桑树中miR408-MnBBP模块调节花青苷生物合成的机制

doi:10.16420/j.issn.0513-353x.2023-0571

摘要/Abstract

摘要：

为探究桑树（Morus alba L.）miR408靶向MnBBP基因调控花青苷合成的分子机制，利用荧光定量PCR技术（qRT-PCR）、生物信息技术、5′-RACE及转基因技术分析了桑树miR408对桑葚花青苷的生物代谢途径的影响。序列比对发现桑树miR408序列与双子叶植物序列完全一致，与单子叶植物序列在第1个核苷酸位置存在单核苷酸多态性；qRT-PCR分析发现miR408在桑树不同组织差异表达，在叶片和果实表达水平最高；使用在线网站Omicstudio预测分析，发现桑树蓝铜结合蛋白基因MnBBP是miR408的靶基因，降解组测序结果也发现MnBBP能够被miR408靶向切割，利用5'-RACE技术进一步验证切割位点位于与miR408互补序列的第9至第10位碱基之间；拟南芥超表达miR408发现，转入桑树miR408提高了拟南芥叶片中花青苷水平；运用转录组测序技术分析转基因和野生型植株基因差异表达，发现差异表达基因富集在花青苷合成途径上，其中5个基因在超表达miR408植株上调得到了qRT-PCR验证。研究结果确定了MnBBP为桑树miR408的靶基因，miR408通过抑制MnBBP表达促进桑葚花青苷的积累。

关键词: 桑, 果实, miR408, MnBBP, 转基因, 花青苷

Abstract:

To elucidate the molecular mechanisms by which mulberry miR408 targets the MnBBP gene to modulate anthocyanin biosynthesis，the effect of mulberry miR408 on anthocyanin metabolism was analyzed by quantitative real-time PCR（qRT-PCR），bioinformatics analysis，5′-rapid amplification of cDNA ends（5′-RACE），and transgenic methodologies. Sequence alignment revealed that the miR408 sequence is identical to that of dicotyledonous plants，while featuring a single nucleotide polymorphism （SNP）at the first nucleotide position in monocotyledons. Expression profiling via qRT-PCR demonstrated differential expression patterns of miR408 across various mulberry tissues，with high expression levels observed in leaves and fruits. Through predictive analysis using the Omicstudio online platform，the mulberry blue-copper binding protein gene（MnBBP）was identified as a target gene of miR408. Degradome sequencing further corroborated that MnBBR is subject to specific cleavage by miR408. 5′-RACE analysis pinpointed the cleavage site between the 9th and 10th bases of the sequence complementary to miR408. Overexpression of mulberry miR408 in Arabidopsis thaliana led to elevated anthocyanin levels in the leaves. Transcriptomic analysis identified differentially expressed genes（DEGs）between the transgenic and wild-type plants，with a notable enrichment of DEGs in the anthocyanin biosynthetic pathway. The up-regulation of five key genes within this pathway was subsequently validated by qRT-PCR. Collectively，this study not only identifies MnBBP as a target gene of miR408 in mulberry，but also elucidates the regulatory roles of miR408 and MnBBP in anthocyanin biosynthesis.

Key words: mulberry, fruit, miR408, MnBBP, transgenic, anthocyanin

孙志超, 郭新淼, 王晖, 谢岩, 高妍夏, 李季生, 高玉军. 桑树中miR408-MnBBP模块调节花青苷生物合成的机制[J]. 园艺学报, 2024, 51(6): 1216-1226.

SUN Zhichao, GUO Xinmiao, WANG Hui, XIE Yan, GAO Yanxia, LI Jisheng, GAO Yujun. Mechanism of miR408-MnBBP Module Regulating Anthocyanin Biosynthesis in Mulberry[J]. Acta Horticulturae Sinica, 2024, 51(6): 1216-1226.

图/表 7

表1 基因的RT-qPCR引物

Table 1 List of RT-qPCR primer sequences used for genes

基因名称 Gene name	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
miR408a	ATGCACTGCCTCTTCCCTGGC
AtCHS（At5G13930）	ACATGCCTGGTGCTGACTAC	AAAAGAGCCTGACCGACGAG
AtANS（At4G22880）	GAGTCTAGCAAAAAGCGGAA	GATTGTGGGAACTTGAGGAC
AtDFR（At5G42800）	CGCCGAAGAGAAAGGATTAG	TATGAGCGTTGCATAAGTCG
AtUFGT（At1G07250）	TCGTCCATATTGAATTCGCC	GAATGGAGGAGGATCTTGGA
AtFLS（At5G63590）	TGGAATCACACTCCTAGTCG	CCCTATGCTCCACACTCTTA
AtARPN（AT2G02850）	GACTCTGGTATCTGGACCTT	CAGTTGTTGTAACTTCCGCT
AtCSD1（At1G08830）	TTCAACCCCGATGGTAAAAC	ACAGCCCTACCAACAATAGA
AtCSD2（At2G28190）	GGCCTCATGGATTTCATCTC	ATCGGCATTGGCATTTATGT
AtSPL7 (At5G18830)	GTATGCTTCCTTGTTCGTGT	AATATCCGCTTCACAATCCG
U6（LOC21407692）	GATAAAATTGGAACGATACAG	ATTTGGACCATTTCTCGATTT
Ribosomal protein L15 （LOC21390050）	GGCTATGTGATTTACCGTGTT	TTGGTCCAGTATGAGTTGAGAA
Actin 2（AT3G18780）	GCCATCCAAGCTGTCTC	GCTCGTAGTCAACAGCAACAA

图1 桑树miR408序列比对（A）及表达分析（B）

Fig. 1 Sequence alignment of miR408（A）and expression analysis（B）in mulberry

图2 桑树中miR408靶基因MnBBP验证 A：降解组测序法测定MnBBP降解转录片段的切割位点，图中红点表示与miR408定向切割一致的读取计数；B：5′-RACE定位MnBBP的miR408定向切割位点，箭头表示切割位点，数字表示测序克隆的频率。

Fig. 2 miR408 targets MnBBP in mulberry A：Mapping of cleavage sites of degraded transcript fragments of MnBBP by degradome sequencing. The red dot indicating read counts consistent with miR408-directed cleavage；B：Mapping of miR408-directed cleavage sites of MnBBP using 5'-RACE. Arrow indicates cleavage site，and the numbers show the frequency of sequenced clones.

图3 转基因（OE）和野生型（WT）拟南芥中miR408表达水平和花青苷含量

Fig. 3 Detection of miR408 expression and anthocyanin content in transgenic and wild type（WT）Arabidopsis ** P < 0.01；* P < 0.05（ANOVA）.

图4 拟南芥转基因株系miR408-OE与野生型差异表达基因的火山图（A）和GO富集散点图（B）

Fig. 4 Advanced Volcano Plot（A）and GO enrichment（B）of differentilly expressed genes between the miR408-OE and WT in Arabidopsis

图5 拟南芥转基因株系miR408-OE与野生型差异表达基因的KEGG富集散点图（A）和MnBBP调控模块构建（B）

Fig. 5 KEGG pathway（A）and miR408-MnBBP regulatory module（B）of differentilly expressed genes between the miR408-OE and WT in Arabidopsis

图6 拟南芥转基因株系miR408-OE及野生型（WT）花青苷合成相关基因表达的转录组数据热图（A）和qRT-PCR定量（B、C）

Fig. 6 Heatmap of transcriptome（A）and qRT-PCR（B，C）of differentilly expressed genes related to anthocyanin synthesis between the miR408-OE and wild type（WT）in Arabidopsis * P < 0.05.

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