人工miRNA干扰共转化法获得无筛选标记转基因香稻
摘要
水稻是世界上最重要的粮食作物之一,全球将近一半的人口以水稻为主食。随着人们生活水平的日益提高,优质稻米的需求量成倍增长。香味是水稻重要的食味品质性状,具有独特香味特征的稻米在市场上的价格也都高于非香稻米,因此香稻育种已成为现代水稻育种的重要内容之一。
本研究中,我们利用网站WMD3(Web MicroRNA Designer3) http://wmd3.weigelworld.org设计了针对控制水稻香味的甜菜碱醛脱氢酶基因BADH2的amiRNA。分别以Ubi组成表达启动子和GluC胚乳特异表达启动子驱动,将其整合进共转化表达载体pSB130,转化非香稻品种日本晴和明恢86。经过香味鉴定和香味物质2AP测定确认Ubi::BADH2amiRNA转基因植株成功获得带有香味,但GluC::BADH2amiRNA转基因植株中未能检测到香味。在Ubi::BADH2amiRNA转基因香味植株中,amiRNA特异地干扰了BADH2的表达,BADH2表达量显著下降,仅相当于未转化植株20-40%的表达量,而近似基因BADH1的表达量没有显著差异。并且在Ubi::BADH2amiRNA转基因植株中检测到了明显的香味物质2AP,而在未转化植株中未检测到该物质,但转基因香稻中2AP的含量显著低于香稻栽培品种。
通过在转基因Ubi::BADH2amiRNA植株T0代和后代T1代、T2代中对目的片断amiRNA和筛选标记基因潮霉素连续进行检测,确认在T1代获得了双T-DNA区发生分离,仅带有amiRNA的无筛选标记转基因香味植株。部分T1株系后代中,amiRNA检测有无分离比接近1：3,其中的amiRNA是单拷贝插入。
脯氨酸含量测定结果表明在Ubi::BADH2amiRNA再生香味植株中,叶片中脯氨酸含量略高于未转基因植株,且与未转基因植株相比,其脯氨酸代谢途径的关键酶基因ProDH的表达量出现显著下降,而P5CS的表达量显著上升。
Rice is the most important food crops in the world, and is the staple food for nearly half the population on the earth. With the improvement of people's living standard, the demand for high quality rice is growing exponentially. Fragrance is an important eating quality property of rice. The price of fragrant rice with an unique flavor character is higher than unfragrant rice in the market. Therefore, fragrant rice breeding is one of the important contents of the modern rice breeding
In this study, we used WMD3(Web MicroRNA Designer3) http://wmd3.weigelworld.org to design amiRNA for betaine aldehyde dehydrogenase gene BADH2controlling rice fragrance. amiRNA driven by Ubi constitutive promoter and GluC endosperm specific promoter respectively was integrated into the co-transformed expression vector pSB130, then transformed into rice varieties of Nipponbare and Minghui86. After the smell identification and the determination of fragrance substance2AP, fragrance was detected in the Ubi::BADH2amiRNA transgenic plants but not in the GluC::BADH2amiRNA transgenic plants. In the Ubi::BADH2amiRNA transgenic fragrant plants, the expression of BADH2was specifically interfered by amiRNA. The expression of BADH2was significantly decreased to almost20-40%of the level of the expression in untransformed plants. The difference in the expression of a close homolog gene BADH1did not achieve significantly level. In the Ubi::BADH2amiRNA transgenic plants, fragrance substance2AP was detected definitely, however, the content was significantly lower than that of fragrant rice cultivars. while2AP was not detected in untransformed plants.
Through continuous detection on targeted fragment amiRNA and selective marker gene hygromycin in the Ubi::BADH2amiRNA transgenic TO generation and its offspring T1, T2generation, the segregation of double T-DNA regions was observed in T1generation. amiRNA was detected in some transgenic frangrant lines without selective marker gene. The segregation ratio of amiRNA in offspring of part of the T1lines was fit to1:3statistically. Therefore, amiRNA in these lines was a single-copy insertion.
The results of proline content determination showed that proline content in leaf of Ubi::BADH2amiRNA regeneration fragrance plants was slightly higher than that of untransformed plants in transgenic plants and the expression of PRODH and P5CS in transgenic plants were down-regulated and up-regulated respectively compared to untransformed plants
本研究中,我们利用网站WMD3(Web MicroRNA Designer3) http://wmd3.weigelworld.org设计了针对控制水稻香味的甜菜碱醛脱氢酶基因BADH2的amiRNA。分别以Ubi组成表达启动子和GluC胚乳特异表达启动子驱动,将其整合进共转化表达载体pSB130,转化非香稻品种日本晴和明恢86。经过香味鉴定和香味物质2AP测定确认Ubi::BADH2amiRNA转基因植株成功获得带有香味,但GluC::BADH2amiRNA转基因植株中未能检测到香味。在Ubi::BADH2amiRNA转基因香味植株中,amiRNA特异地干扰了BADH2的表达,BADH2表达量显著下降,仅相当于未转化植株20-40%的表达量,而近似基因BADH1的表达量没有显著差异。并且在Ubi::BADH2amiRNA转基因植株中检测到了明显的香味物质2AP,而在未转化植株中未检测到该物质,但转基因香稻中2AP的含量显著低于香稻栽培品种。
通过在转基因Ubi::BADH2amiRNA植株T0代和后代T1代、T2代中对目的片断amiRNA和筛选标记基因潮霉素连续进行检测,确认在T1代获得了双T-DNA区发生分离,仅带有amiRNA的无筛选标记转基因香味植株。部分T1株系后代中,amiRNA检测有无分离比接近1：3,其中的amiRNA是单拷贝插入。
脯氨酸含量测定结果表明在Ubi::BADH2amiRNA再生香味植株中,叶片中脯氨酸含量略高于未转基因植株,且与未转基因植株相比,其脯氨酸代谢途径的关键酶基因ProDH的表达量出现显著下降,而P5CS的表达量显著上升。
Rice is the most important food crops in the world, and is the staple food for nearly half the population on the earth. With the improvement of people's living standard, the demand for high quality rice is growing exponentially. Fragrance is an important eating quality property of rice. The price of fragrant rice with an unique flavor character is higher than unfragrant rice in the market. Therefore, fragrant rice breeding is one of the important contents of the modern rice breeding
In this study, we used WMD3(Web MicroRNA Designer3) http://wmd3.weigelworld.org to design amiRNA for betaine aldehyde dehydrogenase gene BADH2controlling rice fragrance. amiRNA driven by Ubi constitutive promoter and GluC endosperm specific promoter respectively was integrated into the co-transformed expression vector pSB130, then transformed into rice varieties of Nipponbare and Minghui86. After the smell identification and the determination of fragrance substance2AP, fragrance was detected in the Ubi::BADH2amiRNA transgenic plants but not in the GluC::BADH2amiRNA transgenic plants. In the Ubi::BADH2amiRNA transgenic fragrant plants, the expression of BADH2was specifically interfered by amiRNA. The expression of BADH2was significantly decreased to almost20-40%of the level of the expression in untransformed plants. The difference in the expression of a close homolog gene BADH1did not achieve significantly level. In the Ubi::BADH2amiRNA transgenic plants, fragrance substance2AP was detected definitely, however, the content was significantly lower than that of fragrant rice cultivars. while2AP was not detected in untransformed plants.
Through continuous detection on targeted fragment amiRNA and selective marker gene hygromycin in the Ubi::BADH2amiRNA transgenic TO generation and its offspring T1, T2generation, the segregation of double T-DNA regions was observed in T1generation. amiRNA was detected in some transgenic frangrant lines without selective marker gene. The segregation ratio of amiRNA in offspring of part of the T1lines was fit to1:3statistically. Therefore, amiRNA in these lines was a single-copy insertion.
The results of proline content determination showed that proline content in leaf of Ubi::BADH2amiRNA regeneration fragrance plants was slightly higher than that of untransformed plants in transgenic plants and the expression of PRODH and P5CS in transgenic plants were down-regulated and up-regulated respectively compared to untransformed plants
引文
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