甜樱桃磷酸蔗糖合酶基因PavSPS的功能分析

doi:10.16420/j.issn.0513-353x.2020-0630

摘要/Abstract

摘要：

从甜樱桃基因组中鉴定了4个磷酸蔗糖合酶（sucrose phosphate synthase,SPS）基因（PavSPSA1、PavSPSA2、PavSPSB和PavSPSC）。实时荧光定量PCR分析表明,PavSPSA1在甜果实发育和成熟过程中表达量最高,其次是PavSPSC,PavSPSA2和PavSPSB表达量较低;PavSPSA1和PavSPSA2在果实成熟软化过程中均上调表达,与甜樱桃果实的成熟软化过程吻合。通过利用病毒诱导的基因沉默（VIGS）技术沉默PavSPSA1,甜樱桃果实蔗糖含量降低,果实着色和果实成熟软化延迟;沉默PavSPSA2、PavSPSB和PavSPSC,甜樱桃果实蔗糖含量、果实着色和果实成熟软化与对照无差异,推测PavSPSA1或许是调控果实蔗糖合成与积累的关键基因,影响果实着色和成熟软化。

关键词: 甜樱桃, 磷酸蔗糖合酶, PavSPSA1, 基因表达, 果实成熟, 基因沉默

Abstract:

In order to ascertain the function of sucrose-phosphate synthase genes（SPS）in the regulation of sweet cherry ripening,four full-length cDNA of sweet cherry SPS genes（PavSPSA1,PavSPSA2,PavSPSB,and PavSPSC）were cloned in sweet cherry genome. Gene expression analysis showed that the expression levels of PavSPSA1 was the highest,followed by PavSPSC,whereas PavSPSA2 and PavSPSB were relatively weakly expressed during the fruit growth and development,and ripening using quantitative real-time PCR. The expression levels of PavSPSA1 and PavSPSA2 coincided with fruit ripening and softening. Furthermore,we used TRV-mediated virus-induced gene silencing（VIGS）to evaluate the function of PavSPSA1,PavSPSA2,PavSPSB,and PavSPSC in the regulation of ripening. The results showed that silencing PavSPSA1 gene decreased sucrose content,delayed fruit coloring,ripening and softening. However,The fruit phenotypes for the PavSPSA2,PavSPSB,and PavSPSC-silenced fruits showed no visible defects when compared with the control fruits. Taken together,our findings determined that PavSPSA1 may be a key gene regulating sucrose synthesis and accumulation in sweet cherry fruit and play an indispensable role in the regulation of sweet cherry fruit ripening and softening.

Key words: sweet cherry, sucrose-phosphate synthase, PavSPSA1, gene expression, fruit ripening, VIGS

中图分类号:

S662.5

齐希梁, 刘聪利, 宋露露, 李明. 甜樱桃磷酸蔗糖合酶基因PavSPS的功能分析[J]. 园艺学报, 2021, 48(8): 1446-1456.

QI Xiliang, LIU Congli, SONG Lulu, LI Ming. Functional Analysis of Sucrose-phosphate Synthase Genes（SPS）in Sweet Cherry[J]. Acta Horticulturae Sinica, 2021, 48(8): 1446-1456.

图/表 8

表1 本研究中所用的引物信息

Table 1 Primers used in this study

引物名称 Primer name	序列（5′-3′） Sequence	引物名称 Primer name	序列（5′-3′） Sequence
PavSPSA1-F	ATGGCGAGCAACGATTGGATAAAC	PavSPSA1-J-F	CAGGTTCATGCCCCGCATGGTGA
PavSPSA1-R	CTACGTCTTGACAACTCCGAGTT	PavSPSA1-J-R	ATTGTGGATGCCTTGGTTTGC
PavSPSA2-F	ATGGCGGGAAACGACTGGGTGAAC	PavSPSA2-J-F	GAGGCAGCAGCTTATGGTCTACCT
PavSPSA2-R	CTACCGCTTGAGAATCCCTAGT	PavSPSA2-J-R	CTTGTCCTTCCCTGCTGCCTCA
PavSPSB-F	ATTTGGCATCTTACCCGTAAGAAG	PavSPSB-J-F	ATGAAGGAGGTGGAAATAAGCTGCT
PavSPSB-R	TCACATTCGAGCAGCAGATTTAGAG	PavSPSB-J-R	AGTACATTGGATGGCAACGCAG
PavSPSC-F	ATGGCGGGAAACGACTGGTTAAACAG	PavSPSC-J-F	GAGATTCTGATACAGGTGGTCAGGT
PavSPSA1-q-F	GCAAACCAAGGCATCCACAAT	PavSPSC-J-R	AGCTCCTCAGCCTCGATCCTCTTC
PavSPSA1-q-R	GAACCCTACAGAGCCTTCAGT	PaNCED1-F	CATGTCGGAGGACGACTTGCCGT
PavSPSA2-q-F	TGAGGCAGCAGGGAAGGACAAG	PaNCED1-R	GCGCCGTCTGGAGAGACGTGGA
PavSPSA2-q-R	AGCCCCAACGGACATACAGATACC	PaPG1-F	ATCACCTTCCGCATTGCTG
PavSPSB-q-F	GGCTTGCTTGTGGACCCTCAT	PaPG1-R	TCACCTTAATGTTGTTGGAG
PavSPSB-q-R	AGCAGCTTATTTCCACCTCCTTC	PaXYL1-F	ACAACTGGAACGGTGTCGAT
PavSPSC-q-F	ACCCCACCACCCGCTTTCA	PaXYL1-R	TCCTGGTGTAATGTTGCTCG
PavSPSC-q-R	GACCCTCGCCATTGCCTACG	PaPL-1-F	GATGGTCGCTTCTATGTTGTCA
PavSPSA2-RNAi-F	AGTAAGGTTACCGAATTCTGAGGCAGCAGGGAAGGACAAG	PaPL-1-R	TGAGATGGAGAGCTCCTCACC
PavSPSA2-RNAi-R	GAGCTCGGTACCGGTACCAGCCCCAACGGACATACAGATACC	PaPAL-F	GCCTCACCAGGCAACAAGAGCA
PavSPSB-RNAi-F	AGTAAGGTTACCGAATTCGGCTTGCTTGTGGACCCTCAT	PaPAL-R	TCTGGCCATCTGGTCCAACAGC
PavSPSB-RNAi-R	GAGCTCGGTACCGGTACCAGCAGCTTATTTCCACCTCCTTCAT	PaCHS-F	GTATGTGCGAGTACATGGCA
PavSPSC-RNAi-F	AGTAAGGTTACCGAATTCACCCCACCACCCGCTTTCA	PaCHS-R	GCTTAGTGAGCTGATAGTC
PavSPSC-RNAi-R	GAGCTCGGTACCGGTACCGACCCTCGCCATTGCCTACG	PaDFR-F	CTGCACCGGAGTGTTCCATGT
Histone2-F	GGTGTGCTTCCGCAGATAA	PaDFR-R	CTGGTGCTCTTCGACGTTCAC
Histone2-R	TCCTCCTTGGGTGGTGAAT	PaANS-F	ATCTCCGATGAGCTCATGG
		PaANS-R	CTCAATGTAATCAGCAGGTG

表1 本研究中所用的引物信息

Table 1 Primers used in this study

引物名称 Primer name	序列（5′-3′） Sequence	引物名称 Primer name	序列（5′-3′） Sequence
PavSPSA1-F	ATGGCGAGCAACGATTGGATAAAC	PavSPSA1-J-F	CAGGTTCATGCCCCGCATGGTGA
PavSPSA1-R	CTACGTCTTGACAACTCCGAGTT	PavSPSA1-J-R	ATTGTGGATGCCTTGGTTTGC
PavSPSA2-F	ATGGCGGGAAACGACTGGGTGAAC	PavSPSA2-J-F	GAGGCAGCAGCTTATGGTCTACCT
PavSPSA2-R	CTACCGCTTGAGAATCCCTAGT	PavSPSA2-J-R	CTTGTCCTTCCCTGCTGCCTCA
PavSPSB-F	ATTTGGCATCTTACCCGTAAGAAG	PavSPSB-J-F	ATGAAGGAGGTGGAAATAAGCTGCT
PavSPSB-R	TCACATTCGAGCAGCAGATTTAGAG	PavSPSB-J-R	AGTACATTGGATGGCAACGCAG
PavSPSC-F	ATGGCGGGAAACGACTGGTTAAACAG	PavSPSC-J-F	GAGATTCTGATACAGGTGGTCAGGT
PavSPSA1-q-F	GCAAACCAAGGCATCCACAAT	PavSPSC-J-R	AGCTCCTCAGCCTCGATCCTCTTC
PavSPSA1-q-R	GAACCCTACAGAGCCTTCAGT	PaNCED1-F	CATGTCGGAGGACGACTTGCCGT
PavSPSA2-q-F	TGAGGCAGCAGGGAAGGACAAG	PaNCED1-R	GCGCCGTCTGGAGAGACGTGGA
PavSPSA2-q-R	AGCCCCAACGGACATACAGATACC	PaPG1-F	ATCACCTTCCGCATTGCTG
PavSPSB-q-F	GGCTTGCTTGTGGACCCTCAT	PaPG1-R	TCACCTTAATGTTGTTGGAG
PavSPSB-q-R	AGCAGCTTATTTCCACCTCCTTC	PaXYL1-F	ACAACTGGAACGGTGTCGAT
PavSPSC-q-F	ACCCCACCACCCGCTTTCA	PaXYL1-R	TCCTGGTGTAATGTTGCTCG
PavSPSC-q-R	GACCCTCGCCATTGCCTACG	PaPL-1-F	GATGGTCGCTTCTATGTTGTCA
PavSPSA2-RNAi-F	AGTAAGGTTACCGAATTCTGAGGCAGCAGGGAAGGACAAG	PaPL-1-R	TGAGATGGAGAGCTCCTCACC
PavSPSA2-RNAi-R	GAGCTCGGTACCGGTACCAGCCCCAACGGACATACAGATACC	PaPAL-F	GCCTCACCAGGCAACAAGAGCA
PavSPSB-RNAi-F	AGTAAGGTTACCGAATTCGGCTTGCTTGTGGACCCTCAT	PaPAL-R	TCTGGCCATCTGGTCCAACAGC
PavSPSB-RNAi-R	GAGCTCGGTACCGGTACCAGCAGCTTATTTCCACCTCCTTCAT	PaCHS-F	GTATGTGCGAGTACATGGCA
PavSPSC-RNAi-F	AGTAAGGTTACCGAATTCACCCCACCACCCGCTTTCA	PaCHS-R	GCTTAGTGAGCTGATAGTC
PavSPSC-RNAi-R	GAGCTCGGTACCGGTACCGACCCTCGCCATTGCCTACG	PaDFR-F	CTGCACCGGAGTGTTCCATGT
Histone2-F	GGTGTGCTTCCGCAGATAA	PaDFR-R	CTGGTGCTCTTCGACGTTCAC
Histone2-R	TCCTCCTTGGGTGGTGAAT	PaANS-F	ATCTCCGATGAGCTCATGG
		PaANS-R	CTCAATGTAATCAGCAGGTG

表2 甜樱桃基因组中SPS基因的相关信息

Table 2 Information of sucrose-phosphate synthase（SPS）genes identified in sweet cherry

基因名称 Gene name	cDNA/ bp	氨基酸 Amino acid	GenBank登录号 ID in GenBank	染色体位置 Chromosome location	拟南芥同源基因 Homologous gene in Arabidopsis
基因名称 Gene name	cDNA/ bp	氨基酸 Amino acid	GenBank登录号 ID in GenBank	染色体位置 Chromosome location	TAIR位置 Locus in TAIR	氨基酸相似度/% Similarity of amino acid
PavSPSA1	3 174	1 057	XM_021953379.1	Chr7：18 650 177 ~ 18 655 575	AtSPS1F（At5g20280）	77.69
PavSPSA2	3 180	1 059	XM_021966658.1	Chr1：35 009 999 ~ 35 016 230	AtSPS2F（At5g11110）	71.36
PavSPSB	2 844	947	XM_021946233.1	chrUn：91 085 245 ~ 91 090 791	AtSPS3F（At1g04920）	67.35
PavSPSC	3 072	1 023	XM_021957268.1	Chr8：324 800 ~ 330 670	AtSPS4F（At4g10120）	72.10

图1 甜樱桃果实不同发育时期的表型

Fig. 1 Phenotypes of sweet cherry fruit at different growth and developmental stages

图2 甜樱桃各发育时期果糖、葡萄糖和蔗糖的含量变化（A）及PavSPS的表达（B）

Fig. 2 Changes in fructose,glucose and sucrose contents（A）and expression proﬁle of PavSPS（B）during fruit growth and development of sweet cherry

图3 PavSPS沉默后甜樱桃果实PavSPSA1、PavSPSA2、PavSPSB和PavSPSC基因相对表达量变化（A）及果实表型（B）

Fig. 3 Quantify of PavSPSA1,PavSPSA2,PavSPSB,and PavSPSC genes（A）in gene silenced sweet cherry fruit and phenotype（B）

图4 沉默PavSPSA1、PavSPSA2、PavSPSB和PavSPSC的甜樱桃果实中花青素和ABA含量显著性差异分析采用One-way ANOVA方法对每个变量进行邓肯氏检验,** 表示达到显著性差异水平P < 0.01。下同。

Fig. 4 Effect of silencing of PavSPSA1,PavSPSA2,PavSPSB,and PavSPSC in sweet cherry fruits on anthocyanin and ABA Statistically significant differences between means were determined using one-way analysis of variance（ANOVA）at the 1% signiﬁcance level. ?? indicates significant differences P-value < 0.01. The same below.

图5 沉默PavSPSA1、PavSPSA2、PavSPSB和PavSPSC的甜樱桃果实中可溶性糖（蔗糖、葡萄糖和果糖）含量和果实硬度的变化

Fig. 5 Effect of silencing of PavSPSA1,PavSPSA2,PavSPSB and PavSPSC in sweet cherry fruits on soluble sugar content （sucrose,fructose and glucose）and fruit firmness

图6 沉默PavSPSA1、PavSPSA2、PavSPSB和PavSPSC后的甜樱桃果实成熟相关基因PavNCED1、PavANS、PavDFR、PavCHS、PavPAL、PavPG1、PavXYL1和PavPL1表达量的变化

Fig. 6 Changes of expression levels of PavNCED1,PavANS,PavDFR,PavCHS,PavPAL,PavPG1,PavXYL1 and PavPL1 in PavSPSA1-,PavSPSA2-,PavSPSB- and PavSPSC-silencing sweet cherry fruit PaNCED1（9-cis-epoxycarotenoid dioxygenase,Pav_sc0003135.1_g490.1.mk）,PavANS（anthocyanidin synthase,Pav_sc0000107.1_g100.1.mk）,PavDFR（dihydro flavonol 4-reductase,Pav_sc0002208.1_g840.1.mk）,PavCHS（Chalcone synthase,Pav_sc0000045.1_g280.1.mk）,PavPAL（phenylalanine ammonia-lyase,Pav_co4071347.1_g010.1.mk）,PavPG1（polygalacturonase,Pav_sc0000557.1_g320.1.br）,PavXYL1（β-D-xylosidase,Pav_sc0001014.1_g030.1.mk）,PavPL1（Pectate lyase 1,Pav_sc0000207.1_g1250.1.mk）.

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