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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (7): 1871-1881.doi: 10.3724/SP.J.1006.2023.24134

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Analysis of ASPAT gene family based on drought-stressed transcriptome sequencing in Vicia faba L.

WEI Zheng-Xin1(), LIU Chang-Yan2(), CHEN Hong-Wei2, LI Li2, SUN Long-Qing2, HAN Xue-Song2, JIAO Chun-Hai2,*(), SHA Ai-Hua1,*()   

  1. 1College of Agriculture, Yangtze University / Engineering Research center of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434025, Hubei, China
    2Hubei Academy of Agricultural Sciences Institute of Food Crops / Hubei Provincial Key Laboratory of Germplasm Innovation and Genetic Improvement of Food Crops, Wuhan 430064, Hubei, China
  • Received:2022-06-03 Accepted:2022-11-25 Online:2023-07-12 Published:2022-12-01
  • Contact: *E-mail: aihuasha@163.com; E-mail: jiaoch@hotmai.com E-mail:2545439813@qq.com;Liucy0602@163.com;jiaoch@hotmai.com;aihuasha@163.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    The National Key Research and Development Program of China(2019YFD1001303);The National Key Research and Development Program of China(2019YFD1001300);The China Agriculture Research System of MOF and MARA(CARS-08);The Hubei Agricultural Science and Technology Innovation Center Project(2021-620-000-001-01)

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Abstract:

Fava beans (Vicia faba L.) is an important food bean crop, and drought can lead to lower fava bean yields. The identification of drought tolerance genes of fava beans is beneficial to breed the drought-resistant varieties through molecular marker-assisted breeding and genetic engineering. In a previous study, we identified a class of aspartate aminotransferase genes (ASPAT) in response to faba bean drought stress by transcriptome sequencing. ASPAT catalyzes the reversible reaction of transamination between aspartate and α-oxoglutarate to generate oxaloacetate and glutamate, and plays a key role in plant metabolism. Based on transcriptomic sequencing data, ASPAT members were identified in genome-wide level. The physicochemical properties, subcellular localizations, gene structures, protein domains, conserved motifs, phylogeny, protein interaction, and gene expression patterns of each member were analyzed. The results indicate that eight ASPAT genes were identified from the transcriptomes, which included three eukaryotype ASPAT (AAT) genes and five prokarytoype ASPAT (PAT) genes with mitochondria and chloroplast location, respectively. Phylogenetic analysis showed that ASPAT could be divided into two subfamilies Iα and Iβ. Iα is AAT protein including VfASPAT1-VfASPAT3, and Iβ is PAT protein consisting of VfASPAT4-VfASPAT8. ASPAT family members of Vicia faba contain motif 1, motif 3, and motif 9, and had similar gene structures and common protein domain aminotran-1-2. VfASPATs protein interaction network suggested that the members might be involved in stress metabolism. Transcriptome and qRT-PCR indicated that he expression patterns of the eight members were different at 16 hours and 64 hours under drought stress. All the members were up-regulated with the exception of VfASPAT4 and VfASPAT6, implying that VfASPATs may have a positive regulation effect on drought stress. In this study, eight ASPAT genes of Vicia faba were identified, and the structures, distribution, evolutionary relationship, and the relative expression patterns of ASPAT gene family members were analyzed. The results of this study laid a foundation for further analysis of drought resistance function of VfASPATs, and the application of ASPAT to improve drought resistance of fava bean.

Key words: Vicia faba L., aspartate aminotransferase (ASPAT), gene family, drought stress, gene expression

Table 1

Primers used for qRT-PCR"

基因Gene name 正向引物序列Forward sequence (5'-3') 反向引物序列 Reverse sequence (5'-3')
VfASPAT1 AAGGATAAACGAATAGCCGCAGT TCGGGATGATAGTAACGGAATGT
VfASPAT2 GGCTTCTCACATTTCTCCTTCTC CAACTCGCCTAACTACATCCAAC
VfASPAT3 TGAAGAATTGCAGCCGTATG TGGCAACTCTTTGCTGTTTG
VfASPAT4 ATTGAGGTGGAGCAGAACAGA CGCCAAGCGAATAACAGGAAC
VfASPAT5 GGTGATGAGGTTATTGTGTTTGC CTTTCCAGTAGGATTGTGAGGG
VfASPAT6 AAGCCCTCGCAGATCGGAACAC CCAAAAACTCCCATAGGCACAA
VfASPAT7 AAAATGGAAGGTAAACTGTCGC CCTGTACTGATTGAGGTCGGAA
VfASPAT8 CTGCAGCAACAAGGGAACAAC TGGCAACTCTTTGCTGTTTG
VfNADHD4 AGGGTTAGTGAGCACCATGC ATAGCCAAAGGGAATACGCC

Table 2

Comparison of germination rate between two faba bean varieties under drought stress (%)"

处理时间
Processing time		 鄂蚕豆1号-T
Ecandou 1-T		 CDAS105-T 鄂蚕豆1号-CK
Ecandou 1-CK		 CDAS105-CK
48 h 0 0 47.6 cd 61.9 bc
64 h 0 4.8 e 78.6 a 76.2 a
72 h 0 9.5 e 83.3 a 81.0 a
96 h 9.5 e 38.1 d 88.1 a 85.7 a

Table 3

Prediction of physicochemical properties and subcellular localization prediction of ASPAT in Vicia faba L."

蛋白质
Protein		 氨基酸数目Number of amino acids 等电点
pI		 相对分子质量
Molecular weight		 脂融指数Aliphatic index 不稳定指数Instability index 疏水性分值GARVY 亚细胞定位
Subcellular
localization
VfASPAT1 424 8.42 46,958.64 82.38 39.68 -0.261 线粒体Mitochondrion
VfASPAT2 417 7.24 45,448.99 96.91 43.76 -0.052 叶绿体Chloroplast
VfASPAT3 453 7.73 49,658.54 84.86 36.02 -0.197 叶绿体, 线粒体
Chloroplast, mitochondrion
VfASPAT4 481 7.60 52,060.70 96.36 36.72 0.039 叶绿体Chloroplast
VfASPAT5 394 6.22 43,469.05 85.91 29.62 -0.063 叶绿体Chloroplast
VfASPAT6 407 5.59 45,024.06 96.61 35.48 0.030 叶绿体Chloroplast
VfASPAT7 409 6.02 44,829.10 89.51 41.14 -0.077 叶绿体Chloroplast
VfASPAT8 459 6.56 49,923.62 79.48 50.90 -0.147 叶绿体Chloroplast

Table 4

Prediction of secondary structure of ASPAT protein in Vicia faba L. (%)"

蛋白质
Protein		 α-螺旋
Alpha helix		 β-转角
Beta turn		 延伸链
Extended strand		 无规则卷曲
Random coil
VfASPAT1 45.28 7.55 14.39 32.78
VfASPAT2 43.65 6.95 15.11 34.29
VfASPAT3 40.84 7.06 17.44 34.66
VfASPAT4 39.09 7.07 18.50 35.34
VfASPAT5 42.39 8.63 17.51 31.47
VfASPAT6 43.49 7.13 14.99 34.40
VfASPAT7 41.08 7.33 19.07 32.52
VfASPAT8 35.29 8.28 20.04 36.38

Fig. 1

Phylogenetic analysis of ASPAT protein in Vicia faba L."

Fig. 2

VfASPAT gene structure, protein domain, and conserved motif A: gene structure; B: protein domain; C: motif; D: amino acid sequence of motif."

Fig. 3

Pyridoxal phosphate binding site of ASPAT protein in faba bean and Arabidopsis thaliana The red rectangle box indicates the pyridoxal phosphate binding sites of AAT and PAT."

Fig. 4

Protein-protein network of ASPAT in Vicia faba L."

Fig. 5

Relative expression pattern of ASPAT genes in Vicia faba based on transcriptome CK1-16 /CK1-64 represents 16 h/64 h normal treatments of Ecandou 1; CK2-16/CK2-64 represents 16 h/64 h normal treatments of CDAS105; T1-16/T1-64 represents 16 h/64 h drought stress of Ecandou 1; T2-16/T2-64 represents 16 h/64 h drought stress of CDAS105."

Fig. 6

Relative expression level of VfASPAT between Ecandou 1 and CDAS105 by qRT-PCR CK-16 and CK-64 indicates the expression ratio of CDAS105/Ecandou 1 at 16 h and 64 h under normal condition; T-16 and T-64 indicates the expression ratio of CDAS105/Ecandou 1 at 16 h and 64 h under drought stress. Lowercase letters indicate significant difference in the 0.05 probability level among the treatments."

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