苹果磷酸果糖激酶基因MdPFPβ调控果实可溶性糖积累的功能

doi:10.16420/j.issn.0513-353x.2022-0423

园艺学报 ›› 2022, Vol. 49 ›› Issue (10): 2223-2235.doi: 10.16420/j.issn.0513-353x.2022-0423

苹果磷酸果糖激酶基因MdPFPβ调控果实可溶性糖积累的功能

于建强¹^,², 顾凯迪¹, 王传增³^,^**(), 胡大刚¹^,^**()

¹山东农业大学园艺科学与工程学院,作物生物学国家重点实验室,山东泰安 271018
²扬州大学园艺与植物保护学院,江苏扬州 225009
³山东省农业科学院黄河三角洲现代农业研究院,山东东营 257000

收稿日期:2022-04-18 修回日期:2022-07-27 出版日期:2022-10-25 发布日期:2022-10-31
通讯作者: 王传增,胡大刚 E-mail:fap_296566@163.com;dazeng123321@163.com
基金资助:
国家自然科学基金项目(32122080);国家自然科学基金项目(31972375);山东省自然科学基金项目(ZR2020YQ25);国家重点研发计划项目(2018YFD1000200)

Functional Characterization of An Apple Pyrophosphate-dependent Phosphofructokinase Gene MdPFPβ in Regulating Soluble Sugar Accumulation

YU Jianqiang¹^,², GU Kaidi¹, WANG Chuanzeng³^,^**(), HU Dagang¹^,^**()

¹State Key Laboratory of Crop Biology,College of Horticultural Science and Engineering,Shandong Agricultural University,Tai'an,Shandong 271018,China
²College of Horticulture and Plant Protection,Yangzhou University,Yangzhou,Jiangsu 225009,China
³Modern Agriculture Research Institute of Yellow River Delta,Shandong Academy of Agricultural Sciences,Dongying,Shandong 257000,China

Received:2022-04-18 Revised:2022-07-27 Online:2022-10-25 Published:2022-10-31
Contact: WANG Chuanzeng,HU Dagang E-mail:fap_296566@163.com;dazeng123321@163.com

摘要/Abstract

摘要：

为阐明苹果（Malus × domestica Borkh.）磷酸果糖激酶基因MdPFPβ（Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit beta,基因序列号MD09G1112800）的生物学功能,首先分析了基因启动子和全长序列信息,该基因包含全长为483 bp完整的开放阅读框,编码161个氨基酸。利用PlantCare数据库进行基因启动子顺式作用元件分析发现,MdPFPβ启动子序列中含有与脱落酸（abscisic acid,ABA）、低温及干旱信号相关的调控元件。荧光定量PCR分析发现,MdPFPβ在苹果叶片和成熟果实中表达量较高,并且受外源ABA的诱导表达。在外施ABA的条件下,MdPFPβ过量表达的苹果愈伤组织中可溶性糖含量明显上升。此外,MdPFPβ转基因番茄植株的光合性能增强,MdPFPβ可能通过调控糖代谢酶的活性最终影响果实中的可溶性糖含量。

关键词: 苹果, MdPFPβ, 可溶性糖, 脱落酸

Abstract:

To elucidate the biological function of the apple（Malus × domestica Borkh.）phospho- fructokinase gene MdPFPβ（Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit beta,gene sequence number MD09G1112800）,its sequence and promoter information were analyzed. The results showed that its open reading frame was 483 bp in length,encoding 161 amino acids. Cis-acting element analysis of its promoter using the PlantCare database revealed that regulatory elements related to abscisic acid（ABA）,low temperature,and drought signaling were present in the promoter sequence of MdPFPβ. Quantitative real-time PCR analysis revealed that MdPFPβ was highly expressed in apple leaves and mature fruits. It was also induced by external application of ABA in apple leaves. Overexpressing MdPFPβ significantly increased soluble sugar content in apple calli compared to that in wild-type under ABA treatment. Moreover,ectopic expression of MdPFPβ promoted photosynthetic performance in tomato leaves,as well as the content of soluble sugar in tomato fruits,indicating MdPFPβ plays a key role in regulating soluble sugar content in fruits.

Key words: apple, MdPFPβ, soluble sugar, abscisic acid

中图分类号:

S661.1

于建强, 顾凯迪, 王传增, 胡大刚. 苹果磷酸果糖激酶基因MdPFPβ调控果实可溶性糖积累的功能[J]. 园艺学报, 2022, 49(10): 2223-2235.

YU Jianqiang, GU Kaidi, WANG Chuanzeng, HU Dagang. Functional Characterization of An Apple Pyrophosphate-dependent Phosphofructokinase Gene MdPFPβ in Regulating Soluble Sugar Accumulation[J]. Acta Horticulturae Sinica, 2022, 49(10): 2223-2235.

图/表 9

表1 本研究中所用的引物

Table 1 Primers used in this study

用途Application	引物名称Primer name	引物序列（5′-3′）Primer sequence
cDNA clone	MdPFPβ-F	ATGTACTCCATGAATGGCAAGG
	MdPFPβ-R 18S-F 18S-R	TGCTTGTGCTCCAAGTTCC ACACGGGGAGGTAGTGACAA CCTCCAATGGATCCTCGTTA
内参基因Reference gene
qRT-PCR			MdPFPβ-For1	TCTCGACGTTCCGCTTCCTCTC
qRT-PCR	MdPFPβ-Rev1	CTGGCCGTAGCATTGCTGAAGG

表2 MdPFPβ的启动子元件分析

Table 2 Some important cis-acting regulatory elements in the promoter of MdPFPβ

调控序列	序列	位点功能	位置
Regulatory sequence	Sequence	Function of site	Location
ABRE	ACGTG	参与ABA响应元件cis-acting element involved in the abscisic acid responsiveness	-619,+1 719
ARE	AAACCA	参与厌氧响应元件cis-acting regulatory element essential for the anaerobic induction	+485,-1 210
G-box	TACGTG	参与光响应元件cis-acting regulatory element involved in light responsiveness	-1 739,+1 926
LTR	CCGAAA	参与低温响应元件cis-acting regulatory element essential for the anaerobic induction	+415,-697
MBS	CAACTG	参与干旱胁迫响应元件MYB binding site involved in drought-inducibility	+43
TC-rich repeats	GTTTTCTTAC	参与防御和胁迫响应元件MYB binding site involved in drought-inducibility	+1 269
CGTCA-motif	CGTCA	参与茉莉酸响应元件cis-acting regulatory element involved in the MeJA-responsiveness	-1 099

图1 MdPFPβ基因信息（A）及其在各组织（B,根作为对照）和ABA处理下的表达（C,0 h水处理作为对照）下同。The same below.

Fig. 1 Genetic information of MdPFPβ（A）and its expression in various tissues（B,root as control）and under ABA treatment（C,0 h water treatment as control） * α = 0.05;** α = 0.01。

图2 苹果愈伤组织MdPFPβ过量表达株系（MdPFPβ-7/9/13）在ABA处理下的表型、基因表达水平、鲜质量和糖含量

Fig. 2 Phenotype,relative expression level of MdPFPβ gene,fresh weight and sugar content of wild type（WT）and MdPFPβ overexpressing apple calli（MdPFPβ-7/9/13）under ABA treatment

图 3 番茄MdPFPβ转基因株系（MdPFPβ-22/32/43）叶片的表型、基因表达水平和光合荧光参数

Fig. 3 Phenotypes,relative expression level of MdPFPβ and photosynthetic fluorescence parameters of wild-type（WT） and MdPFPβ transgenic tomato leaves（MdPFPβ-22/32/43）

图4 番茄MdPFPβ转基因株系（MdPFPβ-22/32/43）叶片的光合速率、气孔导度和胞间二氧化碳浓度

Fig. 4 Photosynthetic rate,stomata conductance and intercellular CO2 concentration of wild-type（WT）and MdPFPβ transgenic tomato leaves（MdPFPβ-22/32/43）

图5 番茄MdPFPβ转基因株系（MdPFPβ-22/32/43）叶片的淀粉、蔗糖、果糖和葡萄糖含量

Fig. 5 Starch content,sucrose content,fructose content and glucose content of wild-type（WT）and MdPFPβ transgenic tomato leaves（MdPFPβ-22/32/43）

图6 番茄MdPFPβ转基因株系（MdPFPβ-22/32/43）果实的表型及果糖、葡萄糖、蔗糖含量

Fig. 6 Phenotype,fructose content,glucose content and sucrose content of wild-type（WT）and MdPFPβ transgenic tomato fruits（MdPFPβ-22/32/43）

图7 番茄MdPFPβ转基因株系（MdPFPβ-22/32/43）果实的PFP、SPS、NI、SS-I、和S-AI活性

Fig. 7 PFP,SPS,NI,SS-I and S-AI activities of wild-type（WT）and MdPFPβ transgenic tomato fruits（MdPFPβ-22/32/43）

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苹果磷酸果糖激酶基因MdPFPβ调控果实可溶性糖积累的功能

Functional Characterization of An Apple Pyrophosphate-dependent Phosphofructokinase Gene MdPFPβ in Regulating Soluble Sugar Accumulation

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苹果磷酸果糖激酶基因MdPFPβ调控果实可溶性糖积累的功能

Functional Characterization of An Apple Pyrophosphate-dependent Phosphofructokinase Gene MdPFPβ in Regulating Soluble Sugar Accumulation

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