低氮胁迫诱导木薯幼苗花青素积累及其基因表达

doi:10.13560/j.cnki.biotech.bull.1985.2021-0441

摘要/Abstract

摘要：

为明确氮素浓度和形态与木薯花青素产生和积累的关系,基于氮素胁迫能够在拟南芥等植物中促进花青素产生的研究结果,以木薯品种Arg7为研究对象,研究木薯无菌幼苗在添加了（1）40 mmol/L NO₃^- + 20 mmol/L NH₄⁺,（2）40 mmol/L NO₃^-,（3）20 mmol/L NH₄⁺,（4）0.4 mmol/L NO₃^- + 0.2 mmol/L NH₄⁺,（5）0.4 mmol/L NO₃^-,（6）0.2 mmol/L NH₄⁺,（7）1 mmol/L（N）,（8）5 mmol/L（N）,（9）9 mmol/L（N）,（10）13 mmol/L（N）10种氮素浓度和形态的MS培养基中生长40 d的农艺性状,以及对花青素合成相关结构基因的诱导和花青素积累的差异。结果表明,木薯品种Arg7无菌幼苗在添加有40 mmol/L NO₃^- + 20 mmol/L NH₄⁺,40 mmol/L NO₃^-和20 mmol/L NH₄⁺的MS缺氮培养基中生长至40 d,没有观察到花青素产生;与40 mmol/L NO₃^- + 20 mmol/L NH₄⁺处理条件相比,在40 mmol/L NO₃^-条件下的株高和初生根根长没有显著差异,但初生根根数显著减少,而在20 mmol/L NH₄⁺条件下其株高、初生根根长和初生根根数都受到极显著抑制;木薯品种Arg7无菌苗在含有0.4 mmol/L NO₃^- + 0.2 mmol/L NH₄⁺,0.4 mmol/L NO₃^-和0.2 mmol/L NH₄⁺的MS缺氮培养基中生长至40 d,3种处理条件下的木薯茎秆和叶柄中花青素积累明显,花青素合成相关结构基因被诱导表达;与40 mmol/L NO₃^- + 20 mmol/L NH₄⁺条件下相比,在0.4 mmol/L NO₃^- + 0.2 mmol/L NH₄⁺,0.4 mmol/L NO₃^-和0.2 mmol/LNH₄⁺条件下的株高、初生根根长和根数极显著降低,而花青素含量极显著增加,且花青素含量随着氮素浓度增加呈负相关关系（R²=0.96）。由此表明,木薯品种Arg7无菌幼苗植株中花青素的诱导表达只与氮素浓度有关,而与氮素形态无关。

关键词: 花青素, 木薯, 氮素浓度, 氮素形态, 基因表达

Abstract:

In order to clarify the relationship between the nitrogen and the anthocyanin in cassava,we studied the agronomic traits and the inductions of anthocyanin synthesis-related genes and anthocyanin accumulation differences of cassava Arg7 sterile seedlings growing in the modified MS media for 40 d with 1）40 mmol/L NO₃^- + 20 mmol/L NH₄⁺,2）40 mmol/L NO₃^-,3）20 mmol/L NH₄⁺,4）0.4 mmol/L NO₃^- + 0.2 mmol/L NH₄⁺,5）0.4 mmol/L NO₃^-,6）0.2 mmol/L NH₄⁺,7）1 mmol/L（N）,8）5 mmol/L（N）,9）9 mmol/L（N）,and 10）13 mmol/L（N）,based on the previous research results of that nitrogen deficiency accelerates anthocyanin synthesis and accumulation in Arabidopsisetc. Results showed that no anthocyanin was observed in the seedlings of cassava variety Arg7 in any of the three modified MS media containing 40 mmol/L NO₃^- alone,20 mmol/L NH₄⁺alone or their combination for 40 d. Compared to cassava seedlings in the combination of 40 mmol/L NO₃^- and 20 mmol/L NH₄⁺,the plant height and length of primary roots under 40 mmol/L NO₃^- alone treatment were not significantly different,but the number of primary roots was significantly reduced. And the plant height,the length and number of primary roots of the seedlings in media with 20 mmol/L NH₄⁺ were significantly inhibited. The anthocyanins accumulated significantly in the stem and petiole of seedlings in any of three MS media supplemented with 0.4 mmol/L NH₄⁺,0.2 mmol/L NH₄⁺,or combination of 0.4 mmol/L NO₃^- and 0.2 mmol/L NH₄⁺ for 40 d,as well as genes related to anthocyanin synthesis were induced to be expressed. Compared to the combination of 40 mmol/L NO₃^- and 20 mmol/L NH₄⁺,the plant height,the lengths and the number of primary roots under MS media with 0.4 mmol/L NO₃^-,0.2 mmol/L NH₄⁺,or combination of 0.4 mmol/L NO₃^-,and 0.2 mmol/L NH₄⁺ significantly decreased,but the anthocyanin content significantly increased. Furthermore,there was a negative correlation between anthocyanin content and nitrogen concentration（R²=0.96）. This indicates that the induced expression of anthocyanins in cassava Arg7 seedlings was only related to nitrogen concentration,but not to the form.

Key words: anthocyanin, cassava, nitrogen concentration, nitrogen form, gene expression

邹良平, 郭鑫, 起登凤, 翟敏, 李壮, 赵平娟, 彭明, 牛兴奎. 低氮胁迫诱导木薯幼苗花青素积累及其基因表达[J]. 生物技术通报, 2022, 38(2): 75-82.

ZOU Liang-ping, GUO Xin, QI Deng-feng, ZHAI Min, LI Zhuang, ZHAO Ping-juan, PENG Ming, NIU Xing-kui. Anthocyanin Accumulation and Its Gene Expression Induced by Low Nitrogen Stress in Cassava Seedlings[J]. Biotechnology Bulletin, 2022, 38(2): 75-82.

图/表 5

表1 花青素合成相关基因特异引物

Table 1 Specific primers for the genes related to anthocy-anin biosynthesis

基因名称Gene name	基因号 Gene ID	引物序列 Primer sequence
MePAL	Manes.04G018000	CTCTCCTCCAGGGTTACTCCG
MePAL	Manes.04G018000	GGCTATGTATGAAAAGGGGACTAAG
Me4CL	Manes.09G127000	GAGGATGCTCTACGGAGTAGGG
Me4CL	Manes.09G127000	AGCCAGTTTCAGGGTCAATGAC
MeCHS	Manes.03G150000	TCCGAGATAACAGCCGTGACAT
MeCHS	Manes.03G150000	GCAGAGACAAGTTCAAACAATGGC
MeF3H	Manes.02G104700	GATTGATGATGTTGGTGGGAAG
MeF3H	Manes.02G104700	AATCCCTTAGCAAGACGAGTCATC
MeDFR	Manes.18G022300	TCTGGCGTTTTCCATGTTGCTA
MeDFR	Manes.18G022300	CATCATACACTGGTTTTTTGTGTTCC
MeANS	Manes.01G070200	GGCAATGTTTTCGCAGAAGAGA
MeANS	Manes.01G070200	CCATTCTTTAGCCGCACTTACAA

图1 氮素浓度和形态对木薯Arg7幼苗的影响 A：40 mmol/L NO3- + 20 mmol/L NH4+;B：40 mmol/L NO3-;C：20 mmol/L NH4+;D：0.4 mmol/L NO3- + 0.2 mmol/L NH4+;E：0.4 mmol/L NO3-;F：0.2 mmol/L NH4+;G：0.4 mmol/L NO3- + 0.2 mmol/L NH4+;H：0.4 mmol/L NO3-;I：0.2 mmol/L NH4+。统计分析采用通用单向方差分析,即其他处理与40 mmol/L NO3- + 20 mmol/L NH4+处理比较,极显著差异P﹤0.001。 Bar=1 cm

Fig.1 Influences of the concentration and the forms of nitrogen onsterile cassava variety Arg7 seedling Data analysis was performed using ordinary one-way ANOVA test for multiple comparisons, i.e., extremely difference P<0.001 when other treatmets compared with 40 mmol/L NO3- +20 mmol/L NH4+

图2 不同氮素处理条件下花青素合成相关结构基因的表达

Fig. 2 Expressions of genes related to anthocyan in biosynthesis under different concentration and forms of nitrogen

图3 不同氮素处理条件下的花青素含量

Fig. 3 Content of anthocyanin under different nitrogen treatment conditions

图4 花青素含量随着氮素浓度递增而呈负相关关系

Fig.4 Anthocyanin content negatively correlated with the increase of nitrogen concentration A：1 mmol/L（N）;B：5 mmol/L（N）;C：9 mmol/L（N）;D：13 mmol/L（N）. A and B：bar=0.5 cm;C and D：bar=1 cm

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