少根根霉Δ~6-脂肪酸脱氢酶基因在转基因油菜中的表达  

Expression of Rhizopus arrhizus Δ~6-Fatty Acid Desaturase Gene in Transgenic Rapeseed

作　　者：张琦[1] 李明春[2] 蔡易[2] 苗翠苹[1] 李绍兰[1] 陈有为[1] 邢来君[2] 

机构地区：[1]云南大学云南省微生物研究所,昆明650091 [2]南开大学微生物学系,天津300071

出　　处：《中国农业科学》2006年第3期463-469,共7页Scientia Agricultura Sinica

基　　金：国家自然科学基金(30200176);云南省自然科学基金(2005C0003Q);云南大学理(工)科校级科研项目(2004Q012B)

摘　　要：【目的】验证少根根霉△6-脂肪酸脱氢酶基因在转基因油菜中的表达情况,为进一步基因工程化生产γ-亚麻酸打下基础。【方法】将少根根霉△6-脂肪酸脱氢酶基因和植物表达载体pCPN2301连接,构建重组表达质粒pCPNRAD6,采用农杆菌介导的油菜子叶节转化法,将该基因导入甘蓝型油菜H165,获得转基因植株,最后通过气相色谱检测基因的表达情况。【结果】PCR、GUS组织化学染色和Southern杂交分析结果表明,目的基因已经整合到油菜基因组中,Northern杂交分析进一步表明目的基因在RNA水平获得表达,气相色谱分析检测到转基因油菜叶片中γ-亚麻酸和十八碳四烯酸生成,证明目的基因获得功能表达。同样,用改变少根根霉Δ6-脂肪酸脱氢酶基因的转译起始密码子周边序列的基因RAD6-1所构建的转基因酵母中,也得到类似的结果,而且各种目的脂肪酸的含量均有提高。【结论】初步建立了少根根霉△6-脂肪酸脱氢酶基因在转基因油菜中的表达体系。[Objective] This study is to detect the expression of Rhizopus arrhizus △^6-fatty acid desaturase gene RAD6 in transgenic rapeseed and provide the primary basis for the future application of gene engineering to GLA production. [ Method ] RAD6 was amplified and subcloned introduced expression vector pCPN2301 to generate a recombinant plasmid pCPNRAD6 under the control of CaMV35S promoter, which was subsequently transformed into Brassicu napus H165 by the Agrobacterium-mediated cotylendon node genetic transformation. The expression of target gene was detected by GC analysis. [Result] PCR, GUS staining, DNA dot and southern blot analysis of the transgenic plants showed that the target gene has been integrated into rape genome. Northern blot results proved that the target gene was expressed at mRNA level, and GC analysis indicated that RAD6 expressed in transgenic rapes with the accumulation ofγ-linolenic acid and octadecatetraenoic acid. However, with modification of sequences flanking AUG codon according to the Kozak sequence, a novel fragment （RAD6-1） of Rhizopus arrhizus △^6-fatty acid desaturase gene was obtained and then transformed into Brassicu napus H165 for expression. As a result, compared with the transgenic rapeseed containing RAD6, the expression level of △^6-fatty acid desaturase gene was increased accordingly. [Conclusion ] The expression system of Rhizopus arrhizus △^6-fatty acid desaturase gene RAD6 in transgenic rapeseed was primarily constructed.

关 键 词：少根根霉 △^6-脂肪酸脱氢酶基因 油菜 Γ-亚麻酸 十八碳四烯酸 

分 类 号：S565.4[农业科学—作物学] Q78[生物学—分子生物学]