褪黑素合酶基因AANAT在莱茵衣藻中的表达及其功能研究

In animals, melatonin(also known as the pineal gland) has been identified as a potent free radical scavenger, which not only helps sleep and adjusts the jet lag reaction, but also improves the function of immune system and retards the process of aging. Melatonin has also been found to be a ubiquitous and highly conserved molecule in the plant kingdom. There are reports demonstrating the ability of melatonin to adjust light cycle, induce flowering, remove reactive oxygen molecules and promote vegetative growth. But the function of melatonin in Microalgae barely keeps unknown. Our project is planned to construct a Chlamydomonas expression vector pDBle-AANAT which harboring one of the melatonin syntheses Arylalkaylamine - N-Acetyltransferase, AANAT. An agrobacterium tumefaciens-mediated method will be introduced to deliver AANAT gene into the genome of a mode micro algae, Chlamydomonas reinhardtii by using. Then the transgenic Chlamydomonas reinhardtii strains will be identified in the antibiotic resistance strains by molecular methods, such as PCR and RT-PCR, or by detecting the expression levels of exogenous gene and the content of melatonin. Following analysis of the anti-oxidation abilities, and circadian rhythm phenotypes of transgenic algae strains. This study reveals the influences of over-accumulation of melatonin on the growth of Chlamydomonas reinhardtii. It will also be desired to add more knowledge on the function of plant melatonin by research it on a single cell level.

褪黑素(Melatonin) 也称松果腺素，在动物体中具有促进睡眠、调节时差、抗衰老等生理功能；在植物体内有调节光周期、清除细胞内活性氧分子和促进营养生长等功能，而其在微藻内的生物学功能研究则国内外鲜见。本项目拟构建褪黑素合酶关键酶AANAT (Arylalkaylamine-N-Acetyltransferase)的衣藻表达载体pDBle-AANAT，以莱茵衣藻(Chlamydomonas reinhardtii)为受体，借助农杆菌介导法将该基因导入该模式微藻基因组中；进而经抗性筛选、培养、分子生物学鉴定转化藻株，得到AANAT转基因藻株；检测AANAT基因的表达水平及褪黑素含量变化，分析AANAT过量表达的藻株所具有的抗氧化能力、抗紫外损伤能力及在昼夜节律中的功能。本研究揭示过量积累的褪黑素对莱茵衣藻生长的影响，拓宽褪黑素功能研究领域，对推动单细胞水平上的褪黑素植物功能研究有重要意义。

褪黑素(Melatonin)也称松果腺素，是色氨酸的吲哚衍生物，普遍存在于动物、藻类、真菌和细菌中。褪黑素在动物体中具有促进睡眠、调节时差、抗衰老等生理功能；在植物体内有调节光周期、清除细胞内活性氧分子和促进营养生长等功能。目前，有关褪黑素在藻类中的生物学功能的研究资料还很薄弱，褪黑素仅在少数藻种中推测具有类似动物体内的昼夜调节功能。为了明确褪黑素在在藻类中的生物学功能，本研究首先构建了携带褪黑素合成关键基因AANAT(Arylalkaylamine-N-Acetyltransferase)的衣藻表达载体pDBle-AANAT，并借助农杆菌介导法和玻璃珠转化法对莱茵衣藻(Chlamydomonas reinhardtii)进行了遗传转化。研究中，通过玻璃珠转化法成功将外源基因AANAT导入莱茵衣藻（C. reinhardtii cc503）的基因组DNA；进而通过抗性筛选、培养及分子生物学检测，获得了褪黑素合酶基因AANAT过表达的转基因莱茵衣藻藻株；经过持续鉴定、扩大培养得到AANAT过表达的莱茵衣藻工程藻株。褪黑素功能验证实验结果显示，在AANAT过表达的工程藻株中，褪黑素的含量显著增加，并表现出对外源NaCl、H2O2造成的过氧化胁迫抵抗能力增强，揭示出AANAT基因过表达能够在一定程度上提高转基因微藻细胞对环境非生物胁迫的抗性。目前，项目相关研究正进一步聚焦于AANAT过表达工程藻的抗紫外损伤能力及其在昼夜节律中的表现等方面，为推动在单细胞水平上，深入研究微藻及植物褪黑素功能奠定初步的基础。