Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (18): 3629-3641.doi: 10.3864/j.issn.0578-1752.2023.18.011

• HORTICULTURE • Previous Articles     Next Articles

Identification and Cloning of Ferritin Family Genes in Grape and Response to Compound Amino Acid-Iron Spraying During Different Fruit Developmental Stages

SONG ZhiZhong1,2,3(), WANG JianPing1,2, SHI ShengPeng3,4, CAO JingWen2, LIU WanHao1, XU WeiHua1, XIAO HuiLin1,2(), TANG MeiLing1,2()   

  1. 1 Institute of Grape, Yantai Academy of Agricultural Science, Yantai 264000, Shandong, China
    2 The Engineering Research Institute of Agriculture and Forestry, Universities of Shandong Ludong University/Key Laboratory of Molecular Module-Based Breeding of High Yield and Abiotic Resistant Plants, Yantai 264025, Shandong, China
    3 Department of Plant Science, University of Cambridge, Cambridge CB2 3EA, United Kingdom
    4 Wolfson College, University of Cambridge, Cambridge CB3 9BB, United Kingdom
  • Received:2023-02-23 Accepted:2023-06-05 Online:2023-09-16 Published:2023-09-21
  • Contact: XIAO HuiLin, TANG MeiLing

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Abstract:

【Objective】Ferritin plays an important role in plant growth and development, and its biological function in fruit trees are essentially unknown. Cloning of grape Ferritin family genes and revealing their expression patterns at different fruit developmental stages and their response differences to foliar spraying of amino acid iron (Fe) compound fertilizers could provide a theoretical basis for studying the molecular mechanisms of Fe nutrition and metabolism in fruit trees. 【Method】The Ferritin family genes were screened and identified in grape genome by homologous cloning. The detailed characteristics of Ferritin genes and their encoded proteins were analyzed by using bioinformatical methods. The tissue-specific expression patterns of Ferritin family genes during distinct fruit developmental stages and differential response to foliar spraying of amino acid-iron compound fertilizer were determined by carrying out real-time fluorescent quantitative PCR. 【Result】In total, 4 Ferritin family genes were retrieved and cloned from grape, entitled with VvFer1-VvFer4, which were distributed on No. 6, 8 and 13 chromosomes, containing 7 introns with different lengths. VvFer proteins were mainly located in chloroplast and nucleus. The amino acid sequence identity of Ferritin homologs from 16 plant species was as high as 61.48%. Phylogenetic tree analysis indicated that Ferritin homologs belonging to the same genus, such as Arabidopsis and turnip of Cruciferae, tobacco and potato of Solanaceae, soybeans, peanuts and chickpeas of Leguminosae, rubber trees, cassava and castor of Euphorbiaceae, and apples, peaches and strawberries of Rosaceae, possessed a closer genetic distance during evolution. Grape VvFer3 was closely clustered with Solanaceae homologs. The expression levels of VvFer genes were different among distinct tissues or organs of 5-year-old Mathelan grape trees during different fruit developmental stages. In particular, the expression level of VvFer3 was the most abundant, and the maximum expression was observed in fruits from hard core stage to mature stage, followed by VvFer2 and VvFer4. The content of Fe Marselan fruits was slightly different among distinct grape developmental stages, which was gradually increased from young fruit stage, and reached the highest value at veraison stage, and then slightly decreased until mature stage, but still higher than that of young fruit stage and hard core stage. Foliar spraying treatment significantly enhanced Fe content of fruits at mature stage, accompanied by ACO (aconitase), NIR (nitrate reductase) and SDH (succinate dehydrogenase). Genes of VvFer2-4 were significantly up-regulated by foliar spraying of amino acid-iron compound fertilizer, which was closely related to distinct grape tissues/organs and different fruit developmental stages. In details, the expression of VvFer2 in fruits was sensitive to foliar spraying treatment during the whole period of grape development. The expressions of VvFer3 in all tested tissues were sensitive to foliar spraying treatment from young fruit stage to veraison stage. The expressions of VvFer4 in phloem and leaves were continuously induced by foliar spraying treatment during the whole period of grape development, whereas in fruits from young fruit stage to veraison stage. The expression of VvFer1 was relatively low, but very uniform, and there was no response to foliar spraying treatment at the transcription level. 【Conclusion】Four Ferritin family genes were cloned and identified in grape, whose expression were significantly different among distinct tissues during different fruit developmental stages and were prone to be up-regulated under foliar spraying treatment of amino acid-iron compound fertilizer. The overall expression level of VvFer3 gene was the highest in all tested tissues (especially in fruits) during the whole fruit development stage, and was up-regulated in fruits under foliar spraying treatment from young fruit stage to the verason stage.

Key words: grape, Fe storage and sequestration, Ferritin, compound amino acid-iron fertilizer, foliar spraying

Table 1

Primers used for CDS amplification of grape Ferritin family genes"

基因 Gene 上游引物 Forward primer 下游引物 Reverse primer 产物长度 Product length (bp)
VvFer1 ATGGAACTCTCTCAAAGGGAG TCATGCTGCACCACCCTCTTC 894
VvFer2 ATGCTTGTGGGAGGTGTTTCA CTAAGACTGACCAAGAAAGAG 786
VvFer3 ATGCTTCTCAAAGCTGCTTCA TCACGCAGCAACAACTCCTCCA 798
VvFer4 ATGCTGCTCAAGTCGTCTGCT TCATGCAGCAACCCCATGATT 777

Table 2

Specific expression primers used in this work"

基因
Gene		 上游引物
Forward primer		 下游引物
Reverse primer		 产物长度
Product length (bp)
VvFer1 GCTGAGAAGGGAGATGCATT CATGTCCCTTTCCAACCCTT 217
VvFer2 GATCCCCAGTTGACAGATTT CCACCCTCTTCGAGGAGCATTT 152
VvFer3 GCATTGTCACTGGAGAAGCT GCAGCAACAACTCCTCCATT 230
VvFer4 CGATAATGATGAAAAGGGAG CTTGATAGATTCCACCTGCT 175

Fig. 1

Amino-acid sequence alignment of VvFer proteins"

Table 3

Information of VvFer genes and encoded protein in grape"

基因
Gene		 登录号
Accession No.		 染色体定位
Chromosome location		 内含子数目
Intron number		 氨基酸
Amino acid		 等电点
PI		 跨膜区Transmembrane 总平均亲水性
GRAVY		 不稳定性指数
Instability index
VvFer1 VIT_208s0058g00430 chr8: 9389844..9394079
reverse		 7 298 5.82 3 -0.346 51.73
不稳定Unstable
VvFer2 VIT_208s0058g00410 chr8: 9346913..9351311
reverse		 7 261 5.57 3 -0.407 53.87
不稳定Unstable
VvFer3 VIT_213s0067g01840 chr13: 1003921..1006908
2. reverse		 7 265 5.77 1 -0.244 51.42
不稳定Unstable
VvFer4 VIT_206s0004g07160 chr6: 7932473..7935888
reverse		 7 258 6.20 0 -0.394 39.41
稳定Stable

Fig. 2

The phylogenetic tree of plant Ferritin homologs"

Fig.3

Motif and gene structure analysis"

Fig. 4

Characteristic sequences of different Motifs"

Fig. 5

Tertiary structure prediction of VvFer proteins"

Table 4

Subcellular localization prediction of VvFer proteins"

蛋白 Protein 细胞核 Nucleus 叶绿体 Chlorophyll 线粒体 Mitochondrial 细胞质 Cytosol
VvFer1 78.58% 14.28% 7.14% -
VvFer2 57.14% 25.01% 10.71% 7.14%
VvFer3 14.28% 75.01% 10.71% -
VvFer4 - 92.86% 7.14% -

Fig. 6

Expression pattern analysis of VvFer genes during different fruit developmental stages YFS: Young fruit stage; HCS: Hard core stage; VS: Veraison stage; SES: Second expanding stage; MS: Mature stage. The same as below"

Fig. 7

Fruit iron content during different grape developmental stages *: Significant difference (P<0.05); **: Extremely significant difference (P<0.01). The same as below"

Table 5

Effect of foliar spraying on enzyme activities of Fe-S proteins in mature stage fruits"

对照
Control		 叶面喷施
Foliar spraying
顺乌头酸酶活性
ACO activity (U/mg protein)		 0.53±0.06 0.79±0.07**
亚硝酸还原酶
NiR activity (U/mg protein)		 2.49±0.32 3.65±0.38**
琥珀酸脱氢酶
SDH activity (U/mg protein)		 5.97±0.62 7.59±0.64*

Fig. 8

Differential response of VvFer genes to foliar spraying of iron fertilizer in transcriptional level"

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