羊踯躅八氢番茄红素脱氢酶基因的克隆及表达分析

doi:10.11924/j.issn.1000-6850.casb2020-0434

中国农学通报 ›› 2021, Vol. 37 ›› Issue (15): 99-105.doi: 10.11924/j.issn.1000-6850.casb2020-0434

所属专题：生物技术

羊踯躅八氢番茄红素脱氢酶基因的克隆及表达分析

肖政(), 苏家乐, 刘晓青, 孙晓波, 何丽斯, 陈尚平, 周惠民, 李畅()

江苏省农业科学院休闲农业研究所/江苏省高效园艺作物遗传改良重点实验室,南京 210014

收稿日期:2020-09-08 修回日期:2020-12-18 出版日期:2021-05-25 发布日期:2021-05-18
通讯作者: 李畅
作者简介:肖政,男,1984年出生,福建三明人,副研究员,博士,主要从事花卉遗传育种研究。通信地址：210014 江苏南京玄武区钟灵街50号江苏省农业科学院园艺研究所,Tel：025-84390223,E-mail： xzforestry@163.com。
基金资助:
国家自然科学基金项目“基于转录组学挖掘调控羊踯躅花瓣黄色性状形成的关键基因”(31600570);江苏省自然科学基金“调控羊踯躅花色突变体花瓣呈色的关键基因挖掘”(BK20150548);江苏省林业发展专项资金“江苏省农科院杜鹃花林木种质资源库”(苏财资环[2020] 26号)

Cloning and Expression Analysis of PDS Gene from Rhododendron molle

Xiao Zheng(), Su Jiale, Liu Xiaoqing, Sun Xiaobo, He Lisi, Chen Shangping, Zhou Huimin, Li Chang()

Institute of Leisure Agriculture, Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014

Received:2020-09-08 Revised:2020-12-18 Online:2021-05-25 Published:2021-05-18
Contact: Li Chang

摘要/Abstract

摘要：

旨在研究羊踯躅八氢番茄红素脱氢酶基因的功能,为其花瓣着色的分子机理研究提供一定的理论依据。以羊踯躅瓣为材料,采用RT-PCR和RACE方法从花瓣cDNA中克隆获得到2098 bp的八氢番茄红素脱氢酶基因cDNA序列,命名为RmPDS。序列分析表明,RmPDS基因包含一个长度为1743 bp编码580个氨基酸的开放阅读框。利用NCBI分析该基因的氨基酸序列,Blast序列分析结果显示RmPDS与其他植物PDS蛋白氨基酸的相似性达到87.80%。用MEGA软件构建系统进化树,结果显示羊踯躅PDS与茶树PDS蛋白的亲缘关系最近。荧光定量PCR检测结果表明RmPDS基因在羊踯躅花蕾期和膨大期表达量较低,在初花期表达量最高,盛花期表达量有所降低。RmPDS基因参与羊踯躅花色的形成,研究结果为进一步探究该基因在花色形成中的作用机制奠定基础,同时也为构建杜鹃VIGS体系提供报告基因。

关键词: 羊踯躅, PDS基因, 花瓣着色, 实时荧光定量PCR, 基因克隆

Abstract:

We aim to study the function of PDS gene in Rhododendron molle, and provide a theoretical basis for studying the molecular mechanism of petal coloring. R. molle was used as material, a PDS gene was cloned from R. molle cDNA using RT-PCR and RACE methods. The full-length cDNA sequence of the PDS gene is 2098 bp, named RmPDS. The sequence analysis showed that the RmPDS contained a 1743 bp open reading frame encoding 580 amino acids. The homologous alignment showed that it shared 87.80% of amino acid identities with PDS from the other plant plants using NCBI blast tool. The phylogenetic tree constructed by MEGA software indicated that the genetic relationship between R. molle and Camellia sinensis was the most intimate. The results of real-time fluorescence quantitative PCR showed that the RmPDS gene expression level was lower in the bud stage and expansion stage, and reached to the highest level in the early flowering stage, but decreased in the blooming stage. RmPDS was involved in the formation of R. molle flower color. This study may provide references for further research on the molecular mechanism of RmPDS involved in the formation of R. molle flower color. Meanwhile, the study provides a reporter gene for the construction of the VIGS system in Rhododendorn.

Key words: Rhododendron molle, PDS gene, petal coloring, real-time PCR, gene cloning

中图分类号:

S685.21

肖政, 苏家乐, 刘晓青, 孙晓波, 何丽斯, 陈尚平, 周惠民, 李畅. 羊踯躅八氢番茄红素脱氢酶基因的克隆及表达分析[J]. 中国农学通报, 2021, 37(15): 99-105.

Xiao Zheng, Su Jiale, Liu Xiaoqing, Sun Xiaobo, He Lisi, Chen Shangping, Zhou Huimin, Li Chang. Cloning and Expression Analysis of PDS Gene from Rhododendron molle[J]. Chinese Agricultural Science Bulletin, 2021, 37(15): 99-105.

图/表 8

图1. 羊踯躅花瓣总RNA电泳图 M：DNA分子量标准;1：样品

图2. 羊踯躅RmPDS基因的PCR扩增结果 M：DNA分子量标准;A：3’RACE扩增产物;B：5’RACE扩增产物;C：全长序列

图3. 羊踯躅RmPDS基因序列及推导的氨基酸序列推导的氨基酸序列标于相应核苷酸序列下面;方框为起始密码子;*表示终止密码子

图4. 羊踯躅PDS与其他物种PDS氨基酸序列的同源性比较

图5. 羊踯躅RmPDS基因疏水性/亲水性预测

图6. 羊踯躅PDS蛋白氨基酸序列二级结构预测

图7. 不同植物的PDS氨基酸序列的系统进化树

图8. RmPDS基因在羊踯躅花瓣不同发育时期的表达量

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羊踯躅八氢番茄红素脱氢酶基因的克隆及表达分析

Cloning and Expression Analysis of PDS Gene from Rhododendron molle

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