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乙酰辅酶A羧化酶(ACCase)是脂肪酸生物合成的限速酶,在油料作物脂肪酸代谢和调控基因工程中具有重要的应用价值。本研究利用同尾酶反复酶切的方法,构建了拟南芥Rubisco SSU小亚基转运肽与大肠杆菌异质型ACCase各亚基基因的融合串联种子特异表达载体,在根癌农杆菌(Agrobacterium tumefaciens)介导的下胚轴转化甘蓝型油菜高油品种CY2,研究异质型ACCase转入油菜对含油量的影响。经过抗生素筛选获得11株转化再生植株,PCR、Southern分子检测证明四个目的基因已整合到T0代再生植株油菜基因组中。RT-PCR检测其中4株,说明在转基因株系中目标基因在籽粒中特异表达。对T2代转基因油菜含油量进行测定,研究表明转基因株系的含油量比对照提高了约5.7%。
Acetyl-Coenzyme A Carboxylase (ACCase) is the rate-limiting enzyme in fatty acid biosynthesis and has important application value in the genetic engineering of fatty acid metabolism and regulation in oilseed crops. In this study, we constructed a tandem seed-specific expression vector of Rubisco SSU small subunit transit peptide of Arabidopsis thaliana and the heterogeneous ACCase subunit gene of Escherichia coli, tumefaciens) mediated transformation of hypocotyls in Brassica napus high oil CY2 to study the heterogeneous type of ACCase into rapeseed on oil content. Eleven transformed and regenerated plants were obtained through antibiotic screening. PCR and Southern blot showed that the four target genes were integrated into T0 regenerated plant canola genome. Four of them were detected by RT-PCR, indicating that the target gene was specifically expressed in the grain in transgenic lines. The oil content of transgenic rapeseed (T2) generation was determined. The results showed that the oil content of transgenic lines increased by 5.7% compared with the control.