Molecular cloning of rice cDNAs related to pollen development using the subtraction hybridization technique

The meiotic stage of pollen mother cell is a very important stage in controlling the development and formation of pollen. In order to clone the rice cDNA(s) of this stage, a normal rice, Annong N and its thermosensitive mutant, Annong S-1 were used as the plant material. The mRNA has been extracted from the young panicle at the meiotic stage. By using the cDNA subtraction hybridization technique, three cDNA fragments, RP-1, RP-2 and RP-3 have been successfully cloned from Annong N. Northern blot analysis reveals that the mRNA of these three clones are expressed only in anthers, and not leaves. The mRNA levels of these clones are lower in anthers of Annong S-1 than in Annong N. Furthermore, the amount of mRNA extracted from anthers of Annong S-1 growing under high temperature (28℃) is lower than plants growing at lower temperature (25℃). Sequence analysis and homology search indicate that these three clones display no similarity to the current database. It is concluded that the three novel cDNA cloned are related to pollen development in rice.     

Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization （FISH） using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells （PMCs）. It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-o-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.     

An anther-specific chalcone synthase-like geneD5 related to rice pollen development

It was shown in a previous analysis that D5 gene from rice (Oryza sativa L.) was an anther-specific gene encoding a chalcone synthase-related protein. In this study, D5 gene was found specifically expressed in tapetum cells as well as in the peripheral cells of the vascular bundle of rice anthers by RNA in situ hybridization. In order to study its function, D5 was transformed into rice in both sense and antisense directions driven by a rice Actin 1 promoter. It has been observed that the pollen grains from the antisense D5 transgenic rice plants are abnormal, indicating that D5 plays a critical role in rice pollen development.     

云南松小孢子母细胞减数分裂的研究

以小孢子材料为对象,全程观察并分析了云南松减数分裂各个时期染色体行为及特征.云南松减数分裂构型为11.72II0 0.24II1 0.04I,配对指数为98.67%,环状二价体的频率和配对指数高于其他裸子植物.染色体交叉平均数目(2.42)和染色体异常分裂现象频率(4%)远低于其他裸子植物.云南松在减数分裂过程中染色体的行为表现出高度保守性,与其高度保守的有丝分裂核型较一致.这也说明云南松所在的松属是松科中较原始的类群.     

一粒小麦小孢子发生过程中的细胞并合

经检测,在正常的一粒小麦(TriticummonococumL.)中有细胞并合发生.细胞并合仅存在于减数分裂的凝线期,核物质从一个花粉母细胞到邻近花粉母细胞的转移是通过胞质通道进行的.细胞并合的发生是自发的,且在花粉母细胞中有方向性.本次调查中未观察到花粉败育现象,具细胞并合的植株结实也未受影响.中期Ⅰ一些花粉母细胞的染色体数目有变化,这造成一些花粉母细胞在后期Ⅰ时出现非正常分离.由于异常花粉母细胞很少,孟德尔分离比例仍然成立.这被认为是在常规遗传分析中不能检测出细胞并合的效应的原因.     

Protection of repetitive DNA borders from self-induced meiotic instability

DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, owing to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination. In the repetitive ribosomal DNA (rDNA) array of the budding yeast Saccharomyces cerevisiae, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin formation. Here we show that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity in the rDNA array. We find that this localized DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination increased specifically in the outermost rDNA repeats, leading to NAHR and rDNA instability. Notably, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2Δ cells. Thus, although the activity of Sir2 globally prevents meiotic DSBs in the rDNA, it creates a highly permissive environment for DSB formation at the junctions between heterochromatin and euchromatin. Heterochromatinized repetitive DNA arrays are abundant in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, the protection of which may be a universal requirement to prevent meiotic genome rearrangements that are associated with genomic diseases and birth defects.     

Development and characterization of interspecific hybrids between <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Subscript>O. latifolia</Subscript> by <Subscript>in situ</Subscript> hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization （GISH） and fluorescent in situ hybridization （FISH） analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase Ⅰ of the hybrid pollen mother cell, poor chromosome pairing was identified and most of the chromosomes were univalent, which resulted in complete male sterility in the hybrid.     

褐飞虱羧酸酯酶基因cDNA片段的克隆及表达分析

利用反转录多聚酶链式反应(RT-PCR)技术克隆了褐飞虱羧酸酯酶基因编码区的cDNA片段,并进行了序列测定.结果表明,所克隆到的cDNA片段长度为396 bp,经BLAST查找比对发现,该片段所编码的氨基酸序列与来自铜绿蝇、家蝇、沟鼠、黑腹果蝇、线虫和埃及伊蚊的羧酸酯酶的片段存在高度同源性.Northern杂交分析显示,在褐飞虱取食抗性水稻后,羧酸酯酶基因表达水平明显升高.以上结果表明,羧酸酯酶基因的表达受抗性水稻的诱导,该基因在有毒化学物质解毒及增强褐飞虱对抗性水稻的耐受性方面可能起着重要作用.     

The cytogenetics and fiber properties of an allohexaploid betweenGossypium hirsutum andG. somalense

Cytogenetics and fiber properties were studied on the Gossypium hirsutum×G. somalense F₂ hybrid and later generations. The cytological analyses of pollen mother cells (PMCs) were made at meiotic metaphase Ⅰ. The results indicated that the hybrid was a new allohexaploid, its chromosome number determined as 2n=6x=78, and genome group as 2[(AD)₁E₂]. Chromosomal configurations of the hexaploid averaged 0.15Ⅰ+ 38.72Ⅱ+ 0.11Ⅲ + 0.02Ⅳ per cell, 85.09% PMCs having 39 bivalents, and only 11.84% PMCs having 1 to 2 multivalents, indicating that heterogenetic recombinations of chromosomes occurs between (AD)₁ and E₂, nevertheless, the frequency is lower. The hexaploids are self-fertile and the progenies remain the hexaploids, whose fibers are light brown and have higher strength by measurement of HVI 900 system. The fiber strength increases 42% than that of upland cotton variety. Therefore, it is possible for the hexaploid to be an important germplasm resource to improve fiber strength of cotton.     

Identification of genes associated with cotyledon senescence in upland cotton

Leaf senescence in plants is an essential develop- mental phase, and an understanding of senescence is important not only for pure scientific reasons, but also for practical purposes. During the last decade, a number of senescence-associated genes (SAGs) …     

褐飞虱细胞色素P450基因cDNA片段的克隆、测序及表达分析

利用反转录多聚酶链式反应(RT-PCR)技术克隆了褐飞虱细胞色素P450基因编码区的cDNA片段,并进行了序列测定.结果表明,所克隆到的cDNA片段长度为237bp,经BLAST查找比对发现,该片段所编码的氨基酸序列与来自烟草天蛾、棉铃虫、埃及伊蚊、家蝇、黑腹果蝇和线虫的CYP6家族的P450的氨基酸序列存在同源性.Northern杂交分析显示,在褐飞虱取食抗性水稻后,P450基因的表达水平明显升高.以上结果表明,P450基因的表达受抗性水稻的诱导,该基因在褐飞虱对抗性水稻的耐受性和解毒方面可能起着重要作用.