Chin Bull Bot ›› 2017, Vol. 52 ›› Issue (5): 568-578.doi: 10.11983/CBB16225

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Expression Analysis of β-1,3-Glucanase Gene from Winter Brassica rapa Under Low Temperature Stress

Li Ma1, Wancang Sun1,*(), Jinhai Yuan1, Zigang Liu1, Junyan Wu1, Yan Fang1, Yaozhao Xu2, Yuanyuan Pu1, Jing Bai1, Xiaoyun Dong1, Huili He1   

  1. 1Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement, Rapeseed Engineering Research Center of Gansu Province, College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China

    2College of Agronomy and Biotechnology, Hexi University, Zhangye 734000, China
  • Received:2016-11-23 Accepted:2017-03-24 Online:2017-07-10 Published:2017-09-01
  • Contact: Wancang Sun E-mail:18293121851@163.com

Abstract:

This study examined the function of β-1,3-glucanase in Brassica rapa with low temperature stress, whose encoding protein was identified by protein mass spectrometry analysis. The cDNA sequence of β-1,3-glucanase was cloned by RT-PCR of winter rapeseed from Longyou6 and Tianyou4 cultivars. Semi-quantitative PCR and real-time fluorescence quantitative PCR were used to study the expression pattern of β-1,3-glucanase in response to low temperature stress. The open reading frame of the β-1,3-glucanase gene was obtained at a length of 1 032 bp, and encoded 343 amino acid. The molecular weight was 38.102 kDa and the isoelectric point 6.63, with 93.94% amino sequence similarity to B. rapa subsp. chinensis and B. napus. The protein encoded by this gene is a hydrophilic protein with one signal peptide and two transmembrane domains. The prediction of the secondary structures indicated that β-1,3-glucanase is a protein with more α-helices. The predicted β-1,3-glucanase contains a conserved amino acid sequence corresponding to the plant Glycol-Hydro 17 superfamily. RT-PCR and semi-quantitative RT-PCR results showed that β-1,3-glucanase was upregulated in response to 4°C, and the gene was upregulated to peak in expression at -4°C; However, the expression was inhibited at lower temperature (-8°C). The β-1,3-glucanase gene cloned from the winter B. rapa cv. ‘Longyou6’ might play a role in cold tolerance.

Key words: β-1, 3-glucanase gene, low temperature, molecular cloning, expression analysis

Figure 1

Apoplast protein map of Brassica rapa cv. ‘Long- you6’ leavesM: Protein marker; 1: Subtilisin-like protease; 2: Chloroplast heat shock protein 70-1; 3: Myrosinase; 4: S-adenosyl-L- homocystein hydrolase; 5: Basic glucanase; 6: β-1,3-gluca- nase; 7: Pathogenesis- related protein-1"

Table 1

Mass spectrometry results of apoplast proteins in Brassica rapa cv. ‘Longyou6’ leaves"

Spot
No.
NCBI accession
No.
Protein name Organism Score Sequence coverage
(%)
1 gi|757534 Subtilisin-like protease Arabidopsis thaliana 146 3
2 gi|166919370 Chloroplast heat shock protein 70-1 Ipomoea nil 291 6
3 gi|11034734 Myrosinase Raphanus sativus 98 6
4 gi|32967699 S-adenosyl-L-homocystein hydrolase A. thaliana 290 29
5 gi|118763538 Basic glucanase Brassica juncea 315 53
6 gi|62361691 β-1,3-glucanase B. rapa subsp. 348 48
7 gi|722274 Pathogenesis-related protein-1 B. juncea 78 41

Figure 2

Electrophoresis results of bacterial PCRM: DNA marker III; 1: Bacterial PCR product"

Figure 3

Nucleotide sequence and deduced amino acid sequence of β-1,3-glucanase cDNAAn upstream in-flame start codon ATG is boxed; * indicates the positions of termination codon TAA."

Figure 4

Bioinformatic analysis of β-1,3-glucanase protein in Brassica rapa cv. ‘Longyou6’(A) Predicted protein hydrophobicity by ProtScale; (B) Predicted signal peptide by Signal P4; (C) Transmembrane helical segments predicted by TMpred; (D) Predication of domains; (E) Predicted secondary structure; (F) Predicted 3-D structure"

Table 2

Predicted physicochemical properties of β-1,3-glucanase gene encoding proteins in cruciferae plants"

Cruciferous plants No. of
amino
acids
Molecular
weight
(kDa)
Theoretical
isoelectric
point
Protein instability
index
Grand average of
hydropathicity
Composition of amino
acid
The main amino acid (%)
Longyou6 343 38.102 6.63 39.58 -0.334 20 Asn (9.6), Leu (8.7)
Tianyou4 343 38.130 7.68 39.02 -0.337 20 Asn (9.6), Leu (8.7)
Brassica rapa 342 38.180 7.69 42.99 -0.370 20 Asn (9.6), Leu (8.8)
B. rapa subsp. chinensis 363 40.659 9.27 39.84 -0.335 20 Asn (8.8), Leu (8.3)
B. napus 363 40.599 9.22 39.96 -0.325 20 Asn (9.1), Leu (8.3)
B. oleracea 351 38.925 6.85 40.48 -0.304 20 Asn (8.8), Ser (8.5)
B. oleracea var. oleracea 365 41.075 9.27 37.84 -0.390 20 Asn (8.8), Leu (7.7)
B. juncea 346 38.087 9.13 38.03 -0.188 20 Gly (9.5), Leu (9.0)
Raphanus sativus 345 38.243 6.20 40.33 -0.326 20 Leu (10.1), Asn (8.4)
Arabidopsis thaliana 355 39.380 5.47 47.15 -0.318 20 Ser (9.3), Leu (8.7)

Figure 5

Phylogenetic analysis of Longyou6 β-1,3-glucanase protein"

Figure 6

Sequence comparison of the β-1,3-glucanase proteins"

Figure 7

Semi-quantitative RT-PCR analysis of β-1,3-glucanase gene in winter Brassica rapa under low temperature stress(A) Longyou6; (B) Tianyou4"

Figure 8

The relative expression level of β-1,3-glucanase gene in leaves of winter Brassica rapa under low temperature stress"

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