Cloning and Expression Analysis of HbRPW8 Gene from Hevea brasiliensis

doi:10.3969/j.issn.1000-2561.2022.12.001

Abstract

Abstract:

RPW8 (resistance to powdery mildew locus 8) is a broad-spectrum resistance gene locus to many plant diseases, especially powdery mildew. In this study, the cloning and expression analysis of RPW8 gene in Hevea brasiliensis were carried out to lay the foundation for the analysis of powdery mildew resistance mechanism and molecular breeding. HbRPW8 gene coding region (CDS) was cloned from rubber tree ‘Reyan 73397’ by RT-PCR. Bioinformatics analysis was carried out, and qRT-PCR method was used to determine the expression of RPW8 gene after seven hormones and powdery mildew infection at different times and disease grades. The full-length cDNA of HbRPW8 was 570 bp, encoding 189 amino acids. The molecular weight of HbRPW8 was 21.73 kDa, the isoelectric point was 9.23, the fat coefficient was 86.30, and the total average hydrophilicity index was -0.404, which was a hydrophilic protein with eight phosphorylation sites. The protein was located in the nucleus without transmembrane domain and signal peptide, and had a conserved domain of RPW8. The main components of the secondary structure of HbRPW8 protein were α-helix and random coil, accounting for 70.90% and 22.22% of the amino acid sequence, respectively. Phylogenetic relationships analysis showed that HbRPW8 gene had the highest similarity with cassava RPW8 gene. Hydrogen peroxide, salicylic acid, ethephon, gibberellin and abscisic acid could rapidly induce the expression of HbRPW8 transcript, and reached the highest level at 0.5 h after treatment, which was about 14, 6, 75, 2.5 and 4 folds of the control, respectively; methyl jasmonate treatment reached the highest level at 6 h after treatment, about 10 times that of the control; HbRPW8 transcript was significantly down-regulated after auxin treatment; with the increase of infection time and disease grade of powdery mildew, the expression level of HbRPW8 increased first and then decreased. The results of this study preliminarily showed that HbRPW8 may be involved in the resistance response mechanism of rubber tree, providing a reference for the study of rubber tree resistance breeding.

Key words: Hevea brasiliensis, HbRPW8 gene, qRT-PCR, bioinformatics, RPW8

CLC Number:

S794.1

GUAN Xin, TU Min, CAI Haibin, WANG Yunyue, HU Yanshi, ZENG Xia. Cloning and Expression Analysis of HbRPW8 Gene from Hevea brasiliensis[J]. Chinese Journal of Tropical Crops, 2022, 43(12): 2395-2404.

Figures/Tables 9

Tab. 1 Primer sequences in the study

引物名称 Primer name	上游引物序列（5′-3′） Forward primer sequence (5′-3′)	下游引物序列（5′-3′） Reverse primer sequence (5′-3′)
HbRPW8	TCCCAAGACTCTTCTGTCATCA	TGACGAGAAAAGCGAACCTACA
HbRPW8-RT	GCTTATGATGCGGGATTT	GCAGGACTGGATGGCTTA
Hb18S	GCTCGAAGACGATCAGATACC	TTCAGCCTTGCGACCATAC

Fig. 1 PCR amplification of HbRPW8 in H. brasiliensis M: DL2000 DNA marker.

Fig. 2 Nucleotide sequence and deduced amino acid sequence of HbRPW8 CDS region

Fig. 3 Prediction of signal peptide (A), transmembrane domain (B) and phosphorylation site (C) of HbRPW8

Fig. 4 Structural analysis of HbRPW8 A: Conserved domain of HbRPW8 protein; B: Prediction of secondary structure of HbRPW8 protein; C: Prediction of tertiary structure of HbRPW8 protein.

Fig. 5 Multiple sequence alignment of HbRPW8 and RPW8 from other plant species Identity of amino acid sequences with 100% was highlighted with black, identity≥75% was highlighted with pink, identity > 50% was highlighted with blue.

Fig. 6 Phylogenic tree of different plants on HbRPW8 amino acid sequences

Tab. 2 Analysis of cis-elements within HbRPW8 promoter regions in H. brasiliensis

顺式作用元件Cis-acting element	核心序列 Core sequence	数量Number	功能 Function
ABRE	ACGTG	1	参与脱落酸反应的顺式作用元件
ARE	AAACCA	2	厌氧诱导所必需的顺式作用调节元件
CAAT-box	CCAAT	19	启动子和增强子区域中常见的顺式作用元件
G-Box	CACGTT	1	参与光反应的顺式作用调节元件
GT1-motif	GGTTAA	2	光响应元件
TATA-box	TATATA/ATATAT/TATA/TATAA/ ATATAA/TATAAA/TATAAAA/ TATACA	122	转录启始元件
TATC-box	TATCCCA	1	参与赤霉素反应性的顺式作用元件
TCA-element	CCATCTTTTT	1	参与水杨酸反应的顺式作用元件
TC-rich repeats	ATTCTCTAAC	1	参与防御和应激反应的顺式作用元件
TGA-element	AACGAC	1	生长素反应元件

Fig. 7 Expression patterns of HbRPW8 under exogenous hormones and powdery mildew treatments Different lowercae letters indicate significant difference among treatments.

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