土肉桂桂皮醛及芳樟醇相關之生合成基因的選殖及功能分析

Abstract

本研究利用退化性引子釣取土肉桂桂皮醛和芳樟醇生合成之相關基因全長，其中phenylalanine ammonia-lyase (PAL)基因之5’ UTR是11 bp，coding region是2127 bp可轉譯出908個胺基酸，3’UTR是192 bp；trans-cinnamate-4-hydroxylase (C4H)基因之5’UTR是76 bp，coding region是1518 bp可轉譯出505個胺基酸，3’UTR是129 bp；4-coumarate:CoA ligase 1 (4CL1)基因之5’UTR是69 bp，coding region是1620 bp可轉譯出539個胺基酸，3’UTR是378 bp；4-coumarate:CoA ligase 3 (4CL3)基因之5’UTR是232 bp，coding region是1698 bp可轉譯出565個胺基酸，3’UTR是293bp；cinnamoyl-CoA reductase (CCR)基因之5’UTR是95 bp，coding region是993 bp可轉譯出330個胺基酸，3’UTR是325 bp；芳樟醇合成酶基因之5’UTR是34 bp，coding region是1755 bp可轉譯出584個胺基酸，3’UTR是209 bp。隨後將PAL、C4H、4CL1、4CL3和CCR等五個基因轉入阿拉伯芥中，利用具有35S promoter的pBI121載體大量表現基因，並於35S promoter和轉殖基因上之序列設計引子，確認基因是否成功轉入阿拉伯芥，成株後利用固相微萃取吸附，於GC-MS分析主成分，試驗結果發現在C4H、4CL1、4CL3和CCR等四個基因轉殖株中皆有發現桂皮醛增加的情形，證實C4H、4CL1、4CL3和CCR等四個基因確實與桂皮醛的生合成相關。 針對土肉桂不同化學品系之14種源進行主成分分析，以反轉錄聚合酶連鎖反應進行檢測結果，得知土肉桂不同種源之桂皮醛和芳樟醇含量與相對應基因表現量並無相關性，而釣取不同種源之4CL3、CCR和LIS之基因全長，發現在4CL3基因片段中有一個桂皮醛種源特定的胺基酸，可用來區分是否為桂皮醛種源；在芳樟醇合成酶基因上，也有發現多個可以用來區分芳樟醇、桂皮醛和樟腦種源之核酸位置。

Phenylalanine ammonia-lyase (PAL), trans-cinnamate- 4-hydroxylase (C4H), 4-coumarate: coenzyme A ligase 1 and 3 (4CL1 and 4CL3), cinnamoyl-CoA reductase (CCR) and linalool synthase (LIS) in Cinnamomum osmophloeum were cloned. The PAL has a 11 bp 5’ UTR, a 2127 bp ORF predicting 908 amino acid, and a 192 bp 3’UTR. The C4H has a 76 bp 5’UTR, a 1518 bp ORF predicting 505 amino acid, and a 129 bp 3’UTR. The 4CL1 has a 69 bp 5’UTR, a 1620 bp ORF predicting 539 amino acid, and a 378 bp 3’UTR. The 4CL3 has a 232 bp 5’UTR, a 1698 bp ORF predicting 565 amino acid, and a 293 bp 3’UTR. The CCR has a 95 bp 5’UTR, a 993 bp ORF predicting 330 amino acid, and a 325 bp 3’UTR. The LIS has a 34 bp 5’UTR, a 1755 bp ORF predicting 585 amino acid, and a 209 bp 3’UTR. Then these genes (PAL, C4H, 4CL1, 4CL3 and CCR) were transformed into Arabidopsis using pBI121 vector. After the genomic DNA and RNA of these transgenic plants were certificated, the volatile compounds were analyzed by solid phase microextration (SPME) and gas chromatography-mass spectrometry (GC-MS). These results demonstrate that the expressions of C4H, 4CL1, 4CL3 and CCR were correlated with cinnamaldehyde biosynthesis. The expression level of genes involved in cinnamaldehyde and linalool synthsis from 14 different sources were analyzed by RT-PCR. The result indicates the major compound of chemotype have no relationship with RNA expression. Comparison the amino acid sequence of 4CL3, CCR, and LIS genes on various type of Cinnamomum osmophloeum, indicated some specific residues of 4CL3 were strongly related with cinnamaldehyde type and some nucleotides of LIS gene were correlated with specific chemotype.