浙贝母淀粉、蔗糖代谢相关基因的克隆与表达分析

Cloning and Expression Analysis of Genes Related to Starch and Sucrose Metabolism in Fritillaria thunbergii

为了培育高品质浙贝母鳞茎,本研究以浙贝3号为材料,采用蒽酮法、高效液相色谱(HPLC)技术和试剂盒法测定浙贝母鳞茎发育过程中淀粉与蔗糖的含量变化;采用同源克隆、cDNA末端快速扩增技术(RACE)克隆蔗糖代谢关键酶基因,使用实时荧光定量PCR(qRT-PCR)测定各基因表达量并进行相关性分析。结果发现,鳞茎中淀粉含量在成熟期达到最大值,可溶性总糖和蔗糖含量在鳞茎发育初期和末期最高,AGP2、AGP3、GBSS、SSS在鳞茎中表达量最高,AGP1在根中表达量最高,5个基因均与淀粉含量显著相关(相关系数分别为0.930、0.839、0.582、0.561和0.826),SS主要表达部位为鳞茎,SPS为叶片,二者均与蔗糖含量呈显著相关性(相关系数为0.575和0.536)。本研究为淀粉、蔗糖代谢基因功能验证,以及浙贝母分子育种与分子调控鳞茎发育相关研究奠定了基础。

In order to cultivate high-quality bulblet of Fritillaria thunbergii, the changes of starch and sucrose during bulb development were determined by anthrone method, High Performance Liquid Chromatography(HPLC) and kit method, and the key enzyme genes involved in sucrose metabolism were cloned by homologous cloning and rapid amplification of cDNA ends(RACE). Using real time quantitative PCR(qPCR) were used to detect the expression of each gene and correlation analyses were performed. The results showed that the starch content in bulb reached the maximum at the mature stage, the soluble sugar and sucrose content were the highest at the early and late stage of bulb development, the expression levels of AGP2, AGP3, GBSS and SSS were the highest in bulb, and the expression level of AGP1 was the highest in root. The expressions of five genes were significantly correlated with starch content(correlation coefficients were 0.826, 0.930, 0.839, 0.582 and 0.561, respectively). SS was expressed mainly in the bulb and the SPS gene was in the leaf. The expressions of both genes were significantly correlated with sucrose content(correlation coefficient was 0.575 and 0.536). This study laid a foundation for functional verification of genes participated starch and sucrose metabolism, and for molecular breeding and molecular regulation of bulb development in F. thunbergii.