莱茵衣藻“细菌型”pepc2基因叶绿体表达及其对油脂蛋白质含量的影响

Regulation and expression of bacteria type pepc2 in Chlamydomonas reinhardtiiand and its effect on protein and lipid content

磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PEPC,EC 4.1.1.31)在植物碳代谢中有重要作用,高等植物中PEPC可以调控蛋白质和脂肪酸含量,但是对绿藻PEPC的研究还较少。莱茵衣藻是绿藻中的模式生物,其基因工程中外源基因的转化以叶绿体转化效率最高,为了提高外源蛋白表达和获得高产油莱茵衣藻,利用叶绿体转化法能进一步提高表达效率。因此,本研究首先克隆了莱茵衣藻"细菌型"PEPCCSⅠ(conserved sequenceⅠ)的639 bp,构建了p ASap I-reve-Crpepc2和p ASap I-forw-Crpepc2正反向叶绿体表达载体,并用基因枪法转化莱茵衣藻cc-400获得了空质粒型,正向型和反向型转基因藻株。其次,应用RTq PCR方法检测Crpepc2的相对表达量,结果表明反向型Crpepc2的表达量减少了89.97%,而正向型增加了201.16%。最后,检测了总蛋白和脂质含量的变化,正向型突变株的总蛋白增加了37.03%;而反向型突变株的脂质含量增加了70.32%,且不饱和脂肪酸含量明显增加。以上结果证明莱茵衣藻"细菌型"PEPC的叶绿体表达不仅可以进一步提高蛋白质与脂肪酸含量,也可为今后外源蛋白的表达和高产油工程藻的应用做贡献。

Phosphoenolpyruvate carboxylase（ PEPC,EC 4. 1. 1. 31） plays an important role in plant carbon metabolism,which can affect protein and lipid metabolism in higher plant,while studies on green alga PEPC are limited. Chlamydomonas reinhardtii is the model organism in green alga,and its chloroplast transformation is mature and efficient. In order to get high expression quality of protein and lipid content,chloroplast transformation method could be applied. In this study,the sequence with 639 bp（ about 1/7 length） of C.reinhardtii CS I was cloned,then C. reinhardtii was transformed by the empty plasmid p ASap I,vectors p ASap I-reve-Crpepc2 and p ASap I-forw-Crpepc2,to generate ＂blank,＂＂forward,＂and＂reverse＂mutants by particle bombardment,respectively. The relative expression of Crpepc2 were detected with RT-q PCR,it showed that Crpepc2 expression of reverse mutant decreased by 89. 97%,and that of forward mutant increased by 201. 16%. Mean while the contents of protein and lipid were detected,the data showed that total protein of forward mutant rose by 37. 03%,and the lipid content of reverse mutant enhanced by 70. 32%,moreover,fatty acid of reverse mutant increased obviously. In conclusion,bacteria type PEPC in Chlamydomonas reinhardtii not only can improve protein and lipid content,but also can make contribution to foreign protein expression and high lipid content engineered microalgae.