中國農業科學 ?? 2008, Vol. 41 ?? Issue (5): 1410-1415 .doi: 10.3864/j.issn.0578-1752.2008.05.019

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BcA9啟動子調控的反義BcMF12對白菜基因沉默的效果

宋江華,朱熠鵬,余小林,向 珣,曹家樹   

  1. 浙江大學蔬菜研究所
  • 收稿日期:2007-01-18 修回日期:2007-10-26 出版日期:2008-05-10 發布日期:2008-05-10
  • 通訊作者: 曹家樹

Expression of the Antisense BcMF12 Gene Driven by the BcA9 promoter in transgenic Chinese cabbage

Jiang-Hua SONG Jia-Shu CAO Xiao-Lin YU Xun Xiang   

  1. 浙江大學蔬菜研究所
  • Received:2007-01-18 Revised:2007-10-26 Online:2008-05-10 Published:2008-05-10
  • Contact: Jia-Shu CAO

摘要: 【目的】探討花藥絨氈層特異啟動子BcA9調控的反義BcMF12基因對白菜(Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis)基因沉默的效果,明確其對花粉發育的影響。【方法】在從白菜花蕾cDNA中克隆BcMF12基因的保守區段的基礎上,將白菜花藥特異表達啟動子BcA9與反義BcMF12基因融合,構建反義表達載體,并經農桿菌介導導入到白菜基因組中,利用抗生素篩選和分子檢測篩選出轉基因植株。【結果】PCR和Southern檢測結果表明,BcA9啟動子驅動的反義BcMF12-GUS報告基因已整合到白菜基因組中;Northern雜交顯示轉基因植株花蕾和花中的BcMF12在mRNA水平的表達明顯受到抑制,而且花粉萌發率顯著下降。【結論】BcA9調控的反義BcMF12基因明顯抑制了BcMF12的表達,從而影響白菜花粉的發育。

關鍵詞: 白菜, BcMF12, BcA9啟動子, 反義RNA, 基因表達, GUS活性

Abstract: 【Objective】The study analyzed the expression of BcMF12 gene regulated by BcA9 promoter in the transgenic Chinese cabbage, and confirmed the effect of antisense BcMF12 gene on the pollen development.【Method】An conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in Chinese cabbage (Brassica campestris L. ssp. chinensis Makino), and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into Chinese cabbage via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis.【Result】The analysis of GUS activity revealed that the expression level of antisense BcMF12-GUS fusion gene regulated by the BcA9 promoter was strongest in the anther, while very weak or no activity could be detected in other organs of transgenic plants. It showed that antisense BcMF12-GUS fusion genes could be tissue-specifically expressed in transgenic Chinese cabbage when driven by the BcA9 promoter. Northern blot suggested that the expression of BcMF12 gene in pollen was down-regulated significantly. The pollen germinated rate of transgenic plants contained the antisense BcMF12 gene decreased as compared with that of the control plants.【Conclusion】The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by the anther-specific BcA9 promoter, which affected the pollen development in Chinese cabbage.

Key words: Brassica campestris ssp. chinensis, BcMF12, BcA9 promoter, antisense RNA, gene expression, GUS activity

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