclear genome are significantly different.     

A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.     

Duplication and DNA segmental loss in the rice genome: implications for diploidization

* Large-scale duplication events have been recently uncovered in the rice genome, but different interpretations were proposed regarding the extent of the duplications. * Through analysing the 370 Mb genome sequences assembled into 12 chromosomes of Oryza sativa subspecies indica, we detected 10 duplicated blocks on all 12 chromosomes that contained 47% of the total predicted genes. Based on the phylogenetic analysis, we inferred that this was a result of a genome duplication that occurred c. 70 million years ago, supporting the polyploidy origin of the rice genome. In addition, a segmental duplication was also identified involving chromosomes 11 and 12, which occurred c. 5 million years ago. * Following the duplications, there have been large-scale chromosomal rearrangements and deletions. About 30-65% of duplicated genes were lost shortly after the duplications, leading to a rapid diploidization. * Together with other lines of evidence, we propose that polyploidization is still an ongoing process in grasses of polyploidy origins.     

稻胚凝集素基因的克隆,序列分析及表达

以水稻基因组ＤＮＡ为模板,以特异引物经聚合酶链式反应方法扩增出稻胚凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＳｍａⅠ位点。序列分析表明,克隆到的基因片段大小为７８１ｂｐ,没有内含子,编码１条长２２７个氨基酸、分子量约２３ｋＤ的肽链,其中Ｎ－端２８个氨基酸是信号肽。与报道的稻胚凝集素ｃＤＮＡ序列进行顺序同源性比较,发现它们之间有很高的同源性（９９．７４％）,其编码区第１６７     

转基因水稻外源基因向近缘种群漂流和渐渗的研究进展

水稻是我国最重要的粮食作物之一,我国有8亿以上的人口以稻米作为主食。但在水稻生产中,由于病、虫、草害及不良气候等逆境因子的影响,严重制约了水稻的高产、稳产。转基因生物技术的迅速发展,为水稻抗性育种提供了新途径。自20世纪80年代以来,我国全方位地开展了转基因水稻的研发,目前已经培育出大量的抗病、抗虫、抗除草剂和抗逆的转基因水稻品种,这将为提高我国水稻的生产力和确保粮食安全做出重要的贡献。但转基因水稻的基因漂流及其可能带来的生物安全问题备受关注。已有报道证明,外源转基因可以通过异交向非转基因品种和野生近缘种漂流。在不同的试验条件下,抗除草剂基因有0．05％-0．53％逃逸的可能,其向不育系的最大漂移频率可达4．518％。抗虫基因向相邻非转基因水稻的平均漂移频率最高为0．875％。因此,本文对水稻与其近缘野生种的杂交情况,转基因水稻外源基因向非转基因品种、野生近缘种以及野生非近缘种的漂流和渐渗及其潜在的生态环境风险等方面进行了简要分析,并对转基因水稻的发展进行了展望,以期为转基因水稻的安全应用提供参考。     

DNA uptake by imbibition and expression of a foreign gene in rice

Uptake of DNA by imbibition of dry and viable rice ( Oryza sativa L.) embryos from a DNA solution and expression of a foreign gene were detected using two different vectors contaíning gusA (β-glucuronidase) and hpt (hygromycin phosphotransferase) as reporter genes. The frequency of transient expression of gusA and hpt genes using the CaMV35S promoter was about 30 to 50%. The main sites of gusA gene expression were meristems of roots and vascular bundles of leaves. Also, DNA uptake, integration and expression of the hpt gene in selected rice were investigated by various PCR methods and Southern blot analysis of genomic DNA. It was shown that the hygromycin phosphotransferase (HPT) DNA was present in the rice genome in an integrated form and not as a plasmid form.     

Mitochondrial DNA variation in pearl millet and related species

Summary Mitochondrial DNA (mtDNA) restriction endonuclease fragment patterns and patterns of mtDNA hybridized by mitochondrial gene probes were used to study phylogenetic relationships of seven Pennisetum species, including five P. americanum (pearl millet) ecotypes and a reference species from the distantly related genus, Panicum. The restriction patterns of the pearl millet ecotypes were uniform with the exception of the ecotype collected in Ethiopia. The probe hybridization method revealed more variability, with both the Rhodesian and Ethiopian ecotypes differing from the others and from each other. Considerable restriction pattern polymorphism was noted among different species of Pennisetum, and Panicum. Significant relationships were noted of Pennisetum polystachyon to P. pedicellatum and of P. purpureum to P. squamulatum using the restriction pattern method. In addition to those relationships, the hybridization method showed relationships of pearl millet to P. purpureum and to P. squamulatum. The relationships noted between species by the hybridization method agreed more closely to the cytological data than those indicated by the restriction pattern method. Therefore, the hybridization method appeared to be the preferred method for studying species relationships. The mitochondrial genome size of pearl millet was calculated to be 407 kb and the mitochondrial genome sizes of other Pennisetum species ranged from 341 to 486 kb.Florida Agricultural Experiment Station Journal Series No. 8485.     

CG hypomethylation leads to complex changes in DNA methylation and transpositional burst of diverse transposable elements in callus cultures of rice

CG methylation (^mCG) is essential for preserving genome stability in mammals, but this link remains obscure in plants. OsMET1‐2, a major rice DNA methyltransferase, plays critical roles in maintaining ^mCG in rice. Null mutation of OsMET1‐2 causes massive CG hypomethylation, rendering the mutant suitable to address the role of ^mCG in maintaining genome integrity in plants. Here, we analyzed ^mCG dynamics and genome stability in tissue cultures of OsMET1‐2 homozygous (?/?) and heterozygous (+/?) mutants, and isogenic wild‐type (WT). We found ^mCG levels in cultures of ?/? were substantially lower than in those of WT and +/?, as expected. Unexpectedly, ^mCG levels in 1‐ and 3‐year cultures of ?/? were 77.6% and 48.7% higher, respectively, than in shoot, from which the cultures were initiated, suggesting substantial regain of ^mCG in ?/? cultures, which contrasts to the general trend of ^mCG loss in all WT plant tissue cultures hitherto studied. Transpositional burst of diverse transposable elements (TEs) occurred only in ?/? cultures, although no elevation of genome‐wide mutation rate in the form of single nucleotide polymorphisms was detected. Altogether, our results establish an essential role of ^mCG in retaining TE immobility and hence genome stability in rice and likely in plants in general.