生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 139-146.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0616
崔吉洁1(), 蔡文波1, 庄庆辉1, 高爱平2, 黄建峰2, 陈亚辉3, 宋志忠1,3()
收稿日期:
2022-05-16
出版日期:
2023-02-26
发布日期:
2023-03-07
作者简介:
崔吉洁,女,研究方向:果树分子生物学;E-mail: 基金资助:
CUI Ji-jie1(), CAI Wen-bo1, ZHUANG Qing-hui1, GAO Ai-ping2, HUANG Jian-feng2, CHEN Ya-hui3, SONG Zhi-zhong1,3()
Received:
2022-05-16
Published:
2023-02-26
Online:
2023-03-07
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。
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
处理Treatment | 对照Control | 缺铁Fe depletion | ||||
---|---|---|---|---|---|---|
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** |
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 | ||||
---|---|---|---|---|---|---|
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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