梨果糖激酶基因PpyFRK5在果实蔗糖积累中的作用

doi:10.16420/j.issn.0513-353x.2021-0445

摘要/Abstract

摘要：

以‘丰水’梨为试材,对果实果糖激酶基因PpyFRK5进行克隆、表达分析和功能研究。结果表明,PpyFRK5编码的蛋白属于磷酸果糖激酶B亚家族（pfkB）,具有高度保守的pfkB家族特征性结构域及果糖激酶特有的ATP结合域和糖结合域;PpyFRK5在果实不同发育时期和不同器官中存在差异表达,在成熟的果实和种子中表达量最高;PpyFRK5定位于质膜和细胞质中;在果实发育的整个过程中,PpyFRK5的表达与蔗糖含量呈显著正相关,瞬时超表达PpyFRK5的梨果实蔗糖含量显著升高,瞬时沉默PpyFRK5的梨果实蔗糖含量显著降低;PpyFRK5的启动子区域包含多个参与光响应、激素诱导以及胁迫响应的顺式作用元件。

关键词: 梨, 果糖激酶, 表达模式, 基因功能, 蔗糖积累

Abstract:

As one of the important members of fructokinase gene family,the gene of PpyFRK5 was cloned utilizing‘Hosui’pear（Pyrus pyrifolia Nakai）fruit as the experimental material in the study. Simultaneously,its expression patterns and biological functions were studied. The results indicated that the protein encoded by PpyFRK5 belongs to the phosphofructokinase B subfamily（pfkB）,and contains the highly conserved pfkB characteristic domain and the ATP binding domain and sugar binding domain unique to fructokinase. The expression level of PpyFRK5 exhibited significant distinctions in different pear organs and fruit development stages,and reached the highest in fruits and seeds at the mature stage. PpyFRK5 was localized in the plasma membrane and cytoplasm. During the whole development of the pear fruit,there was a significant positive correlation between the expression level of PpyFRK5 and the sucrose content,and the sucrose content of pear fruits with transient overexpression of PpyFRK5 was significantly increased,while that of pear fruits with transient silencing of PpyFRK5 was significantly decreased;the promoter region of PpyFRK5 contained multiple cis-acting elements involved in light response,hormone induction,and stress response.

Key words: pear, fructokinase, expression pattern, gene function, sucrose accumulation

中图分类号:

S661.2

陶鑫, 朱荣香, 贡鑫, 吴磊, 张绍铃, 赵建荣, 张虎平. 梨果糖激酶基因PpyFRK5在果实蔗糖积累中的作用[J]. 园艺学报, 2022, 49(7): 1429-1440.

TAO Xin, ZHU Rongxiang, GONG Xin, WU Lei, ZHANG Shaoling, ZHAO Jianrong, ZHANG Huping. Fructokinase Gene PpyFRK5 Plays an Important Role in Sucrose Accumulation of Pear Fruit[J]. Acta Horticulturae Sinica, 2022, 49(7): 1429-1440.

图/表 10

表1 本研究使用的引物序列

Table 1 Primer sequences used in this study

用途 Use	名称 Name	序列（5′-3′） Sequence
实时荧光定量PCR	PpyFRK5-F	TTCGTTGGCAAGGTTGGAG
Quantitative real-time PCR	PpyFRK5-R	TCACCGTCGTTCTTCAAGGTC
	Tubulin-F	TGGGCTTTGCTCCTCTTAC
	Tubulin-R	CCTTCGTGCTCATCTTACC
基因克隆、超表达和亚细胞定位 Cloning,overexpression,and subcellular localization	35S:PpyFRK5-GFP-F	GAGAACACGGGGGACTCTAGAATGGCGAATTCAGAGAGTCCAC
	35S:PpyFRK5-GFP-R	GCCCTTGCTCACCATGGATCCTTTGGCTTTGGACTTGGAAATG
病毒诱导的基因沉默	pTRV2-PpyFRK5-F	GAAGGCCTCCATGGGGATCCTGTGGTTCTGTCCCTGTGG
Virus induced gene silencing	pTRV2-PpyFRK5-R	GCCTCGAGACGCGTGAGCTCCATCAGACTGGGTTGGAAGA
启动子扩增 Promoter amplification	PpyFRK5-pro-F	GTCGACGGTATCGATAAGCTTCCTTATATTTGTCGACTGTAGGTCGA
	PpyFRK5-pro-R	CGCTCTAGAACTAGTGGATCCTCTCTCTTAACTCTTGCGATTGAGA

图1 PpyFRK5和PbrFRK5的核酸序列比对

Fig. 1 Nucleic acid sequence alignment of PpyFRK5 and PbrFRK5

图2 PpyFRK5基因的结构组成白色框表示5′和3′端;黄色框表示外显子;灰色框表示内含子。

Fig. 2 Gene structure analysis of PpyFRK5The white boxes indicate the 5′ and 3′ ends;the yellow boxes indicate exons and the gray boxes indicate introns.

图3 PpyFRK5氨基酸序列分析A:在染色体上的位置和保守结构域;B:编码氨基酸序列比对。（a1）、（a2）和（a3）为pfkB家族的标志结构;（b）为果糖激酶家族特征性bac_FRK结构域;α代表糖结合位点,β代表ATP结合位点; Ppy:砂梨;Pbr:白梨;Md:苹果;Pp:桃;Rc:月季;Fv:草莓;Sl:番茄;At:拟南芥。

Fig. 3 Amino acid sequence analysis of PpyFRK5A:A schematic diagram of the position of PpyFRK5 on the chromosome and the position of conserved domains in PpyFRK5;B:Amino acid sequence alignment of PpyFRK5;（a1）,（a2）and（a3）are the landmark structures of the pfkB family and（b）is the characteristic bac_FRK domains of the fructokinase family;α represents the sugar binding site,and β represents the ATP binding site;Ppy:Pyrus pyrifolia; Pbr:Pyrus bretschneideri;Md:Malus × domestica;Pp:Prunus persica;Rc:Rosa chinensis; Fv:Fragaria vesca;Sl:Solanum lycopersicum;At:Arabidopsis thaliana.

图4 PpyFRK5的系统发育树

Fig. 4 Phylogenetic tree of PpyFRK5

图5 PpyFRK5的亚细胞定位

Fig. 5 Subcellular location of PpyFRK5

图6 PpyFRK5在梨果实不同发育时期（A）和不同器官中（B）的相对表达量

Fig. 6 Relative expression level of PpyFRK5 at different stages of fruit development（A）and different organs（B）in pear

图7 梨果实发育过程中PpyFRK5表达与蔗糖含量之间的线性回归分析

Fig. 7 Linear regression analysis between PpyFRK5 expression and sucrose content during development of pear fruit

图8 超表达（A）和沉默表达（B）PpyFRK5的梨果实中的PpyFRK5相对表达量和可溶性糖含量

Fig. 8 Relative expression level of PpyFRK5 and soluble sugar content in pear fruits with overexpression（A）and silencing（B）of PpyFRK5* α = 0.05,** α = 0.01.

表2 PpyFRK5启动子区顺式作用元件分析

Table 2 Analysis of the main cis-elements in promoter region of PpyFRK5

顺式作用元件 cis-element	序列 Sequence	功能 Probable function
ABRE	ACGTG	脱落酸响应元件Involved in the abscisic acid response
ARE	AAACCA	厌氧诱导响应元件Essential for the anaerobic induction
AE-Box	ACAAACAA	光响应元件Part of a module for light response
CGTCA-motif	CGTCA	MeJA响应元件Involved in the MeJA response
Box-4	ATTAAT	光响应元件Involved in light response
CAAT-Box	CCAAT;CAAT	启动子和增强子区域元件Element in the promoter and enhancer regions
G-Box	CACGTT	光响应元件Involved in light response
LTR	CCGAAA	低温响应元件Involved in low-temperature response
MBS	CAACTG	MYB结合位点MYB binding site
TATA-Box	TATA;TATAAA	转录起始位点Approximately -30 from the transcription start
RY-element	CATGCATG	种子特异性调控响应元件Involved in seed-specific regulation
TC-rich repeats	ATTCTCTAAC	防御和胁迫响应元件Involved in defense and stress response
TCT-motif	TCTTAC	光响应元件Part of a light responsive element
TGACG-motif	TGACG	MeJA响应元件Involved in the MeJA response
as-1	TGACG	转录结合位点Transcription factor-binding site
circadian	CAAAGATATC	昼夜节律控制响应元件Involved in circadian control

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