垂丝海棠MhMYB114-Like的克隆及其在苹果愈伤组织中的抗缺铁功能鉴定

doi:10.16420/j.issn.0513-353x.2020-0208

摘要/Abstract

摘要：

从苹果砧木垂丝海棠（Malus halliana Koehne）中克隆得到了MhMYB114-Like基因（序列号:LOC103405832）。该基因包含全长为 602 bp的完整开放阅读框,编码200个氨基酸,蛋白质等电点为9.38。系统进化树分析表明,MhMYB114-Like与野草莓该家族蛋白亲缘关系最近。分析MhMYB114-Like的启动子区域发现包含干旱、光、生长素和黄化响应等顺式作用元件。荧光定量 PCR 分析表明,在缺铁胁迫下MhMYB114-Like在垂丝海棠叶和根中表达水平显著上升,MhMYB114-Like的表达明显受缺铁胁迫的诱导。将MhMYB114-Like遗传转化‘王林’苹果愈伤组织,通过测定相关生理指标进行抗性分析发现,过量表达MhMYB114-Like可明显提高转基因愈伤组织对缺铁胁迫的抗性。结果表明 MhMYB114-Like可响应缺铁胁迫,在抵抗缺铁胁迫中具有重要作用。

关键词: 苹果, MhMYB114-Like, 功能鉴定, 缺铁胁迫, 基因表达

Abstract:

The MhMYB114-Like gene（GenBank accession number:LOC103405832）was cloned from apple rootstock Malus halliana. The gene contains a full open reading frame of 602 bp,which encodes 200 amino acids,and the protein has an isoelectric point of 9.38. The phylogenetic tree analysis showed that apple MhMYB114-Like was the closest relative to wild strawberry. Promoter cis-element prediction of apple MhMYB114-Like showed that there were drought,light,auxin,yellowing and other responsive regulatory elements. Fluorescence quantitative PCR analysis demonstrated that the expression level of MhMYB114-Like gene was greatly increased in the leaves and roots of Malus halliana under iron deficiency condition,and the expression was obviously induced by iron deficiency stress. In addition,The genetic transformation of MhMYB114-Like into apple‘Orin’callus and resistance analysis by measuring related physiological indexes showed that the overexpression of MhMYB114-Like could significantly improve the resistance of transgenic callus to iron deficiency stress. Therefore,MhMYB114-Like is involved in the response to iron deficiency and play a crucial role in resisting iron deficiency stress.

Key words: apple, MhMYB114-Like, functional identification, iron deficiency stress, gene expression

中图分类号:

S661.1

张仲兴, 程丽, 王双成, 张德, 刘兵, 王延秀. 垂丝海棠MhMYB114-Like的克隆及其在苹果愈伤组织中的抗缺铁功能鉴定[J]. 园艺学报, 2021, 48(1): 127-136.

ZHANG Zhongxing, CHENG Li, WANG Shuangcheng, ZHANG De, LIU Bing, WANG Yanxiu. Cloning of MhMYB114-Like from Malus halliana and Its Functional Identification of Iron Deficiency Tolerance in Apple Callus[J]. Acta Horticulturae Sinica, 2021, 48(1): 127-136.

图/表 9

表1 本研究中所用到的引物

Table 1 Primers used in this study

基因 Gene	序列（5′-3′）Sequence
MYB114-like（qRT-PCR） MYB114-like（gene clone）	F:TGATCAGACCTCAACCCCGA;R:GACGACGACGTTTGTGGTGA F:ATGGAGGGATATAACGTTAACTTGA;R:CTAACTCAAGACTGGGACATACA

图1 垂丝海棠MhMYB114-Like蛋白与其他物种中的该蛋白序列比对

Fig. 1 The MhMYB114-Like protein in Malus halliana was compared with that of other species

图2 垂丝海棠MhMYB114-Like与其他物种家族该蛋白的系统进化分析进化树分支处的数字表示该分支的置信度,数值越大,可靠性越高。

Fig. 2 Phylogenetic analysis of this protein of MhMYB114-like from Malus halliana and other species families The number at the branch of the evolutionary tree indicates the confidence of the branch,the larger the value,and the higher the reliability.

表2 MhMYB114-Like基因上游调控序列重要顺式作用元件分析

Table 2 Some important cis-acting regulatory elements in the upstream regulatory sequences of MhMYB114-Like

元件名称 cis-Acting element	核心序列 Sequence	位点功能 Function of site	位置 Location
GT1CONSENSUS	GRWAAW	光响应元件cis-Acting element involved in light responsiveness	-586
DPBFCOREDCDC3	ACACNNG	脱落酸响应cis-Acting element involved in abscisic ascid responsiveness	-619
LTRECOREATCOR15	CCGAC	耐寒响应 cis-Acting element involved in resistance cold responsiveness	-1 549
CIACADIANLELHC	CAANNNNATC	生理节律响应cis-Acting element involved in circadian responsiveness	-1 203
ACGTATERD1	ACGT	黄化响应元件cis-Acting element involved in chlorisis responsiveness	-804
DOFCOREZM	AAAG	碳代谢响应元件cis-Acting element involved in carbon metabolism	+ 704
CATATGGMSAUR	CATATG	生长素响应元件cis-Acting element involved in the auxin responsiveness	+ 1 217
SREATMSD	TTATCC	糖响应元件cis-Acting element involved in sugar responsiveness	+ 946
PYRIMIDINEBOXHVEPB1	TTTTTTCC	激素调控元件cis-Acting element involved in hormone responsiveness	+ 1 877
ARR1AT	NGATT	细胞分裂素响应cis-Acting element involved in cytokinin responsiveness	+ 1 991
MYB2CONSENSUSAT	YAACKG	耐旱响应cis-Acting element involved in drought-tolerant responsiveness	+ 1 049

图3 MhMYB114-Like在缺铁胁迫下的表达水平 * P < 0.05,表示与对照相比差异显著水平。

Fig. 3 Expression level of MhMYB114-Like under iron deficiency stress * < 0.05,represents a significant level of difference compared to the control group.

图4 转MhMYB114-Like愈伤的qRT-PCR验证及其在缺铁胁迫下的相对表达量

Fig. 4 qRT-PCR verification of transgenic MhMYB114-Like callus and the relative expression under iron deficiency stress * P < 0.05.

图5 ‘王林’苹果野生型和转MhMYB114-Like基因愈伤组织在加铁和缺铁条件下的生长情况

Fig. 5 Growth of wild-type and transgenic MhMYB114-Like callus of‘Orin’Apple under iron and iron deficiency conditions

图6 MhMYB114-Like过量表达提高‘王林’苹果愈伤组织L1、L2、L3及其野生型WT中的抗氧化酶活性

Fig. 6 Overexpression of MhMYB114-Like improves enzyme activity in‘Orin’apple callus L1,L2,L3 and wild type（WT） * P < 0.05.

图7 MhMYB114-Like过量表达提高‘王林’苹果愈伤组织L1、L2、L3及其野生型WT中的Fe2+含量和FCR活性

Fig. 7 Overexpression of MhMYB114-Like improves Fe2+ content and FCR activity in‘Orin’apple callus L1,L2,L3 and wild type（WT） * P < 0.05.

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