利用转基因技术提高山银花木犀草苷含量的研究

Galuteolin, belonging to flavonoids, has arose more recognition because of its pharmacological activity in antioxidation, antibacteria, antivira, and reducing blood lipid. The difference in the content of galuteolin has been the main reason to take Lonicera confusa DC. from Lonicera japonica Thunb. by Chinese Pharmacopoeia. If we improve pharmacological value of Lonicera confuse DC. by increasing the content of galuteolin through genetic engineering, it will have great significance to solve the imbalance between supply and demand of Lonicera japonica Thunb. and to relieve ecological pressure of Karst Rocky Desertification Region by enlarging area of Lonicera confusa DC.which is the traditional plant locally..In this study, the flavonoids synthesis(FNS) and flavanone 3-hydroxylase (F3H), would be cloned from lonicera japonica Thunb., the function of the two enzymes in the pathway of flavonodis biosynthesis in Caprifoliaceae would be explained by the correlation analysis between the content of galuteolin and the gene expression difference in the callus treated differently and in the flowers of different developmental stage in Lonicera japonica Thunb and Lonicera confusa DC.,respectively. Then, the two genes would be transferred into Lonicera confusa DC. through expression vector and interference vector by Agrobacterium- mediated transformation to improve the content of galuteolin in Lonicera confuse DC..And pharmacodynamics evaluation and acute toxicity test of trangenic plant would be carried out to screen the germplasm with good quality. What we would do will be the new way of development and utilization of Lonicera confusa DC..

木犀草苷是一种天然黄酮，以其强抗氧化性、抗菌、抗病毒、降血脂的药理作用而引起国内外学者的广泛重视。木犀草苷含量差异是中国药典2005年后将山银花从金银花分列出来的主要原因。利用遗传工程提高山银花木犀草苷含量，进而提高其药用价值，对于缓解金银花的供需矛盾，大面积推广山银花种植面积、缓解石漠化地区生态压力重要意义。.本项目运用基因克隆、植物组织培养、荧光定量PCR等技术，研究不同诱导条件下金银花愈伤组织和不同花发育时期的金银花、山银花黄酮合成酶（FNS）和黄烷酮3-羟化酶（F3H）基因表达差异以及对应材料中的木犀草苷含量变化，分析这两个基因对木犀草苷合成的影响，阐明它们在忍冬属植物黄酮类生物合成调控机制中的功能；再以农杆菌介导的方式调控它们在山银花中的表达，以提高山银花的木犀草苷含量，并对转基因植株进行药效学评价和急毒实验，期望筛到优良种质，为进一步开发利用山银花探索新途径。

山银花和金银花之争焦点在于黄酮化合物木犀草苷的含量。为了探讨忍冬属不同种植物木犀草苷含量差异产生的原因，本项目克隆了黄酮合成途径中的FNS和F3H全长基因，并用q-PCR检测了他们在忍冬和华南忍冬不同发育时期花组织的表达量，结合各自组织的木犀草苷含量进行相关分析，发现在忍冬中，F3H和FNS基因与木犀草苷含量的相关系数分别为0.8668和0.6389，证明这两个基因对于木犀草苷合成都具有重要影响。为了通过调控黄酮合成途径基因表达量调控黄酮类化合物合成，并从中筛选到木犀草苷含量高的种质，本项目构建了FNS的过表达载体和F3H-RNAi载体，建立了红腺忍冬的遗传转化体系，并以农杆菌介导的方式将这两个基因成功导入到红腺忍冬的基因组。到目前为止，本项目得到了这两个基因转化的阳性苗，其中转化FNS基因阳性继代苗为32株，转化F3H基因的阳性继代苗为15株。由于红腺忍冬芽诱导至少需要50天时间，且芽生长缓慢，阳性植株目前只处于瓶苗阶段，没能进行种质筛选。本项目的科学意义在于合成途径中直系同源基因的表达量相对高低可以解释至少是能部分解释同属不同种植物中同种化合物合成量高低。本项目所建立的红腺忍冬遗传转化体系对忍冬属其它植物遗传转化体系的建立具有重要借鉴价值。除了为红腺忍冬种质资源创新提供方法以外，此遗传转化体系还为忍冬属植物新基因的鉴定和验证提供了实验体系，这对于开展大宗药材忍冬分子生物学研究来说具有重要意义。