参与蓖麻油酸转移的磷脂酶基因的挖掘与功能研究

Ricinoleic acid (12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid（90%） in castor oil. This hydroxy fatty acid (HFA) is used in many industrial applications. Due to the presence of the toxic ricin and hyperallergenic 2S albumins in the seed, castor is not suitable for large-scale agriculture, and development of transgenic oilseed crops for production of ricinoleic acid in high amounts by genetic engineering is desirable. This hydroxy fatty acid (HFA) is synthesized on the sn-2 position of phosphatidylcholine (PC) by fatty acid △12-hydroxylase which encoded by the castor bean FAH12 gene. However, expression of RcFAH12 in Arabidopsis resulted only 17% HFAs in the seed oil. The mobilizing of HFA from HFA-PC to TAG represents a major bottleneck for the accumulation of HFAs in TAG of transgenic Arabidopsis eeds. The phospholipase-mediated pathways may be important for mobilizing HFAs from PC. Phospholipases are a complex and important group of enzymes that hydrolyze phospholipids and are involved in a wide range of physiological processes in plants, while their roles in lipid biosynthesis and metabolism were overlooked. It has often been suggested, but remains to be demonstrated that phospholipases could be an important biotechnological target for modification of seed oils, particularly in relation to engineering oilseeds with unusual fatty acids of industrial value. We did many preliminary works, and identified eight castor phospholipase genes which expressed specificly in developing seeds by using bioinformatics analysis and gene expression profiling.In this proposal, we will analyze the enzymatic activity of the eight selected phospholipase genes on HFA, and then transform the canditate genes to yeast and Arabidopsis thaliana trangenic line CL7 to identify the gene's role in removing HFA from PC by detecting the amounts and structures of HFA-TAG,HFA-DAG and HFA-PC, and by performing the transcriptome profiling of transgenic lines. The aim is to discover the special castor phospholipase homological genes involved in HFA removal and 3HFA-TAG assembly in castor bean, which will be useful for circumventing the bottlenecks in engineering of oilseed plants to accumulate HFA in seed TAG.

蓖麻油含高达90%的羟基化脂肪酸（HFA），工业用途广泛而特殊。脂肪酸的羟基化在细胞膜磷脂PC上完成，在能产生HFA的转基因植物中，HFA不能从PC有效的转移到种子储存油脂甘油三酯（TAG）中是利用其它作物生产蓖麻油的瓶颈。磷脂的水解是实现HFA有效转移的重要基础,磷脂酶是一类磷脂水解酶，水解产物有游离脂肪酸（FFA）、与磷脂含相同脂肪酸的磷脂酸（PA）和甘油二酯（DAG）,它们都是TAG合成的主要前体物。蓖麻能合成HFA且能在种子中高效累积，表明在物种的进化过程中，蓖麻保留了一些与HFA转移相关的特殊磷脂酶基因。本项目在已得到8个在蓖麻种子中特异表达的磷脂酶基因的前期工作基础上，拟通过酶活性分析、酵母系统表达分析、能产生HFA的转基因拟南芥植物系统表达分析以及转基因植株的表达谱分析，挖掘蓖麻种子中与蓖麻油累积相关的重要磷脂酶基因，旨在解决基因工程方法大量生产蓖麻油的瓶颈问题。

蓖麻油具有重要的工业价值，由于蓖麻在农业生产上的局限性，大量研究试图揭示植物种子高效累积蓖麻油的分子机制，以通过基因工程方法大量生产蓖麻油。本项目在推测磷脂酶在蓖麻油累积过程中具有重要作用的基础上，从蓖麻基因组中筛选出8个磷脂酶基因，通过酶活性分析、酶母系统表达、植物系统表达等方法，研究了RcPLA1-5、RcPAT-PLA1-6和RcPLD9三个磷脂酶基因的功能。结果表明，RcPLA1-5和RcPAT-PLA1-6酶对PC具有一定的水解活性，而RcPLD9酶对底物没有明显的水解活性；在酵母表达系统中，在不同的培养温度下，三个酶对酵母油含量及脂肪酸组成的影响存在差异，在32模式培养时，RcPLA1-5和RcPAT-PLA1-6基因均能使酵母的HFA含量增加，而RcPLD9在不同培养条件下均减少HFA的累积，RcPLA1-5和RcPAT-PLA1-6减少HFA在极性脂（PL）中的积累，而RcPLD9增加了HFA在PL中的积累，在三个转基因酵母菌株中，HFA主要存在于TAG1和TAG2中，RcPLA1-5菌株中TAG2含量增加，RcPAT-PLA1-6菌株中TAG1含量增加，而RcPLD9菌株中均显著减少；在CL7中表达时，三个基因均使种子油含量及PL中的HFA含量下降，RcPLA1-5增加种子HFA的含量，TAG1和TAG2的含量没有明显差异，但TAG3含量显著增加，RcPAT-PLA1-6和RcPLD9均减少HFA的含量，RcPAT-PLA1-6种子中HFA在TAG1、TAG2和TAG3中的含量均下降，RcPLD9种子中HFA在TAG2中的含量均增加，在TAG1和TAG3中的含量下降。以上结果表明，RcPLA1-5、RcPAT-PLA1-6和RcPLD9三个磷脂酶水解部位不同，对HFA累积的作用也不同，RcPLA1-5有利于HFA从极性脂向TAG的转移和积累，而RcPLD9阻碍了HFA从极性脂向TAG的转移和积累，RcPAT-PLA1-6有利于HFA从极性脂向TAG的转移但不能促使其在TAG中积累。另外，本项目增加了对另一种高效累积蓖麻油酸植物风筝果（Hiptage benghalensis）种子发育时期的转录组学分析，对蓖麻油酸累积过程中油脂代谢相关基因的差异表达、可变剪接和lncRNA进行了筛选与分析。本项目的实施，为揭示蓖麻油酸累积的分子机制向前迈进了一步。