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植物研究 ›› 2019, Vol. 39 ›› Issue (3): 441-449.doi: 10.7525/j.issn.1673-5102.2019.03.014

• 研究报告 • 上一篇    下一篇

P119驱动amiRNA介导的PL基因沉默对草莓果实硬度的影响

周敏, 蒋丹, 刘玥秀, 王小蓉, 汤浩茹, 陈清   

  1. 四川农业大学园艺学院, 成都 611130
  • 收稿日期:2019-01-02 出版日期:2019-05-05 发布日期:2019-05-11
  • 通讯作者: 陈清 E-mail:supnovel@sicau.edu.cn
  • 作者简介:周敏(1997-),女,本科生,主要从事园艺植物育种研究。
  • 基金资助:
    国家级大学生创新训练计划资助项目(201710626049);国家自然科学基金(31600232)

amiRNA-mediated PL Gene Silencing Driven by the Fruit-specific Promoter P119 in Strawberry and Its Effect on Fruit Firmness

ZHOU Min, JIANG Dan, LIU Yue-Xiu, WANG Xiao-Rong, TANG Hao-Ru, CHEN Qing   

  1. College of Horticulture, Sichuan Agricultural University, Chengdu 611130
  • Received:2019-01-02 Online:2019-05-05 Published:2019-05-11
  • Supported by:
    National Students’ Innovation and Entrepreneurship Training Program(201710626049);National Natural Science Foundation of China(31600232)

摘要: 果胶裂解酶基因(Pectate lyase,PL)是调控果实软化的重要靶点。本研究测定了红颜草莓果实在发育过程中果胶裂解酶活性变化和硬度变化规律,并通过人工小干扰RNA(artificial micro-interfering RNA interference,amiRNA)技术,以拟南芥miR390a前体序列作为沉默PL基因的骨架,在番茄果实特异性启动子P119的驱动下,通过农杆菌介导转入草莓果实中瞬时表达,通过RT-PCR分析草莓瞬时转染后PL基因的表达量,并检测了PL沉默对果实硬度的影响。结果表明,随着草莓果实发育的推进,果胶裂解酶活性呈逐渐上升趋势,与果实硬度呈负相关;构建了基于拟南芥miR390a为骨架的amiRNA靶向栽培草莓中的PL基因;在番茄果实特异性启动子P119驱动下,miRNA前体可以在草莓果实中瞬时表达;草莓果实中总PL基因表达量下降达34.5%;基因沉默组果实硬度比对照组更高,且沉默对果实颜色发育进程无明显影响。该结果表明:果胶裂解酶主要在果实发育后期调节细胞壁的解离,采用amiRNA沉默PL基因可以延缓草莓果实软化进程。

关键词: 草莓, 果胶裂解酶, amiRNA干扰, P119果实特异性启动子

Abstract: Pectate lyase(PL) is one of the most profitable candidate enzymes for improving the fruit firmness and delaying the softening process in strawberry(Fragaria×ananassa Duch.) by molecular modifying. The role of PL in strawberry fruit softening was studied by amiRNA-mediated silencing. The changes of pectate lyase activity and fruit firmness were determined across the fruit development of cultivated strawberry ‘Benihoppe’. Candidate PLs were submitted toWMD3(http://wmd3.weigelword.org) for miRNA target site screening(target Fragaria×ananassa PUT v183, minimum number of included targets:one). Those candidate miRNA target sites were then manually checked, ensuring that the target loci exist in all PL transcripts. This target sequence was introduced into the Arabidopsis thaliana miR390a precursor sequence as the backbone. The validated construct was transformed into the Agrobacterium GV3101. Transient expression in strawberry fruits was conducted by agroinfilltration. Expression level of the PL genes was monitored by RT-PCR. Fruit firmness was examined too. With the development of strawberry fruit, the PL activity gradually increased, especially the stages after turning red. The PL activity increased sharply and reached the maximum in the full red period. In contrast, fruit firmness gradually decreased during fruit development especially during the process from big green to color transition, and then the rate of hardness reduction slowed. The P119 promoter from tomato was able to drive amiRNA gene expression, which can reduce the expression of PL gene in strawberry fruit by 34.5%. The knock down of PL gene does not interfere with the accumulation of anthocyanins in strawberry, and the silencing group had higher fruit hardness than the control group. In a comprehensive analysis, PL regulates the dissociation of cell wall mainly in the late fruit developmental stage. Obviously, from our results, there are also other factors that affect the softening of strawberry. The p119 fruit-specific promoter has transcriptional activity in silencing PL gene of strawberry in the fruit, which lays a foundation with the application in future molecular modification. To this end, stable transformation is underway. Therefore, PL regulates plants cell wall dissociation at the late development stages of strawberry. Silencing the gene with P119 using amiRNA can delay fruit softening process.

Key words: strawberry, Pectate lyase, AmiRNA, P119 fruit-specific promoter

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