葡萄NHX基因家族的鉴定和表达分析
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摘要
【目的】探究葡萄NHX基因家族生物信息学特性及在非生物胁迫下的表达情况,以期筛选出与非生物胁迫相关的家族成员,从而为葡萄抗逆性研究提供理论依据。【方法】以拟南芥、水稻及胡扬NHX基因序列为基础,对葡萄NHX基因进行同源克隆、染色体定位、氨基酸组成成分、理化性质、motif、蛋白质二级结构以及基因芯片表达等分析,同时利用qRT-PCR技术分析葡萄NHX基因家族表达情况。【结果】葡萄NHXs可分为2个亚族,主要分布在第1、5、7、14、15、19号染色体上,其外显子数为12～23个;VvNHX06的氨基酸数最少,有291个,VvNHX07的氨基酸数最多,有1141个。Motif分析发现,N端含有典型的锌指结构,蛋白质二级结构以α-螺旋和无规则卷曲为主。亚细胞定位预测发现,葡萄NHX基因主要分布在细胞膜、内质网、线粒体、液泡和细胞质中。基因芯片表达显示,使用NaCl、ABA和PEG处理后,葡萄叶片中VvNHX02和VvNHX06基因表达量均呈上升趋势。qRT-PCR分析结果表明,在200 mmol·L~(-1)NaCl、100 mmol·L~(-1)ABA处理后的葡萄叶片中,VvNHX06表达量显著增加,分别是对照的30倍和60倍。用10%PEG处理实验材料6 h后,VvNHX05的表达量明显增加,是对照的5倍。【结论】VvNHX05和VvNHX06基因与植物耐盐和抗旱性有密切关系,可为葡萄的逆境胁迫机制研究提供参考。
【Objective】Grape(Vitis vinifera L.) is widely grown the world. China is the second country for grape production in the world. As the increase of cultivation area, it is very important to study the resistance of grape to the abiotic stresses. NHX has the functions of maintaining Na+(K+) concentration,regulating pH in cells, controlling cell expansion, and maintaining cell turgor. NHX is a key factor related to drought and salt tolerance in plants. The bioinformatics characteristics of grape NHX genes family and its expression under abiotic stresses were surveyed to screen out family members related to abiotic stresses.【Methods】The test materials were the seedlings propagated in vitro of‘Red Globe'preserved by the College of Horticulture, Gansu Agricultural University. The single-bud stem segment of the‘Red Globe'grape was cultured on the subculture medium(MS+0.2 mg · L~(-1) IAA) for 35 d. Then the tissue culture seedlings were removed from the containers, the root agar was washed off, and they were transferred into MS liquid medium without hormones. The seedlings wer cultured in incubator at(28±0.5)℃with, a photoperiod of 16 h/8 h(light/dark). After 30 d culture, the seedlings were transfered to the MS media with 10% PEG, 100 mmol · L~(-1) ABA and 200 mmol · L~(-1) NaCl, respectively. The pure water was used as CK. Three biological replicates were set for each treatment, and the treatment duration was 6 h.And based on the NHX gene sequences of Arabidopsis thaliana, rice and poplar, homologous cloning,chromosome localization, amino acid composition, physicochemical properties, motif, protein secondary structure and gene chip expression of grape NHX gene were analyzed. Quantitative analysis of grape NHX gene family expression by qRT-PCR was performed.【Results】Grape NHXs gene could be divided into two sub families, eight members of the grape NHX gene family were obtained by comparison with the NHX genes of existing plants. According to the localization analysis, eight grape NHX genes were distributed on 6 th chromosomes, VvNHX07 was located on the first chromosome, and VvNHX02 on the 5 th chromosome, VvNHX03 on the 7 th chromosome, VvNHX01 and VvNHX08 on the 14 th chromosome, VvNHX05 and VvNHX06 on the 15 th chromosome, and VvNHX04 on the 19 th chromosome. And the number of exons was 12-23. VvNHX06 gene only had 291 amino acids. VvNHX07 gene had 1 141 amino acids. The full-length protein sequence was predicted by MEME software, and 8 domains were obtained. No conserved amino acid sequence was predicted in VvNHX07. Only motif1 was present in VvNHX05, and all 8 domains were existing in VvNHX01, VvNHX02, VvNHX03, VvNHX04 and VvNHX08. Analysis of each domain revealed that motif1, motif2, motif3, motif4, and motif6 all contained 50 conserved amino acids with high conservation. Motif7 and motif8 contained only 41 conserved amino acids, but they were highly conserved. Gene structure analysis revealed that there were significant differences in the number of exons of VvNHXs gene family members, and 8 VvNHXs genes contained introns and exons proton. There were 14 exons proton in VvNHX01, VvNHX02, VvNHX03,VvNHX05 and VvNHX08 genes, 15 exons in VvNHX04, 12 exons in VvNHX06, and 23 exons in VvNHX07. Subcellular localization predicted that grape NHX genes were mainly distributed in cell membrane, endoplasmic reticulum, mitochondria, vacuole and cytoplasm. VvNHX05 was expressed in both peroxisome and cell matrix; VvNHX07 was only expressed in chloroplast. VvNHX01 was only expressed in cell plasmids; VvNHX02 and VvNHX04 were expressed in cell membrane, endoplasmic reticulum, mitochondria, vacuole and cytoplasm; VvNHX05, VvNHX06, VvNHX07 were expressed in the nucleus. The secondary structure of the protein had Alpha helix, Beta turn and Random coil. Therefore,the secondary structure of VvNHXs was mainly composed of Alpha helix and Random coil. The gene chip analysis showed that as increase of the treatment time of salt, ABA and PEG stress the expression levels, of VvNHX02 and VvNHX06 significantly increased in grape leaves. The highest gene expression was found on VvNHX06 at 24 h after treatment. The expression level of VvNHX08 gene significantly increased under 8 h treatment, and then decreased rapidly. The expression level of the VvNHX03 gene reached a maximum at 4 h. After treatment with ABA, the expression levels of VvNHX03, VvNHX04,and VvNHX08 genes decreased as the increase of the treatment time. The results of qRT-PCR analysis showed that there was a difference in the expression of grape NHXs in the leaves with different treatments. Under different stress treatments, the VvNHXs genes families were expressed in different degrees in the leaves. The relative expressions of VvNHX01, VvNHX02, VvNHX03, VvNHX04, VvNHX05, VvNHX06 and VvNHX08 in the leaves under 200 mmol · L~(-1) NaCl were up-regulated to different degrees compared with those in the control group(CK), among which the up-regulated expressions of VvNHX06 were the most significant, 30 times higher than those in the control group(CK). After treatment with 100 mmol · L~(-1) ABA, the relative expressions of VvNHX02, VvNHX03, VvNHX06, VvNHX07 and VvNHX08 were significantly higher than those of the control group(CK). VvNHX06 showed the most significant change compared with the control group, which was 60 times higher than those in the control group. VvNHX01 and VvNHX04 were lower than those in the control group(CK). VvNHX05 did not change significantly compared with those in the control group(CK). Under 10% PEG treatment, the relative expressions of VvNHX01, VvNHX03, VvNHX05 and VvNHX08 in the leaves were up-regulated to different degrees compared with those in the control group. The upregulation of VvNHX05 was the most obvious, 5 times higher than that of the control group. VvNHX02, VvNHX04, VvNHX06 and VvNHX07 were down-regulated.【Conclusion】VvNHX05 and VvNHX06 genes could be closely related to salt tolerance and drought resistance in the grape plants, and it would provide a reference for the research on abiotic stresses to grape.
【Objective】Grape(Vitis vinifera L.) is widely grown the world. China is the second country for grape production in the world. As the increase of cultivation area, it is very important to study the resistance of grape to the abiotic stresses. NHX has the functions of maintaining Na+(K+) concentration,regulating pH in cells, controlling cell expansion, and maintaining cell turgor. NHX is a key factor related to drought and salt tolerance in plants. The bioinformatics characteristics of grape NHX genes family and its expression under abiotic stresses were surveyed to screen out family members related to abiotic stresses.【Methods】The test materials were the seedlings propagated in vitro of‘Red Globe'preserved by the College of Horticulture, Gansu Agricultural University. The single-bud stem segment of the‘Red Globe'grape was cultured on the subculture medium(MS+0.2 mg · L~(-1) IAA) for 35 d. Then the tissue culture seedlings were removed from the containers, the root agar was washed off, and they were transferred into MS liquid medium without hormones. The seedlings wer cultured in incubator at(28±0.5)℃with, a photoperiod of 16 h/8 h(light/dark). After 30 d culture, the seedlings were transfered to the MS media with 10% PEG, 100 mmol · L~(-1) ABA and 200 mmol · L~(-1) NaCl, respectively. The pure water was used as CK. Three biological replicates were set for each treatment, and the treatment duration was 6 h.And based on the NHX gene sequences of Arabidopsis thaliana, rice and poplar, homologous cloning,chromosome localization, amino acid composition, physicochemical properties, motif, protein secondary structure and gene chip expression of grape NHX gene were analyzed. Quantitative analysis of grape NHX gene family expression by qRT-PCR was performed.【Results】Grape NHXs gene could be divided into two sub families, eight members of the grape NHX gene family were obtained by comparison with the NHX genes of existing plants. According to the localization analysis, eight grape NHX genes were distributed on 6 th chromosomes, VvNHX07 was located on the first chromosome, and VvNHX02 on the 5 th chromosome, VvNHX03 on the 7 th chromosome, VvNHX01 and VvNHX08 on the 14 th chromosome, VvNHX05 and VvNHX06 on the 15 th chromosome, and VvNHX04 on the 19 th chromosome. And the number of exons was 12-23. VvNHX06 gene only had 291 amino acids. VvNHX07 gene had 1 141 amino acids. The full-length protein sequence was predicted by MEME software, and 8 domains were obtained. No conserved amino acid sequence was predicted in VvNHX07. Only motif1 was present in VvNHX05, and all 8 domains were existing in VvNHX01, VvNHX02, VvNHX03, VvNHX04 and VvNHX08. Analysis of each domain revealed that motif1, motif2, motif3, motif4, and motif6 all contained 50 conserved amino acids with high conservation. Motif7 and motif8 contained only 41 conserved amino acids, but they were highly conserved. Gene structure analysis revealed that there were significant differences in the number of exons of VvNHXs gene family members, and 8 VvNHXs genes contained introns and exons proton. There were 14 exons proton in VvNHX01, VvNHX02, VvNHX03,VvNHX05 and VvNHX08 genes, 15 exons in VvNHX04, 12 exons in VvNHX06, and 23 exons in VvNHX07. Subcellular localization predicted that grape NHX genes were mainly distributed in cell membrane, endoplasmic reticulum, mitochondria, vacuole and cytoplasm. VvNHX05 was expressed in both peroxisome and cell matrix; VvNHX07 was only expressed in chloroplast. VvNHX01 was only expressed in cell plasmids; VvNHX02 and VvNHX04 were expressed in cell membrane, endoplasmic reticulum, mitochondria, vacuole and cytoplasm; VvNHX05, VvNHX06, VvNHX07 were expressed in the nucleus. The secondary structure of the protein had Alpha helix, Beta turn and Random coil. Therefore,the secondary structure of VvNHXs was mainly composed of Alpha helix and Random coil. The gene chip analysis showed that as increase of the treatment time of salt, ABA and PEG stress the expression levels, of VvNHX02 and VvNHX06 significantly increased in grape leaves. The highest gene expression was found on VvNHX06 at 24 h after treatment. The expression level of VvNHX08 gene significantly increased under 8 h treatment, and then decreased rapidly. The expression level of the VvNHX03 gene reached a maximum at 4 h. After treatment with ABA, the expression levels of VvNHX03, VvNHX04,and VvNHX08 genes decreased as the increase of the treatment time. The results of qRT-PCR analysis showed that there was a difference in the expression of grape NHXs in the leaves with different treatments. Under different stress treatments, the VvNHXs genes families were expressed in different degrees in the leaves. The relative expressions of VvNHX01, VvNHX02, VvNHX03, VvNHX04, VvNHX05, VvNHX06 and VvNHX08 in the leaves under 200 mmol · L~(-1) NaCl were up-regulated to different degrees compared with those in the control group(CK), among which the up-regulated expressions of VvNHX06 were the most significant, 30 times higher than those in the control group(CK). After treatment with 100 mmol · L~(-1) ABA, the relative expressions of VvNHX02, VvNHX03, VvNHX06, VvNHX07 and VvNHX08 were significantly higher than those of the control group(CK). VvNHX06 showed the most significant change compared with the control group, which was 60 times higher than those in the control group. VvNHX01 and VvNHX04 were lower than those in the control group(CK). VvNHX05 did not change significantly compared with those in the control group(CK). Under 10% PEG treatment, the relative expressions of VvNHX01, VvNHX03, VvNHX05 and VvNHX08 in the leaves were up-regulated to different degrees compared with those in the control group. The upregulation of VvNHX05 was the most obvious, 5 times higher than that of the control group. VvNHX02, VvNHX04, VvNHX06 and VvNHX07 were down-regulated.【Conclusion】VvNHX05 and VvNHX06 genes could be closely related to salt tolerance and drought resistance in the grape plants, and it would provide a reference for the research on abiotic stresses to grape.
引文
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[3]郭强,孟林,李杉杉,张琳,毛培春,田小霞.马蔺NHX基因的克隆与基因表达分析[J].植物生理学报,2015,51(11):2006-2012.GUO Qiang,MENG Lin,LI Shanshan,ZHANG Lin,MAO Peichun,TIAN Xiaoxia.Cloning and gene expression analysis of marin NHX gene[J].Journal of plant physiology,2015,51(11):2006-2012.
[4]APSE M P,AHARON G S,SNEDDEN W A,EDUARDOBLUMWALD.Salt tolerance conferred by overexpression of a vacuolar Na+/H+antiport in Arabidopsis[J].Science,1999,5431(285):1256-1258.
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