Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS} alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin a from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3' end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS) alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin α from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3′ end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

肝癌相关基因MEF2D反义RNA的鉴定及分析

鉴定和分析肝癌细胞中新的天然反义RNA MEF2D-AS根据已建立的双链RNA文库筛选与肝癌相关的反义RNA.通过RT-qPCR验证其反义RNA的存在,采用cDNA末端快速扩增技术获得其全长序列,并检测HepG2细胞经5-Aza-dC处理前后MEF2D基因正反义链表达量的变化.结果显示,成功鉴定出1条全长为1 265bp的新的天然反义RNA,并预测其属于长链非编码RNA(long non-coding RNA,简称为lncRNA),命名为MEF2D-AS.经5-Aza-dC处理后,MEF2D-AS的表达量升高,而MEF2DmRNA的表达量降低.证实了MEF2D-AS的存在并预测其属于长链非编码RNA,且与MEF2D正反义RNA是反相关关系.此研究结果可为进一步探讨肝癌发生机制提供参考.     

包台型水稻不育系花药发育过程中的Ca2+分布

用焦锑酸钾沉淀法研究了包台型不育水稻及其保持系花药发育早期细胞中的Ca²⁺分布变化.从花粉母细胞时期开始,不育系与保持系花药中的Ca²⁺分布出现差异,不育系药壁外层和药室内壁及药隔中均有Ca²⁺出现,而保持系花药中基本没有Ca²⁺分布,在减数分裂时期表现更加明显,不育系药壁和药隔中的Ca²⁺相对于花粉母细胞时期明显地有所增加,特别是绒毡层细胞和药隔中木质部细胞的次生加厚壁上Ca²⁺增加尤其明显,而保持系花药中的Ca²⁺较少.笔者从花药发育的更早期去寻找水稻雄性不育的机理.     

Fertility analysis of the Arabidopsis transformed with antisense rice osRACD gene

The full length osRACD cDNA sequence was subcloned into the pBI121 plasmid in the antisense orientation under the control of the CaMV35S promoter to construct the expression vector pBID, and the constructs were introduced into Arabidopsis plants by using the vacuum infiltration method. The siliques of the transformants stopped growing after anthesis, and they turned yellow or died later; and the siliques from the control plants transformed by the pBI continued growing after anthesis and matured normally. In vitro pollen germination demonstrated that the growth and elongation process of the pollens of the transgenic plants was inhibited, the pollen tubes were shorter and slightly fatter than the tubes of the control plants, which grew normally with long cyclindrical tubes. The above results suggest the function of osRACD gene involved in regulation of the growth and elongation process of pollen tube, its encoding protein may be one of the important factors in regulation of fertility transition of the photoperiod-sensitive genic male-sterile rice Nongken 58S.     

同源盒基因Hox2.3在小鼠造血调控中的作用

同源盒基因在造血细胞系细胞中表达,它们的表达具有细胞类型特异性,为了阐明同源盒基因在小鼠造血调控过程中是否有决定性作用,我们采用反义脱氧寡聚核苷酸抑制试验和逆转录-聚合酶链式反应(RT-PCR)试验,证明小鼠同源盒基因(Hox2.3)对正常小鼠髓系血细胞的生长、发育调控起重要的作用。     

Effects of CIITA antisense RNA on the expression of HLA class II molecules

To study the effect of the major histocompatibility complex class II (MHC II) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class II molecules, 5′ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-II was constructed. HeLa cells transfected with pcDNA-II and pcDNA3 were induced by IFN-γ for 3 d. The expression of HLA class II molecules on HeLa/pcDNA-II cells was significantly decreased, while it has no effect on the expression of HLA class I molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class II molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in graft transplantation.     

RAB5A在乳腺癌中的表达及转移相关性分析

目的 探讨RAB5A在乳腺癌转移中的作用与机制,以期为乳腺癌的基因治疗及研究提供新的靶点。方法 检测不同分化程度的乳腺癌组织标本中RAB5A蛋白的表达情况;应用FAM标记的RAB5A反义寡核苷酸,体外转染高转移度的乳腺癌细胞,观察肿瘤细胞的转移相关指标的变化。结果 乳腺癌组织中大多呈现不同程度RAB5A的表达,差异有显著性意义（P<0.05）,在浸润导管癌中RAB5A的表达程度与其他组织类分型差异有显著性意义（P<0.05）,肿瘤组织不同分化程度染色显示乳腺癌分化程度越低（P<0.05）,在细胞基底膜侵袭和运动实验中RAB5A反义核酸封闭后穿膜细胞数均明显减少。结论 RAB5A是一个可能与乳腺癌发生、转移相关的基因,并且还可能与它们的分化程度有关。RAB5A在人乳腺癌细胞的侵袭及转移特性的形成中具有重要的作用,RAB5A的反义分子能够有效地阻断该基因表达的翻译过程。     

Two closely linked rice U14 boxC/D snoRNAs

By analysis of the conserved elements in yeast U14 boxC/D snoRNA. the conserved elements in rice U14 boxC/D snoRNA have been speculated. Through computer search of the international rice genome database, two rice U14 snoRNA gene candidates are obtained. These two putative U14 snoRNA genes are closely linked on rice chromosome 2. The coding sequences of these two snoR-NAs exhibit the hallmark structure of boxC/D antisense snoRNA. They both have conserved boxC and boxD sequences and a 14nt-long complement to the sequence between 414nt and 427nt of rice 18S rRNA (according to GenBank accession no. X00755). The experimental evidence shows that these two snoRNAs are involved in the methylation of the complementary sequence of rice 18S rRNA. The existence and localization of these two snoRNAs are proved by RT-PCR and Northern blot. Further analysis shows that both of the newly found rice snoRNAs have high homology with maize U14 snoRNA. and they are named rice U14.1 snoRNA and U14.2 snoRNA respectively. The gene sequence encoding these two snoRNAs has been deposited in the GenBank database under accession number of AF332622.     

Introducing antisense <Emphasis Type="Italic">waxy</Emphasis> gene into rice seeds reduces grain amylose contents using a safe transgenic technique

Rice (Oryza sativa L.) eating quality is one of themost important traits. Amylose content (AC) in rice en-dosperm is a major index affecting rice eating quality[1,2].It has a negative correlation with gel consistency of rice[3].Based on amylose content, r…     

Introduction of antisense waxy gene into main parent lines of indica hybrid rice

Amylose content in rice endosperm is one of thekey determinants of rice eating and cooking quality, and thepoor quality of indica hybrid rice is closely related to thehigh amylose level in rice grains. In order to improve thegrain quality of the indica hybrid rice by genetic engineering,an antisense fragment of rice waxy gene, driven by theintroduced into three major parent lines of indica hybrid rice,all contain a high amylose level in the grains, via Agrobacte-rium, and more than 100 hygromycin-resistant plants wereregenerated. The analysis of PCR amplification and South-ern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenicrice plants. Most of the primary transgenic rice plants grewnormally, and the mature seeds from these transgenic plantswere performed for analysis of the amylose content. Theresults showed that the amylose content in the endosperm ofsome grains was reduced and the lowest reached 7.02% inone homozygous transgenic line, 72.4% lower than that ofthe wild type. The influence of the altered amylose contenton the gelatinization temperature and gel consistency wasalso observed in several homozygous transgenic rice plants.     

Introduction of antisensewaxy gene into main parent lines ofindica hybrid rice

Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality ofindica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of theindica hybrid rice by genetic engineering, an antisense fragment of ricewaxy gene, driven by the 5′-franking sequences of the ricewaxy gene, was successfully introduced into three major parent lines ofindica hybrid rice, all contain a high amylose level in the grains, viaAgrobacterium, and more than 100 hygromycinresistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains was reduced and the lowest reached 7.02% in one homozygous transgenic line, 72.4% lower than that of the wild type. The influence of the altered amylose content on the gelatinization temperature and gel consistency was also observed in several homozygous transgenic rice plants. The two authors contributed equally to this work.     

花粉愈伤组织群体偏态分离的SSR标记鉴定

利用水稻花粉育性基因S-b的SSR分子标记RMl3，鉴定籼粳稻杂种Fl的花粉愈伤组织的基因型，以确定每块愈伤组织在S-b座位的遗传来源，结果表明该座位的等位基因S^i或S^j在愈伤组织群体中分离比不符合1：1的规律，出现偏态分离现象．选择不同培养基及增加预冷处理不会改变偏态分离的方向，但预冷处理会增大偏分离的趋势．为进一步解释DH群体构建中的某些基因的偏态分离现象提供了依据，同时讨论了将SSR标记引入组织培养体系的可行性．     

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.     

UDP-glucose pyrophosphorylase2 (OsUgp2), a pollen-preferential gene in rice, plays a critical role in starch accumulation during pollen maturation

UDP-glucose pyrophosphorylase (UGPase) is predominantly present and plays significant role in carbohydrate metabolism in plants. Two homologous UGPase genes, OsUgp1 and OsUgp2, exist in rice genome. OsUgp1 has recently been reported to be essential for callose deposition during pollen mother cell and meiosis stages as well as for seed carbohydrate metabolism. In this study, a full-length cDNA of OsUgp2 was isolated from rice anther. Northern blot and RNA in situ hybridization indicated that the expression o...