桃NAC基因家族的鉴定及PpNAC050促进果实果糖积累研究

doi:10.16420/j.issn.0513-353x.2023-0085

摘要/Abstract

摘要：

植物特异性转录因子 NAC（NAM，ATAF1/2，CUC2）在调控果实品质中发挥着重要作用。为探索NAC基因在桃（Prunus persica）果实可溶性糖调控中的作用，在桃基因组中鉴定得到117个NAC基因家族成员（PpNAC），其在桃8条染色体上呈不均匀分布。系统发育关系分析显示，这些PpNAC可归为13个亚类群。克隆了一个在果实发育后期高表达的PpNAC050基因（Prupe.7G014100），其编码蛋白定位于细胞核中，具有转录激活活性。瞬时过表达PpNAC050显著提高了桃果肉中果糖和葡萄糖含量；EMSA和双荧光素酶实验表明，PpNAC050作为转录抑制子，可直接与调控果糖含量的基因——液泡膜单糖外转运蛋白基因PpERDL6-1（PpERDL16）的启动子结合，暗示PpNAC050可通过抑制PpERDL6-1表达来提高果肉中果糖积累。

关键词: 桃, 果糖, NAC, PpNAC050, PpERDL6-1

Abstract:

The plant-specific NAC（NAM，ATAF1/2，CUC2）transcription factors play important roles in regulating fruit quality. To explore the role of the NAC in the regulation of soluble sugars in peach （Prunus persica）fruit quality，a total of 117 potential peach NAC members（PpNAC）were identified with an uneven distribution across all eight chromosomes as well as a scaffold. Phylogenetic analyses indicated that these PpNAC were classified into 13 subgroups. The NAC gene PpNAC050 that is highly expressed during later fruit development were further characterized，and it was in nucleus with transcriptional activation. Transient overexpression of PpNAC050 in peach fruits significantly increased fructose content and glucose content in peach fruit flesh. EMSA and dual-luciferase assays showed that PpNAC050，being a transcriptional repressor，directly bound to the promoters of PpERDL6-1（PpERDL16），a vacuolar membrane monosaccharide transporter gene regulating fructose content，suggesting that PpNAC050 positively regulated fructose accumulation by repressing PpERDL6-1 gene expression.

Key words: peach, fructose, NAC gene family, PpNAC050, PpERDL6-1

刘建豪, 荆彦付, 刘月芯, 徐摇光, 于洋, 葛秀秀, 谢华. 桃NAC基因家族的鉴定及PpNAC050促进果实果糖积累研究[J]. 园艺学报, 2024, 51(9): 1983-1996.

LIU Jianhao, JING Yanfu, LIU Yuexin, XU Yaoguang, YU Yang, GE Xiuxiu, XIE Hua. Identification of Peach NAC Gene Family and Role of PpNAC050 in Promoting Fruit Fructose Accumulation[J]. Acta Horticulturae Sinica, 2024, 51(9): 1983-1996.

图/表 7

表1 引物序列信息

Table 1 Primer sequences

基因 Gene	引物序列（5′-3′） Primer sequence	PCR产物大小/bp PCR product size
PpNAC-BD-F	ATCTCAGAGGAGGACCTGCATATGATGAGTATGGCCAATGCCATG	1 638
PpNAC-BD-R	CGGCCGCTGCAGGTCGACGGATCCAGAACTTTGTGGGAAAAGAGTCGC
PpNAC-F	ATGAGTATGGCCAATGCCATG	1 641
PpNAC-R	TCAAGAACTTTGTGGGAAAAGAGTCGC
PpNAC-1300-F	TAAATACTAGTGGATCCGGTACCATGAGTATGGCCAATGCCATG	1 638
PpNAC-1300-R	TCGCCCTTGCTCACCATGGTACCAGAACTTTGTGGGAAAAGAGTCGC
PpNAC-62SK-F	GGCCGCTCTAGAACTAGTGGATCCATGAGTATGG CCAATGCCAT GT	1 638
PpNAC-62SK-R	ACCGGGCCCCCCCTCGAGGTCGACAGAACTTT GTGGGAAAAG AGTC
q-NAC-F	TGAGTATGGCCAATGCCATGT	181
q-NAC-R	TTGGGCAGCAAATCAAGCAC
PpNAC-AD-F	CCATGGAGGCCAGTGAATTCATGAGTATGGCCAATGCCATGTAT	1 638
PpNAC-AD-R	TGCCCACCCGGGTGGAATTC AGAACTTTGTGGGAAAAGAGTCG
PpERDL6-1-NAC-F	GACATGTGTATAAATACCTCAGAACACGAGCATATATTGCATTGCATCCC	50
PpERDL6-1-NAC-R	GGGATGCAATGCAATATATGCTCGTGTTCTGAGGTATTTATACACATGTC
4T-PpNAC-F	CCGCGTGGATCCCCGGAATTCATGAGTATGGCCAATGCCATG	1 638
4T-PpNAC-R	GTCACGATGCGGCCGCTCGAGAGAACTTTGTGGGAAAAGAGTCGC
PpERDL6-1-800-lucF	GGTCGACGGTATCGATAAGCTT GTCACGTCTG ATGTCCCAGT	1 971
PpERDL6-1-800-lucR	GCAGGAATTCGATATCAAGCTT GTTTTTTTCTC ACTACTCTAA
PpERDL6-1-q-F	TCACTATGGTCGGAGCAACG	183
PpERDL6-1-q-R	ATGTAAGTCAGCACGCCAGA

图1 桃 NAC基因家族的染色体定位

Fig. 1 Chromosomal localization of NAC family genes in peach

图2 桃（Prupe）和拟南芥（AT）NAC家族成员系统进化分析

Fig. 2 Phylogenetic analysis of NAC family members in peach（Prupe）and Arabidopsis（AT） a：ANAC001/ONAC003；b：TIP；c：OSNAC8；d：NAC2；e：ANAC011；f：NAM/NAC1；g：OSNAC7；h：ONAC022/TERN；j：ANAC063/SENU5；k：ANAC104；l：ATAF/AtNAC3；m：NAP；n：ANAC063.

图3 PpNAC050 的基因相对表达量（A）、基因片段扩增（B）和转录激活检测（C） S1：第一次快速生长期；S2：硬核期；S3：第二次快速生长期。A图中不同小写字母表示不同果实发育期间在0.05水平差异显著。

Fig. 3 Expression（A），fragment amplification（B）and transcriptional activation detection（C）of PpNAC050 S1：First rapid growth phase；S2：Lag phase；S3：Second rapid growth phase. Different lowercase letters indicate in Fig. A significant differences at the 0.05 level in different fruit development periods.

图4 PpNAC050亚细胞定位 A：重组载体 pCAMBIA1300-PpNAC050-GFP 示意图； B：共聚焦显微镜观察烟草叶片细胞GFPs和PpNAC050-GFP融合蛋白的瞬时表达。

Fig. 4 The subcellular localization of PpNAC050 A：Schematic diagram the recombinant plasmid pCAMBIA1300-PpNAC050-GFP； B：The GFP fluorescence from PpNAC050-GFP fusion protein was localized to the nucleus of the cells.

图5 瞬时过表达桃果实中的PpNAC050表达水平和果糖、葡萄糖、蔗糖含量 CK-1、CK-2和CK-3：空载 pGreen0029-62SK；TOE-1、TOE-2和TOE-3：过表达。ns：不显著。

Fig. 5 The expression level and fructose，glucose and sucrosecontent in peach fruits transiently overexpressing PpNAC050 CK-1，CK-2和CK-3：Empty pGreen0029-62SK；TOE-1，TOE-2 and TOE-3：Overexpression. ns：Non-significant. * P < 0.05，** P < 0.01（Student’s t-test）.

图6 PpNAC050抑制糖转运蛋白PpERDL6-1的表达 A：PpERDL6-1在瞬时过表达PpNAC050果实中的表达水平；B和C：分别是酵母单杂交和EMSA验证PpNAC050与PpERDL6-1启动子结合；D：PpNAC050对PpERDL6-1启动子活性影响的双荧光素酶分析。误差线代表3次生物学重复标准差。

Fig. 6 PpNAC050 suppresses the expression of sugar transporter PpERDL6-1 A：The expression levels of sugar transporter PpERDL6-1 in transiently overexpressing PpNAC050 fruits；B and C：Yeast one-hybrid and EMSA assay showing PpNAC050 binds to the PpERDL6-1 promoter fragment，respectively. D：The effects of PpNAC050 on the PpERDL6-1 sugar transporter promoter with dual-luciferase reporter assay in tobacco. Error bars represent the standard deviation of three biological replicates. ** P < 0.01，Student’s t-test.

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