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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (8): 1146-1157.doi: 10.3724/SP.J.1006.2019.84124

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

Identification and function analysis of AVP1, VHA-a2, and VHA-a3 genes in Brassica napus L.

YAO Jun-Yue1,2,HUA Ying-Peng1,2,ZHOU Ting1,2,WANG Tao1,2,SONG Hai-Xing1,2,GUAN Chun-Yun3,ZHANG Zhen-Hua1,2,*()   

  1. 1 College of Resource and Environment, Hunan Agricultural University, Changsha 410128, Hunan, China
    2 Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Changsha 410128, Hunan, China
    3 Hunan Branch, National Oil Crops Improvement Center, Changsha 410128, Hunan, China
  • Received:2018-09-20 Accepted:2019-04-15 Online:2019-08-12 Published:2019-07-16
  • Contact: Zhen-Hua ZHANG E-mail:zhzh1468@163.com
  • Supported by:
    This study was supported by the National Key R&D Program of China(2018YFD0200901);This study was supported by the National Key R&D Program of China(2018YFD0200906);the China Agriculture Research System(CARS-13)

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Abstract:

Vacuoles play an important role in regulating plant cell differentiation and growth. AVP1, VHA-a2, and VHA-a3 genes are important carrier elements for regulating the acid-base balance inside and outside the vacuole of plants and the energy provided by ion transport. In this study, nine BnaAVP1, three BnaVHA-a2, and four BnaVHA-a3 genes were identified in Brassica napus genome database and NCBI plant genome annotation database by using the known AVP1, VHA-a2, and VHA-a3 genes of Arabidopsis thaliana as reference sequences. Bioinformatics studies on copy number variation, molecular characteristics, transmembrane domain, conserved motifs, chromosome localization, phylogenetic tree construction, secondary structure and three- dimensional structure prediction of proteins and high-throughput transcriptome sequencing were carried out, indicating that the number of genes of BnaAVP1 and BnaVHA-a3 was significantly higher than that of B. oleracea and B. rapa; the AVP1, VHA-a2 and VHA-a3 proteins of B. napus belonged to a stable protein composed of acidic amino acids. The analysis of evolutionary selection ability showed that after low nitrogen treatment, BnaAVP1 genes were mainly expressed in shoots, and the expression of BnaAVP1 genes was down-regulated in shoots after three hours of low nitrogen treatment, and up-regulated in roots after 72 hours of low nitrogen treatment. BnaVHA-a2 and BnaVHA-a3 genes were expressed in both shoots and roots, while BnaVHA-a2 genes were up-regulated after 72 hours of low nitrogen treatment, and BnaVHA-a3 genes were down-regulated after three hours of low nitrogen treatment. After low phosphorus treatment, the expression of most BnaAVP1 genes was up-regulated in roots, and no difference in shoots. The expression of BnaVHA-a2 genes was almost no difference, and that of BnaVHA-a3 genes up-regulated in both shoots and roots. This result lays a foundation for further study on the biological functions of AVP1, VHA-a2, and VHA-a3 genes in B. napus and the molecular mechanism that AVP1, VHA-a2, and VHA-a3 proteins hydrolyze ATP to provide energy for plant metabolism, and provides references for other species family genetic bioinformatics studies that are known to have large amounts of data.

Key words: Brassica napus L, AVP gene, VHA gene, bioinformatics

Fig. 1

Copy number variation of AVP1, VHA-a2, and VHA-a3 genes in Brassica napus, B. rapa, and B. olerace The number at the top of the histogram is the number of genes copied for that species."

Table 1

Information of AVP1, VHA-a2, and VHA-a3 genes in Brassica napus"

基因名称
Gene name		 基因库编号
Database No.		 基因编号
Gene ID		 基因长度
CDS (bp)		 氨基酸长度
Amino acids (aa)		 分子量
MW (kD)		 理论等电点
pI		 亲水性指数
GRAVY
BnaAVP1-1 GSBRNA2T00010424001 BnaA09g56670D 2409 802 83.97 5.06 0.620
BnaAVP1-2 GSBRNA2T00033628001 BnaA06g38620D 1821 606 64.61 5.00 0.281
BnaAVP1-3 GSBRNA2T00136659001 BnaC08g38730D 3525 1174 126.46 6.29 0.269
BnaAVP1-4 GSBRNA2T00139755001 BnaC08g16730D 2310 769 80.59 5.03 0.611
BnaAVP1-5 GSBRNA2T00068861001 BnaC06g40530D 2310 769 80.61 5.00 0.574
BnaAVP1-6 GSBRNA2T00054245001 BnaC05g11930D 2316 771 80.83 5.08 0.618
BnaAVP1-7 GSBRNA2T00103071001 BnaA08g23780D 2310 769 80.52 5.03 0.616
BnaAVP1-8 GSBRNA2T00146278001 BnaA07g35600D 2310 769 80.70 5.01 0.572
BnaAVP1-9 GSBRNA2T00062272001 BnaA06g10340D 2316 771 80.83 5.08 0.621
BnaVHA-a2-1 GSBRNA2T00137010001 BnaC08g35720D 2466 821 92.97 5.61 0.060
BnaVHA-a2-2 GSBRNA2T00108093001 BnaC04g33510D 2472 823 92.96 5.61 0.056
BnaVHA-a2-3 GSBRNA2T00090255001 BnaA04g12160D 2460 819 92.80 5.56 0.059
BnaVHA-a3-1 GSBRNA2T00061106001 BnaC03g60340D 2463 820 92.85 5.86 0.041
BnaVHA-a3-2 GSBRNA2T00075109001 BnaC01g00150D 2457 818 92.50 5.80 0.080
BnaVHA-a3-3 GSBRNA2T00150888001 BnaA08g16770D 2460 819 92.79 5.75 0.043
BnaVHA-a3-4 GSBRNA2T00073949001 BnaA01g05300D 2454 817 92.32 5.66 0.071

Fig. 2

Synonymous nucleotide substitution rates (Ks) and non-synonymous nucleotide substitution rates (Ka) of the BnaAVP1, BnaVHA-a2, and BnaVHA-a3 proteins in Brassica napus L. "

Fig. 3

Chromosome localization of the BnaAVP1s, BnaVHA-a2s, and BnaVHA-a3s of Brassica napus"

Fig. 4

Distribution of conserved motifs in BnaAVP1s, BnaVHA-a2s, and BnaVHA-a3s of Brassica napus"

Fig. 5

Exon-intron structure of BnaAVP1, BnaVHA-a2, and BnaVHA-a3 of Brassica napus"

Fig. 6

Evolutionary relationships of BnaAVP1, BnaVHA-a2, and BnaVHA-a3 proteins of Brassica napus Blue represents the BnaAVP1 family, green represents the BnaVHA-a3 family, and red represents the BnaVHA-a2 family."

Fig. 7

Secondary structure analysis of BnaVHA-a2-1 protein GOR4 (A) and PSIPRED (B) are two tools for the secondary structure analysis."

Fig. 8

Prediction of three-dimensional structure of BnaAVP1, BnaVHA-a2, and BnaVHA-a3 protein A: BnaVHA-a2-1; B: BnaVHA-a3-1; C: BnaAVP1-1."

Fig. 9

Transcriptome analysis of BnaAVP1, BnaVHA-a2, and BnaVHA-a3 family genes in Brassica napus S: shoot; R: root; 0, 3, and 72 (h) represent the time course of nitrogen deficiency. The results represent the average of three biological replicates. * represent significant differences. (A): BnaVHA-a2s; (B): BnaVHA-a3s; (C): BnaAVP1s."

Fig. 10

Transcriptome analysis of BnaAVP1, BnaVHA-a2, and BnaVHA-a3 family genes in Brassica napus S: shoot; R: root; CK represent normal condition and LP represent low phosphorus stress. The results represent the average of three biological replicates. * represent significant differences. (A): BnaVHA-a2s; (B): BnaVHA-a3s; (C): BnaAVP1s."

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