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-α-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.
To study the efficiency of generating selectable marker-free (SMF) transgenic rice, two transformation methods were employed for four rice varieties (Wuxiangjing 9, Longtefu, Xieqingzao and Zhenshan 97). One method is by using a single twin T-DNA binary vector pYH592 in one Agrobacterium strain, which is composed of two separate T-DNA regions (one carrying an antisense Wx gene and the other carrying a HPT gene). The other one, named as two-strain/ two-vector system, is by using two separate binary vectors in two separate Agrobacterium cultures. The results indicated that the average co-transformation frequencies of the antisense Wx gene and the HPT gene were 10.1% and 45.0%, respectively, for the four rice varieties. And the SMF transgenic plants selected from the offsprings of co-transformants were 55.6% and 60.0% in the two-strain/two-vector and twin T-DNA vector binary systems, respectively.
Yu Heng-xiu Lu Mei-fang CHEN Xiu-hua GONG Zhi-yun LIU Qiao-quan Gu Ming-hong
From the progenies of a telotrisomic of chromosome 9 short arm of an indica rice variety, Zhongxian 3037, a phenotypical variant was selected. The variant plant had rolled leaves, dispersed plant type, as well as a low seed-setting rate. Cytological and molecular cytological investigations revealed two extra chromosomes, which were the shortest in somatic cells of the variant. Fluorescent in situ hybridization (FISH) analysis using a rice centromere specific DNA (RCS2) and a DNA sequence specific for chromosome 9 on premetaphase and pachytene chromosomes showed that these two chromosomes were the short arms of chromosome 9. That is to say, the variant was a telotetrasomic of chromosome 9. Among the 25 pachytene cells, the two telosomic chromosomes paired each other to form a bivalent and didn’t pair with other normal chromosome 9 as multivalents in 96% cells. However, the bivalent was easy to disassociate in advance.
GONG Zhi-yun GAO Qing-song Yu Heng-xiu YI Chuan-deng Gu Ming-hong
依据水稻端粒酶基因的相关生物学信息,构建了含有水稻端粒酶序列RNA干扰结构的植物表达载体pC am 23A-1-2,并利用农杆菌介导法将目的基因导入到粳稻品种日本晴中,获得了78个独立转化子,共165棵转基因植株。通过PCR和Southern杂交分析,证明RNA干扰结构已整合进水稻基因组中;应用染色体末端限制性片段分析法,显示在转基因水稻中端粒DNA序列长度有逐代缩短的趋势,初步证明这些转基因水稻中端粒酶亚基已失活,但是T0和T1代转基因水稻植株的主要农艺性状没有发生明显变化。
