中国水稻科学 ›› 2015, Vol. 29 ›› Issue (2): 126-134.DOI: 10.3969/j.issn.1001-7216.2015.02.003
李晓静1, 徐多多1, 徐益敏1, 翟开恩1, 杨窑龙2, 潘建伟1, 饶玉春1,2,*()
收稿日期:
2014-07-27
修回日期:
2014-12-02
出版日期:
2015-03-10
发布日期:
2015-03-10
通讯作者:
饶玉春
基金资助:
Xiao-jing LI1, Duo-duo XU1, Yi-min XU1, Kai-en ZHAI1, Yao-long YANG2, Jian-wei PAN1, Yu-chun RAO1,2,*()
Received:
2014-07-27
Revised:
2014-12-02
Online:
2015-03-10
Published:
2015-03-10
Contact:
Yu-chun RAO
摘要:
禾本科植物的脆秆突变体是进行次生细胞壁研究的理想材料。对水稻 bc88突变体的前期研究表明, BC88编码一个纤维素合酶催化亚基OsCesA9。通过水稻遗传转化 BC88启动子融合GUS的载体, 检测到BC88在根、茎、叶、鞘、花中均有表达,并且在茎和根中表达量较高,这可能是由于 BC88突变后影响了根系的正常发育及功能,进而影响地上部的生长,最终导致 bc88半矮表型。将 BC88与GFP的融合表达载体转入水稻和烟草表皮细胞中,共聚焦显微观察显示,OsCesA9定位在质膜上。质膜是纤维素合成的主要场所,这充分说明了OsCesA9是水稻次生细胞壁中纤维素合成必不可少的纤维素合酶催化亚基之一。本研究为 BC88执行其生物学功能提供了新依据,对进一步认识该基因在调节次生细胞壁合成等方面的作用具有重要的意义。
中图分类号:
李晓静, 徐多多, 徐益敏, 翟开恩, 杨窑龙, 潘建伟, 饶玉春. 水稻纤维素合酶催化亚基的编码基因BC88的表达分析[J]. 中国水稻科学, 2015, 29(2): 126-134.
Xiao-jing LI, Duo-duo XU, Yi-min XU, Kai-en ZHAI, Yao-long YANG, Jian-wei PAN, Yu-chun RAO. Expression of OsBC88, a Rice Cellulose Synthase Catalytic Subunit Gene[J]. Chinese Journal OF Rice Science, 2015, 29(2): 126-134.
引物名称 Primer | 引物序列(5'-3') Primer sequence(5'-3') |
---|---|
pBC88-2035-HindⅢ-F | CCGAAGCTTCTTTTCCATTTTCATATTTCCCTGCCATCA |
pBC88-3896-BamHⅠ-R | GCCGGATCCGTGTGGGAGACGGTGGCGGGTTGGTTG |
BC88-no ATG-XbaⅠ-F | GGCTCTAGAGAGGCGAGCGCCGGGCTGGTGGCCGG |
BC88-stop codon-XbaⅠ-R | AGCTCTAGATCAGCAGTTGATGCCGCACTGCCTGACGTC |
BC88-600-R | CTCAGACACCGGGTAGGGGTGGATGC |
OsActin-Real-F | TGGCATCTCTCAGCACATTCC |
OsActin-Real-R | TGCACAATGGATGGGTCAGA |
OsBC-104-F | AGGTGTGCGAGATATGCGGCGACGAG |
OsBC-213-R | CTCGTACTCGTAGCAGGGGCGGCACAC |
表1 OsBC88 基因克隆及实时PCR 引物序列
Table 1 Primer sequences for OsBC88 cloning and Real-time PCR.
引物名称 Primer | 引物序列(5'-3') Primer sequence(5'-3') |
---|---|
pBC88-2035-HindⅢ-F | CCGAAGCTTCTTTTCCATTTTCATATTTCCCTGCCATCA |
pBC88-3896-BamHⅠ-R | GCCGGATCCGTGTGGGAGACGGTGGCGGGTTGGTTG |
BC88-no ATG-XbaⅠ-F | GGCTCTAGAGAGGCGAGCGCCGGGCTGGTGGCCGG |
BC88-stop codon-XbaⅠ-R | AGCTCTAGATCAGCAGTTGATGCCGCACTGCCTGACGTC |
BC88-600-R | CTCAGACACCGGGTAGGGGTGGATGC |
OsActin-Real-F | TGGCATCTCTCAGCACATTCC |
OsActin-Real-R | TGCACAATGGATGGGTCAGA |
OsBC-104-F | AGGTGTGCGAGATATGCGGCGACGAG |
OsBC-213-R | CTCGTACTCGTAGCAGGGGCGGCACAC |
图1 PBI121 BC88 pro :: GUS 载体构建 A-PBI121 BC88 pro :: GUS 载体; B-BC88启动子PCR扩增产物; C-PCR阳性菌液鉴定; D-PCR阳性菌落鉴定; M-DNA分子量标记; CK-阳性对照。
Fig.1. Construction of PBI121 BC88 pro :: GUS vector. A, Structure of PBI121 BC88 pro :: GUS vector; B, PCR products of BC88 promoter; C, PCR identification of positive bacterial suspension; D, PCR identification of positive bacterial colony; M, DNA marker; CK, Positive control.
图2 pcambia 1300-2×35s:: eGFP: BC88 载体构建 A-pcambia 1300-2×35s:: eGFP: BC88载体; B-BC88的PCR扩增产物; C-PCR阳性菌液鉴定; D-PCR阳性菌落鉴定; M, DNA分子量标记; CK, 阳性对照。
Fig. 2. Construction of pcambia 1300-2×35s:: eGFP: BC88 vector. A, Structure of pcambia 1300-2×35s:: eGFP: BC88 vector; B, PCR products of BC88; C, PCR identification of positive bacterial suspension; D, PCR identification of positive bacterial colony, M, DNA marker; CK, Positive control.
图3 OsBC88 组织表达分析 A-苗期日本晴的根、茎、叶、鞘GUS染色; B-开花期日本晴小穗花序的GUS染色; C-苗期pBI121: pro BC88:: GUS转基因阳性植株的根、茎、叶、鞘GUS染色; D-开花期pBI121:BC88 pro:: GUS 转基因植株的小穗花序GUS染色。
Fig. 3. Tissue expression analysis of OsBC88. A, GUS staining of root, culm, leaf, sheath of Nipponbare at seedling stage; B, GUS staining of flower of Nipponbare at flowering stage, serving as negative control; C, GUS staining of root, culm, sheath, leaf of pBI121: BC88 pro :: GUS transgenic lines at seedling stage; D, GUS staining of flower of pBI121:BC88 pro:: GUS transgenic lines at flowering stage.
图5 BC88 的亚细胞定位 A-35S:: eGFP 和35S:: eGFP: BC88 在烟草表皮细胞中的亚细胞定位; B-35S:: eGFP 在水稻根尖细胞中的定位, 下部是对上部的放大。GFP代表绿色荧光蛋白, DIC代表微分干涉对比阶段, Merged代表GFP与DIC的合并。
Fig. 5. Subcellular localization of BC88. A, Localization of 35S:: eGFP and 35S:: eGFP: BC88 in tobacco epidermal cells; B, Localization of 35S:: eGFP in rice root tip cells, the bottom is the amplification of the upside. GFP indicates the green fluorescence of proteins, DIC indicates the differential interference contrast phase, and Merged indicates the merging of GFP and DIC.
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