葡萄铁蛋白基因Ferritin的鉴定、克隆及其在果实发育不同时期对氨基酸铁复合肥喷施的响应

doi:10.3864/j.issn.0578-1752.2023.18.011

Abstract

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

SONG ZhiZhong, WANG JianPing, SHI ShengPeng, CAO JingWen, LIU WanHao, XU WeiHua, XIAO HuiLin, TANG MeiLing. Identification and Cloning of Ferritin Family Genes in Grape and Response to Compound Amino Acid-Iron Spraying During Different Fruit Developmental Stages[J].Scientia Agricultura Sinica, 2023, 56(18): 3629-3641.

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References 35

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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

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

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

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%	-

	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*

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

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