Sequence Characteristics of Laccase Gene Cglac3 and Its Expression in Two Infection-Related Gene Mutants from Colletotrichum gloeosporioides on Mango

doi:10.3969/j.issn.1000-2561.2020.06.019

Abstract

Abstract:

In this experiment, a laccase gene Cglac3 was cloned by homologously from Colletotrichum gloeosporioides, which is the fungal pathogen of mango anthracnose, and its expressions during infection process, in laccase gene Cglac1 and exoglucanase gene CgCBH1 deletion knockout mutants, were comparatively analyzed using real-time fluorescent quantitative PCR. Cglac3 was 1842 bp in size with three introns of 56, 51 and 58 bp respectively. The open reading frame of Cglac3 encoded 558 amino acids with a protein molecular weight of 61.38 kDa and an isoelectric point (PI) of 4.50. Cglac3 was highly expressed at 6 h after inoculation when the fungal spores were germing and forming appressoria, increased rapidly at 12 h after inoculation when the fugus were invading. Since then, the expresson of Cglac3 fluctuated during hyphal expansion and leaf necrosis, but both were much higher than the expression level at 0 h. It suggested that Cglac3 played an important role during C. gloeosporioides infection, might be a key virulent factor. Compared with the wild type, the expression of Cglac3 decreased by 74% in the C. gloeosporioides ΔLAC1 mutant, and decreased by 56% in the ΔCgCBH1 mutant. Interestingly, the expression of LAC1 was also decreased by 68% in the ΔCgCBH1 mutant, consistent with the trend of Cglac3 expression, indicating that LAC1 affected the expression of Cglac3, and CgCBH1 affected both the expressions of Cglac3 and LAC1.

Key words: mango, laccase, Cglac3 gene, qRT-PCR

CLC Number:

S432
Q78

ZHONG Changkai,XIAO Chunli,ZHANG He,PU Jinji,WU Qiuyu,LIU Yanli,LIU Xiaomei. Sequence Characteristics of Laccase Gene Cglac3 and Its Expression in Two Infection-Related Gene Mutants from Colletotrichum gloeosporioides on Mango[J]. Chinese Journal of Tropical Crops, 2020, 41(6): 1202-1207.

Figures/Tables 7

Tab. 1 Main primer information in the study

目标片段 Target fragment	引物名称 Primer name	引物序列（5′-3′） Primer sequence (5′-3′)	预期长度 Sequence length/bp	退火温度 Annealing temperature/℃
5lac3	5lac3-F1	CCGTCTGTCATCGTGGAGCAG	1540	59.4
5lac3	5lac3-R1	TCGTTGGAAGAGCAGGTGAGC	1540	59.4
3lac3	3lac3-F1	GATGACGCGACGTGCAGATAA	1493	57.2
3lac3	3lac3-R1	TGAGGCTGGAAAGTCCTACCG	1493	57.2
Rlac3	Rlac3-F1	CTCCTCTTCCCTGCCTTGACG	1662	59.4
	Rlac3-R1	GAGGGCGTAGGCGTTCCAGTT	1662	59.4

Fig. 1 Amplification of DNA and cDNA fragment of laccase gene Cglac3 M: DL2000 DNA Marker; 1: PCR product of 5lac3; 2: PCRproduct of 3lac3; 3: PCR product of Rlac3.

Fig. 2 Secondary structure prediction for protein Cglac3 Blue letters: α-helix; Red letters: Extended strand; Green letters: β-turn; Yellow letters: Random coil.

Fig. 3 Phylogenetic tree of predicted protein Cglac3 and other fungal laccases The name of phylogenetic tree consists of the species name, the gene name and the landing number of GenBank. The number on the branch is bootstrap. Cglac3 is marked with a black triangle.

Fig. 4 Conserved domains of predicted protein Cglac3

Tab. 2 Expression of Cglac3 gene in process of C. gloeosporioides infecting leaves

项目 Item	时间/h
项目 Item	0	6	12	24	36	48	72
相对表达量	1.00^Gg	11.22^Cc	15.03^Aa	8.56^Ff	9.67^Ee	12.79^Bb	10.36^Dd

Fig. 5 Expression of Cglac3 gene in process ofC. gloeosporioides infecting leaves (A) and in mutant ΔLAC1 and ΔCgCBH1 (B)

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