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Archive > Volume 15 Issue 2 > 2023,15(2):291-297. DOI:https://doi.org/10.1016/j.chmed.2022.12.005 Prev Next

紅花查爾酮合酶基因的克隆、表達及活性分析

Original articles

Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius

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    摘要:
    目的：黃酮類化合物是紅花(Carthamus tinctorius L.)中的生物活性化合物，其中查爾酮合成酶（CHS）是第一限制性酶。然而，目前尚不清楚參與紅花中類黃酮生物合成的查爾酮合酶基因（CHS）。本研究以紅花全長轉(zhuǎn)錄組數(shù)據(jù)及模式植物類黃酮代謝研究為基礎，對紅花查爾酮合成酶基因進行克隆、表達分析及酶活性驗證。 方法：根據(jù)課題組前期紅花的全長轉(zhuǎn)錄組數(shù)據(jù)，篩選查爾酮生物合酶基因。利用PCR從紅花的花中克隆紅花查爾酮合成酶基因（CtCHS）。對克隆的CtCHS序列進行生物信息學分析，并采用實時熒光定量PCR（RT-PCR）分析其在紅花不同器官（根、莖、葉、花）及花不同發(fā)育階段的表達模式。通過多克隆抗體蛋白質(zhì)印跡法（Western Blot）分析紅花查爾酮合成酶（CtCHS）在花不同發(fā)育階段的表達情況。構建CtCHS的重組表達載體，誘導并純化CtCHS重組蛋白，用于體外酶促反應檢測（催化對香豆酰輔酶A和丙二酰輔酶A的反應以產(chǎn)生柚皮素查爾酮），反應產(chǎn)物利用高效液相色譜儀（HPLC）和液質(zhì)聯(lián)用儀（LC-MS）進行檢測。 結果：本研究從紅花的花中成功克隆出兩個全長CtCHS基因（CtCHS1和CtCHS3），基因長度分別為1525 bp和1358 bp。RT-PCR分析表明，兩個基因在花中均表達量最高，但在花各發(fā)育階段，CtCHS1的表達均高于CtCHS3。WB分析表明，在花的每個發(fā)育階段只能檢測到CtCHS1蛋白。HPLC和LC-MS結果表明，CtCHS1重組蛋白可以催化底物（對香豆酰輔酶A和丙二酰輔酶A）轉(zhuǎn)化為柚皮素查爾酮，證明CtCHS1重組蛋白具有活性。 結論：CtCHS1參與紅花中柚皮素查爾酮的生物合成。

    Abstract:
    Objective: Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated. Methods: Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS. Results: Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone. Conclusion: CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower.

    關鍵詞:
    紅花;查爾酮合酶基因;表達分析;類黃酮;功能驗證;基因克隆

    Keywords:


    Project Supported:
    This project was supported by grants from the National Natural Science Foundation of China (No. 82274039, 81803669) and China Postdoctoral Science Foundation (No. 2018M643790XB).

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