BcA9启动子调控的反义BcMF12对白菜基因沉默的效果

 【目的】探讨花药绒毡层特异启动子BcA9调控的反义BcMF12基因对白菜（Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis）基因沉默的效果，明确其对花粉发育的影响。【方法】在从白菜花蕾cDNA中克隆BcMF12基因的保守区段的基础上，将白菜花药特异表达启动子BcA9与反义BcMF12基因融合，构建反义表达载体，并经农杆菌介导导入到白菜基因组中，利用抗生素筛选和分子检测筛选出转基因植株。【结果】PCR和Southern检测结果表明，BcA9启动子驱动的反义BcMF12-GUS报告基因已整合到白菜基因组中；Northern杂交显示转基因植株花蕾和花中的BcMF12在mRNA水平的表达明显受到抑制，而且花粉萌发率显著下降。【结论】BcA9调控的反义BcMF12基因明显抑制了BcMF12的表达，从而影响白菜花粉的发育。

Effect of the Antisense BcMF12 Driven by the BcA9 Promoter on Gene Silencing in Brassica campestris L.ssp.chinensis

【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.