RNA干扰20S蛋白酶体α亚基基因对莱茵衣藻油脂代谢的影响

Effect of RNA Interference 20S Proteasome α Subunit A Gene on Lipid Metabolism in Chlamydomonas reinhardtii

摘要:

为了解20S蛋白酶体α亚基（20S proteasome alpha subunit A，POA1）基因对莱茵衣藻（Chlamydomonas reinhardtii）油脂代谢的调控，对莱茵衣藻CC425在低氮胁迫下的CrPOA1表达进行了分析，克隆POA1同源基因片段，构建pMaa7IR/XIR干涉载体并转化莱茵衣藻CC425，对CrPOA1进行有效沉默，测定转基因藻株细胞干质量和油脂含量；克隆全长基因，构建CrPOA1-GFP融合表达载体并转化洋葱表皮细胞，进行亚细胞定位。结果表明，莱茵衣藻在低氮培养下，CrPOA1 mRNA水平比对照（正常培养）显著降低（P<0.01），RNAi转基因藻株CrPOA1 mRNA水平比对照pMaa7IR/XIR（maa7）降低79.36%~85.35%，沉默效果较好；RNAi转基因藻株细胞干质量与对照maa7无显著差异，油脂含量比对照maa7显著降低6.38%~24.63%，CrPOA1正向调控莱茵衣藻油脂；亚细胞定位于细胞核。这表明CrPOA1参与了莱茵衣藻的油脂代谢过程。

Abstract:

To understand the regulation of 20S proteasome α subunit A (POA1) gene on lipid metabolism in Chlamydomonas reinhardtii, the expression of CrPOA1 in C. reinhardtii were analyzed by qRT-PCR under low nitrogen conditions. The fragment of CrPOA1 in C. reinhardtii CC245 was cloned, and then was used to construct a CrPOA1 RNA interference vector and transferred into C. reinhardtii. The cell dry weight and lipid content of transgenic algae strains was measured. The CDs sequence was cloned. The fusion expression vector of CrPOA1-GFP was constructed and transformed into onion epidermal cells for subcellular localization. The results showed that CrPOA1 mRNA level of C. reinhardtii in low nitrogen culture was significantly decreased than that in normal culture (P<0.01). The CrPOA1 mRNA level of RNAi transgenic algae was significantly decreased by 79.36%-85.35%. The cell dry weight of CrPOA1 RNAi transgenic algae was not significantly different from the control (maa7). The lipid content was significantly decreased by 6.38%-24.63% compared with the control (maa7). The CrPOA1 might local in nucleus. So, it was suggested that CrPOA1 could be involved in lipid metabolism of C. reinhardtii.