紫花苜蓿MsJMT基因的克隆及其与育性的关联分析

以实验室前期对紫花苜蓿(Medicago sativa L.)不育系BSA-Seq分析为背景,为探究茉莉酸类对其育性的影响,本试验采用PCR法,成功从紫花苜蓿基因组中克隆了一个全长1047bp,功能为控制茉莉酸O-甲基转移酶合成的基因,命名为MsJMT。分析表达模式后发现,该基因编码氨基酸348个,编码蛋白为不稳定非分泌亲水蛋白。亚细胞定位结果为核质表达。构建基因进化树后,验证其氨基酸序列与5种豆科植物相似度达99%以上。利用qPCR技术分析其对紫花苜蓿花苞育性的时空表达差异,结果显示不育系全时期表达量比保持系高且在Ⅲ时期两者差异极显著(P<0.01)。而茉莉酸含量测定结果则相反,为不育系全时期低于保持系,且保持系Ⅲ时期含量最高。利用外源茉莉酸甲酯喷施处理Ⅲ时期花苞,结果为在0.5mmol·L^(-1),1mmol·L^(-1)水平下花药提前开裂。研究结果表明MsJMT可能通过茉莉酸通路影响紫花苜蓿育性。

Effect of Methyl Jasmonic Acid on Baccatin Ⅲ Biosynthesis

As baccatin Ⅲ is an immediate diterpenoid precursor of taxol, the increase of baccatin Ⅲ is beneficial to the biosynthesis of taxol. Addition of methyl jasmonic acid （M J） enhances the activity of 10- deaceyle baccatin （DAB） iii acetyl transferase which catalyzes the bioconversion from 10-DAB Ⅲ to beccatin Ⅲ. In this paper, the baccatin Ⅲ content was increased by 174% by the addition of 100 IJmol/L MJ in suspension cultures of Taxus cuspidate. Induction by MJ also increased the expression of a 49.0-kDa protein. This paper describes the cell free acetylation of 10-DAB Ⅲ in crude extracts of enzyme from the suspension cultures of Taxus cuspidate. The reaction product was confirmed by high performance liquid chromatograph （HPLC）. About 25.0% of the 10-DAB Ⅲ was acetylized into baccatin Ⅲ on the 4th day with 100 IJmol/L MJ. The 10-DAB Ⅲ acetyl transferase activity reached a peak on the 2nd day with 100 μmol/L MJ, with 54.7% of the 10-DAB Ⅲ transformed into baccatin Ⅲ. The baccatin Ⅲ content increased with the increase of 10-DAB Ⅲ acetyl transferase activity, although the biosynthesis was delayed by more than 24 h. The remarkable induction of MJ on baccatin Ⅲ biosynthesis shows a promising way to increase the production of taxol.

丹参茉莉酸羧甲基转移酶的原核蛋白表达分析

茉莉酸羧甲基转移酶基因(jasmonic acid carboxyl methyltransferase,JMT)是植物茉莉酸甲酯(methyl jasmonate,MeJA)生物合成过程中的关键酶基因,在植物体内可依赖S-腺苷-蛋氨酸(S-adenosyladenylL-methionine,SAM)作为甲基供体,催化茉莉酸(jasmonic acid,JA)形成茉莉酸甲酯。本研究将丹参茉莉酸羧甲基转移酶基因cDNA构建到原核表达载体PET-28a(+)上后转化到大肠杆菌BL21(DE3)并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。SDS-PAGE凝胶电泳检测结果表明该蛋白的分子量大小约44 kD,与预测的蛋白分子质量大小一致,从而证实丹参JMT基因在大肠杆菌中表达成功。丹参JMT基因在原核细胞表达将对此酶基因的蛋白纯化、体外酶活力检测以及基因功能等研究奠定一定的基础。