Cloning and Expression of a Chitinase Gene SgGH19-1 from Stylosanthes and Its Enzymatic Properties Analysis

doi:10.3969/j.issn.1000-2561.2019.12.014

Abstract

Abstract:

Chitin is a major component of the cell wall of fungi, exoskeleton of insects, and crustacean shells. Chitinase is one of the main hydrolase that catalyzes the degradation of chitin. Induction of PR-3 chitinase protein accumulation is an adaptive mechanism for plants to enhance the resistance to fungal diseases. Stylo (Stylosanthes spp.) is an important tropical forage legume. Although anthracnose caused by Colletotrichum gloeosporioides is one of the most destructive fungal diseases of stylo, little information is available regarding the responses of stylo chitinase during the pathogen infection. In the study, the PR-3 chitinase of stylo was characterized, and its expression pattern and biochemical properties were also analyzed. The results showed that a chitinase gene of PR-3 group in stylo was the cloned by homologous gene sequence method, and its coding region was 984 bp. The gene belongs to the Class I chitinase of the glycoside hydrolase 19 family, and was named SgGH19-1. Quantitative PCR analysis showed that the expression level of SgGH19-1 gene in the leaves of stylo was significantly increased after C. gloeosporioides infection, and with the increase of chitinase activity. Subsequently, the recombinant protein of SgGH19-1 was expressed and purified in Escherichia coli. Biochemical properties of SgGH19-1 showed both exochitinase and endochitinase activities, and the activities of endochitinase was 9.1 fold higher than that of exochitinase. Furthermore, the optimum pH and temperature for SgGH19-1 activity was 5.0 and 40 ℃ respectively. Taken together, SgGH19-1 is a chitinase that involved in the response of stylo to C. gloeosporioides attack. These results herein suggest that SgGH19-1 could potentially be employed as a new marker gene for developing cultivars of stylo with great tolerance to anthracnose.

Key words: chitin, chitinase, Stylosanthes, protein purification, enzymatic property

CLC Number:

S541

LIU Pandao,WU Xique,LUO Jiajia,XU Wenrong,LIU Guodao. Cloning and Expression of a Chitinase Gene SgGH19-1 from Stylosanthes and Its Enzymatic Properties Analysis[J]. Chinese Journal of Tropical Crops, 2019, 40(12): 2411-2417.

Figures/Tables 7

Tab. 1 List of primers used in the study

引物名称Primer name	引物序列Primer sequence (5′-3′)
SgPR-3-EST-F	CATGAAACCACAGGAGGATGG
SgPR-3-EST-R	ACATTCAAGCCCGCCGTTAAT
SgPR-3-5′RACE-R	TGTTAGTGATCACTCCATATCCAGG
SgPR-3-3′RACE-F	AAGCAGATTATTGTACTCCTTCCCA
RACE-UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
RACE-NUP	AAGCAGTGGTATCAACGCAGAGT
SgPR-3-ORF-F	ATGGGATACACAAAAATTTCTCATC
SgPR-3-ORF-R	TTAAAATGACTTTTGATTGTTGCAA
SgGH19-1-RT-F	ACCACCAAAGCTGAACAATGTG
SgGH19-1-RT-R	AATGCTACCAACATCACCACCG
SgEF-1a-F	CACTTCAGGACGTGTACAAGATC
SgEF-1a-R	CTTGGAGAGCTTCATGGTGCA
SgGH19-1-GST-F	GGGGCCCCTGGGATCCATGGGATACACAAAAATTTCTC
SgGH19-1-GST-R	GGGAATTCGGGGATCCTTAAAATGACTTTTGATTGTTGC

Fig. 1 Full length cloning of SgGH19-1 M: DL2000 Marker; 1: EST of SgGH19-1; 2: PCR product of 5′ terminal; 3: PCR product of 3′ terminal; 4: ORF of SgGH19-1.

Fig. 2 Phylogenetic tree analysis of SgGH19-1 with chitinase proteins in Arabidopsis thaliana and Oryza sativa The first two letters of each PAP protein label represent the abbreviated species name. At: Arabidopsis thaliana; Os: Oryza sativa; Sg: Stylosanthes guianensis.

Fig. 3 Influence of C. gloeosporioides infection on SgGH19-1 expression in stylo leaves Different lowercase letters indicate significant difference (P<0.05).

Fig. 4 Effect of C. gloeosporioides infection on chitinase activity of stylo leaves Different lowercase letters indicate significant difference (P<0.05).

Fig. 5 Purification and biochemical properties of recombinant SgGH19-1 protein M: Protein Marker (10-120 kDa); A: SDS-PAGE analysis of GST:SgGH19-1 protein in E. coli. 1: Total E. coli protein without IPTG induction; 2: Total E. coli protein induced by IPTG; 3: Purified recombinant SgGH19-1 protein after once elution; 4: Purified recombinant SgGH19-1 protein after twiceelution. B: Enzyme activity analysis of SgGH19-1 protein, the different lowercase letters indicate significant difference (P<0.05).

Fig. 6 Effect of pH and temperature on endochitinase activity of SgGH19-1 protein

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