森林草莓FveD27的表达特性和功能

doi:10.3864/j.issn.0578-1752.2021.10.013

Abstract

Abstract:

【Objective】It is known that the new plant hormone strigolactone is a key factor regulating plant branching development, but the role of strigolactone in the growth and development of strawberry plants is unclear. In this study, the expression characteristics and function of the key gene DWARF27 (D27) for strigolactone biosynthesis in woodland strawberry (Fragaria vesca) was revealed, and the role of FveD27 in strawberry branching growth and development was explored, which would lay a theoretical foundation for studying strawberry plant architecture. 【Method】The coding sequence region of strigolactone synthesis key gene FveD27 was cloned by RT-PCR from woodland strawberry. The phylogenetic relationship between FveD27 and D27 in apple, Arabidopsis and other species was analyzed by using MEGA 6.0. A fusion vector of FveD27 and GFP was constructed to inject tobacco leaves for analyzing the subcellular location of FveD27. The expression level of FveD27 in different organs of woodland strawberry was analyzed by qRT-PCR technology. The FveD27 promoter and GUS fusion expression vector was constructed and transformed into woodland strawberry by Agrobacterium-mediated method, and the expression characteristic of FveD27 was further analyzed by GUS staining. The overexpression vector of FveD27 was constructed and the FveD27 overexpression strawberry lines were obtained by Agrobacterium-mediated transformation with leaf disks of woodland strawberry as explants. 【Result】The coding sequence region of FveD27 with the length of 789 bp was cloned from woodland strawberry. The subcellular localization in tobacco showed that FveD27 was located in the chloroplast. The expression levels of FveD27 in woodland strawberry organs from high to low were followed by young leaves, shoot tips, petiole, mature leaves and roots. The FveD27 promoter sequence with a length of 1670 bp was cloned, and transgenic plants with pFveD27::GUS gene showed that the young leaves and buds had strong GUS activity, the mature leaves and petioles had weak GUS activity, while the roots had little GUS activity. The result of FveD27 expression revealed by GUS analysis was consistent with the result by qRT-PCR. The FveD27 overexpression vector was constructed and the transgenic woodland strawberry lines overexpressing FveD27 were obtained with Agrobacterium-mediated transformation. Overexpressing FveD27 in woodland strawberry inhibited significantly the formation of branch crowns and increased the number of inflorescences. 【Conclusion】FveD27 had the functions of regulating the development of branch crowns and the number of inflorescences in woodland strawberry, and this study provided new ideas for regulating the number of branch crowns and yield of strawberry.

Key words: Fragaria vesca, strigolactone, D27, subcellular localization, branching

SUN HongYing,WANG Yan,LI WeiJia,ZHU TianShu,JIANG Ying,XU Yan,WU QingYue,ZHANG ZhiHong. Expression Characteristics and Function of FveD27 in Woodland Strawberry[J].Scientia Agricultura Sinica, 2021, 54(10): 2179-2191.

Figures/Tables 13

Table 1

Fig. 1

Fig. 2

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Fig. 4

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Fig. 6

Table 2

Prediction of cis-acting elements related to FveD27 promoter"

顺式作用元件 cis-regulatory element	位置（方向） Position（stand）	序列 Sequence	功能 Function
ACA-motif	247(-)	AATCACAACCATA	（gapA-CMA1）中gapA的一部分涉及光响应性 Part of gapA in (gapA-CMA1) involved with light responsiveness
AE-box	405(-)	AGAAACAA	光响应的元件的一部分 Part of a module for light response
GT1-motif	382(+)	GGTTAA	光响应元件 Light responsive element
I-box	345(-), 986(+)	TGATAATGT	光响应元件的一部分 Part of a light responsive element
LAMP-element	181(+)	CTTTATCA	光响应元件的一部分 Part of a light responsive element
TCT-motif	1159(-)	AACGAC	光响应元件的一部分 Part of a light responsive element
MRE	379(-)	AACCTAA	参与光诱导的MYB绑定位点 MYB binding site involved in light responsiveness
CCAAT-box	881(+)	CAACGG	MYBHv1结合位点 MYBHv1 binding site
MBS	1487(-)	CAACTG	参与干旱诱导的MYB绑定位点 MYB binding site involved in drought-inducibility
GC-motif	920(-)	CCCCCG	参与缺氧特异性诱导性的增强子元件 Enhancer-like element involved in anoxic specific inducibility
ARE	1781(+)	AAACCA	厌氧诱导的必要顺式作用调节元件 cis-acting regulatory element essential for the anaerobic induction
TCA-element	-384(-), -1107(-)	CAGAAAAGGA	水杨酸响应的顺式作用元件 cis-acting element involved in salicylic acid responsiveness
P-box	117(+)	CCTTTTG	赤霉素响应元件 Gibberellin-responsive element
TATC-box	1127(-)	TATCCCA	赤霉素响应的顺式作用元件 cis-acting element involved in gibberellin-responsiveness
TGACG-motif	1406(-), 1413(+)	TGACG	MeJA响应的顺式作用元件 cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif	1406(+), 1413(-)	CGTCA	MeJA响应的顺式作用元件 cis-acting regulatory element involved in the MeJA-responsiveness
TATA-box	188(-)	TATAATAAT	转录起始位点上游-30左右位置的核心启动元件 The core activation element at about -30 upstream of the transcription start site

Table 2

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

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引物 Primer	序列（5′→3′） Sequence（5′→3′）	目的 Purpose
FveD27-F FveD27-R	ACGCGTCGACATGGAAGCATCACATTTCT CGCGGATCCCTAACTGGAGCAGTTAT	基因全长克隆 Full-length gene cloning
FveD27-DL-F FveD27-DL-R	AGCAGAATAAGACCGGCAGA TGCAGCTTGGACATTTTGAG	实时荧光定量 Real-time PCR
Fve-26S-F Fve-26S-R	TAACCGCATCAGGTCTCCAA CTCGAGCAGTTCTCCGACAG	内参基因 Reference gene
FveD27(eGFP)-F FveD27(eGFP)-R	TCCCCCGGGATGGAAGCATCACATTT CGGGGTACCACTGGAGCAGTTATTGT	亚细胞定位 Subcellular localization
proFveD27-F proFveD27-R	GCTGCAGGCTGATTCCTAAGGGTATC TGCTCTAGAATGCATGGTGGGGAATTTAAAG	启动子克隆 Promoter cloning

Expression Characteristics and Function of FveD27 in Woodland Strawberry

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