Journal of Nuclear Agricultural Sciences ›› 2021, Vol. 35 ›› Issue (11): 2470-2481.DOI: 10.11869/j.issn.100-8551.2021.11.2470

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

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

LI Ziming1(), PAN Yichen1, FAN Xiaoping1, JIANG Jianming2, WANG Zhi'an2, WANG Zhonghua1,*()   

  1. 1Institute of Biotechnology, Zhejiang Wanli University, Ningbo, Zhejiang 315100
    2Zhejiang Institute of Traditional Chinese Medicine, Hangzhou, Zhejiang 310023
  • Received:2020-08-13 Accepted:2020-10-27 Online:2021-11-10 Published:2021-09-18
  • Contact: WANG Zhonghua

浙贝母淀粉、蔗糖代谢相关基因的克隆与表达分析

李梓铭1(), 泮仪晨1, 范小平1, 江建铭2, 王志安2, 王忠华1,*()   

  1. 1浙江万里学院生物技术研究所,浙江 宁波 315100
    2浙江省中药研究所有限公司,浙江 杭州 310023
  • 通讯作者: 王忠华
  • 作者简介:李梓铭,男,主要从事浙贝母分子生物学研究研究。E-mail: 1273354662@qq.com
  • 基金资助:
    国家重点研发计划项目(2017YFC1700700);现代农业产业技术体系建设专项(CARS-21);浙江省中药材新品种选育重大科技专项(2021C02074);宁波市公益项目(2019C10011)

Abstract:

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.

Key words: Fritillaria thunbergii, bulb, starch, sucrose, gene cloning, gene expression

摘要:

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、GBSSSSS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。

关键词: 浙贝母, 鳞茎, 淀粉, 蔗糖, 基因克隆, 基因表达