杧果Fe-S簇装配基因MiISU1的生物学功能

doi:10.13560/j.cnki.biotech.bull.1985.2022-0616

摘要/Abstract

摘要：

Fe-S簇装配机制相关基因在植物生长发育过程中起重要作用，克隆参与杧果线粒体Fe-S簇装配机制的ISU1，并分析其生物学功能，为研究热带果树Fe-S簇装配的分子机制提供基因资源和理论支撑。利用同源克隆法获得杧果MiISU1，通过实时荧光定量PCR分析该基因的组织特异性表达特征及其在转录水平对不同铁素处理的响应情况，创制MiISU1超表达转基因拟南芥株系并分析其潜在的生物学功能。结果表明，从二倍体杧果‘桂热82’中克隆获得MiISU1，在幼果中的表达水平最高，其次是在成熟果实、盛开期花朵和新生叶片中，而在新生根和新生韧皮部中的表达量较低；缺铁处理显著抑制了MiISU1在一年生杧果嫁接幼苗根中的表达量，而高铁毒害显著增强了MiISU1在整株幼苗不同组织中的表达量。此外，超表达MiISU1显著促进了转基因拟南芥的生长、铁素富集水平、叶片叶绿素含量、硝酸还原酶、乌头酸酶和琥珀酸脱氢酶的酶活力，通过增强转基因植株的根系发育进而提升对缺铁胁迫的耐受能力。MiISU1在杧果铁素营养和代谢中发挥作用，其活性受铁素供应水平的调控。

关键词: 杧果, 铁代谢, Fe-S簇装配机制, ISU1, 缺铁胁迫, 转基因拟南芥

Abstract:

Iron-sulfur（Fe-S）cluster assembly genes play important roles in plant growth and development. We focused on the cloning and functional determination of MiISU1 involved in mitochondrial Fe-S cluster assembly in mango（Mangifera indica）and analyzed its biological functions, which may provide gene resources and theoretical foundation for the molecular mechanisms of Fe-S cluster assembly in tropical crops. The MiISU1 was isolated by homology cloning technology from mango. The tissue-specific expression characteristics and their responses to different Fe supplies were detected by quantitative real-time PCR. Functional verification of MiISU1 was carried out via creating over-expressed transgenic Arabidopsis seedlings. Results showed that MiISU1 was isolated from diploid mango ‘Guire 82’, whose expression was the highest in young fruits, followed by mature fruits, full-bloom flowers and new leaves, but was low in new roots and new phloem. In addition, Fe depletion significantly reduced the expression of MiISU1 in the roots of 1-year-old grafted seedlings, while high Fe toxicity significantly enhanced the expression of MiISU1 in the whole tested seedlings. Moreover, over-expression of MiISU1 in Arabidopsis significantly enhanced plant growth, accompanied with enhanced Fe accumulation, leaf total chlorophyll, nitrite reductase（NIR）, aconitase（ACO）and succinate dehydrogenase（SDH）activities in transgenic lines, and improved plant resistance to Fe depletion via enhancing the root growth and development. MiISU1 plays a key role in Fe nutrition and metabolism of mango, which is regulated by external Fe status.

Key words: Mangifera indica, Fe metabolism, Fe-S cluster assembly machinery, ISU1, Fe deficiency stress, transgenic Arabidopsis

崔吉洁, 蔡文波, 庄庆辉, 高爱平, 黄建峰, 陈亚辉, 宋志忠. 杧果Fe-S簇装配基因MiISU1的生物学功能[J]. 生物技术通报, 2023, 39(2): 139-146.

CUI Ji-jie, CAI Wen-bo, ZHUANG Qing-hui, GAO Ai-ping, HUANG Jian-feng, CHEN Ya-hui, SONG Zhi-zhong. Biological Function of Gene MiISU1 for Fe-S Cluster Assembly in Mangifera indica[J]. Biotechnology Bulletin, 2023, 39(2): 139-146.

图/表 7

图1 13种植物ISU1同源蛋白氨基酸序列一致性分析 At：拟南芥；Bd：短柄草；Cs：柑橘；Es：盐芥；Fv：草莓；Gm：大豆；Mi：杧果；Md：苹果；Os：水稻；Pp：桃；Pt：白杨；Sl：番茄；Vi：葡萄。下同

Fig. 1 Amino acid sequence identity analysis of ISU1 homologs from 13 plant species At: Arabidopsis thaliana; Bd: Brachypodium distachyon; Cs: Citrus sinensis; Es: Thellungiella salsuginea; Fv: Fragaria vesca; Gm: Glycine max; Mi: Mangifera indica; Md: Malus domestica; Os: Oryza sativa; Pp: Prunus persica; Pt: Populus trichocarpa; Sl: Solanum lycopersicum; Vi: Vitis vinifera. The same below

图2 13种植物ISU同源蛋白系统发育树

Fig. 2 Phylogenetic tree of ISU homologs from 13 plant species

图3 杧果MiISU1的组织特异性表达分析

Fig. 3 Tissue-specific expression analysis of MiISU1 gene in mango

图4 杧果MiISU1对不同铁素供应条件的响应差异 *表示在P<0.05水平差异显著；**表示在P<0.01水平差异极显著

Fig. 4 Differential responses of MiISU1 gene to different Fe supplies in mango tissue culture seedlings * indicates significant difference at the P < 0.05 level, ** indicates extremely significant difference at the P < 0.01 level

图5 超表达MiISU1转基因拟南芥创制及验证 A：重组表达载体构建示意图；B：T1代转基因株系PCR鉴定

Fig. 5 Generation and verification of MiISU1 over-expres-sion transgenic Arabidopsis seedlings A: Schematic diagram of recombinant expression vector construction; B: PCR verification of T1 transgenic lines

图6 超表达MiISU1转基因拟南芥表型分析 A：对照条件；B：缺铁处理

Fig. 6 Phenotype analysis of MiISU1 over-expression tran-sgenic Arabidopsis seedlings A: Control conditions; B: Fe deficiency treatment

表1 转基因拟南芥缺铁处理5 d的生理分析

Table 1 Physiological analysis of transgenic Arabidopsis seedlings under Fe depletion for 5 d

处理Treatment	对照Control		缺铁Fe depletion
处理Treatment	野生型Wild type	T₃转基因株系 T₃ transgenic lines	野生型 Wild type	T₃转基因株系 T₃ transgenic lines
整株生物鲜重Fresh weight of whole seedling/g	60.62 ± 5.01	78.24 ± 5.29**	21.75 ± 2.53	28.39 ± 3.21**
地上部生物鲜重Fresh weight of shoots/g	49.42 ± 3.16	62.52 ± 3.44**	16.08 ± 1.92	15.73 ± 1.68
根部生物鲜重Fresh weight of roots/g	10.58 ± 1.44	15.72 ± 1.51**	5.67 ± 0.65	12.66 ± 1.47**
整株铁含量 Fe concentration/（g·Kg^-1 DW）	0.32 ± 0.02	0.47 ± 0.04**	0.08 ± 0.01	0.13 ± 0.01**
叶片叶绿素含量Total leaf chlorophyll/（g·kg^-1 FW）	0.46 ± 0.03	0.51 ± 0.05	0.17 ± 0.02	0.23 ± 0.02**
NiR活性NiR activity/（U·mg^-1·protein^-1）	1.23 ± 0.11	1.51 ± 0.12*	0.32 ± 0.02	0.34 ± 0.02
ACO活性 ACO activity/（U·mg^-1·protein^-1）	0.43 ± 0.02	0.53 ± 0.03*	0.17 ± 0.01	0.23 ± 0.01**
SDH活性SDH activity/（U·mg^-1·protein^-1）	6.97 ± 0.41	9.31 ± 0.46**	2.17 ± 0.22	2.83 ± 0.25**

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