笃斯越桔果皮色泽变异的分子解析及相关基因的克隆

Vaccinium uliginosum is a important wild berry of Ericaceae, Vaccinium. It is mainly located in Da Hinggan Mountain, Inner Mongolia and Changbai Mountain etc. Its berry has a higher economic value, but at present it has not been cultivated and utilized. The colour of peel is an important factor for the appearance and the processing quality of Vaccinium uliginosum fruit. White-fruited Vaccinium uliginosum was a variety found in wild resource, it is a extremely rare germplasm resource, its main mutative traits is that the fruit is silvery white, lack of anthocyanin, and other ingredients content are quite different than that of wild type. In this study, Vaccinium uliginosum and its white-fruited variety were used as material, to be compared with the biological characters and the differences of the nutrient composition between the two fruit; to be compared with the differences of contents like the anthocyanins, proanthocyanidins, flavonoids between the two fruit in the development process; used the transcriptome sequencing technology and bioinformatics method to compare with the differences of the gene expression in the colour-changed period and the fruit maturation period, and also cloning and characteristion analysis of differential gene, structural gene and regulatory gene involved in flavonoid biosynthetic pathway by quantitative RT-PCR, and further research on the mechanism of differential expressions. In this study, morphology, metabolism and gene expressions multi-levels are designed, to study the biological basis on the peel variation of Vaccinium uliginosum deeply, it preliminarily reveals the peel variation mechanism of Vaccinium uliginosum, and it provides theoretical basis for treasuring the utilization of resources and knowing deeply the coloring mechanism of Vaccinium plants.

笃斯越桔是杜鹃花科越桔属野生浆果，主要分布在大兴安岭、内蒙古、长白山区等地，其浆果经济价值极高，但目前还未栽培利用。果皮色泽是笃斯越桔果实外观和加工品质的重要性状。白果笃斯为野生资源中发现的变种，是一种极其稀有的种质资源，其主要变异性状为成熟期果实银白色，几乎不含花青苷，其它成分也与野生型有较大差异。本研究拟以笃斯越桔及其白果变种为试材，比较二者生物学性状及营养成分的差异；比较二者在果实发育过程中花青苷、原花青素、黄酮类色素含量；应用转录组测序技术分析比较转色期及成熟期果实基因表达的差异，克隆并通过定量PCR比较差异基因、类黄酮合成途径相关结构基因、调节基因的时空表达特性，并进一步探讨其差异表达的机理。本研究拟通过形态、代谢、基因表达等多个层面，深入理解笃斯越桔果皮变异的生物学基础，初步揭示笃斯越桔果实着色及果皮变异机理，为珍稀资源的利用及加深对越桔属植物果实着色机理的认识提供理论依据。

白果笃斯越桔为野生资源中发现的变种，几乎不含花青苷，来源于体细胞突变，是花青苷合成机理研究的良好材料。本项目以笃斯越桔白果变种及其野生型为试材，通过营养成分及黄酮类化合物含量分析、转录组测序、qPCR分析等方法对其变异的机理进行了初步研究。结果表明：1. 白果变种成熟果实可滴定酸、还原型抗坏血酸高于野生型，还原糖、可溶性糖、多酚、类黄酮、花青素低于野生型。白果变种中二氢杨梅素、儿茶素、表儿茶素含量高于野生型；山奈酚、杨梅素、槲皮素、二氢槲皮素、原花青素B2、矢车菊素-3-葡萄糖苷低于野生型。2. RNA-seq注释42837个Unigenes，325个差异表达基因中上调表达41个，下调表达284个。3. qPCR验证结果表明，白果变种成熟果实中结构基因VuCHS、VuF3’H、VuDFR、VuANS、VuANR和VuUFGT基因极显著下调表达，VuPOD、VuCCR和VuCHI极显著上调表达，转录因子VuMYB2、VuMYB4、VuMYB7、VuMYB8、VuMYB10、VuTDR4、VuB3和VuMADS-box极显著上调表达，转录因子VuMYB6和VuMYBPA1极显著下调表达。4.在不同发育时期野生型果实中，VuCHS、VuDFR和VuANS基因在果实发育前期与果实开始着色时表达量较高，结构基因VuF3’H、VuF3’5’H、VuUFGT和转录因子VuMYB6、VuMYBPA1、VuMYB7和VuTDR4基因的表达量在果实发育过程中逐渐增加，且在果实开始着色时其表达量迅速上升，与花青苷的积累规律一致。5. 综上所述，转录因子VuMYB6和VuMYBPA1的表达与花青苷积累密切相关，在白果变种成熟果实中极显著下调表达，在野生型果实发育的转色期和果实成熟期显著升高，调控相关结构基因下调表达，导致花青苷不能积累，可能是白果突变体变异的关键基因。本研究结果深入理解了笃斯越桔果皮变异的生物学基础，初步揭示了笃斯越桔果实着色及果皮变异机理，为珍稀资源的利用及加深对越桔属植物果实着色机理的认识提供了理论依据。