Expression and Function of an ACC Oxidase Gene (HbACO7) from the Bark of Hevea brasiliensis

doi:10.3969/j.issn.1000-2561.2021.09.001

Abstract

Abstract:

Natural rubber is an important industrial raw material, mainly harvested from the rubber-producing laticifers residing in the bark of rubber tree (Hevea brasiliensis). Previous studies reveal that ethylene promotes both latex flow and regeneration, and the application of ethrel (an ethylene generator) or ethylene on bark could stimulate latex yield. In this study, based on the Hevea genome and transcriptome database we previously constructed, eight HbACO genes encoding the Hevea ACC oxidase (ACO), a key enzyme in ethylene biosynthesis, were cloned and examined for their expression patterns in seven Hevea tissues. Although the HbACO genes exhibited varying degrees of redundant expression in the seven tissues examined, each retained a distinct pattern of tissue expression. Of the eight HbACOs, HbACO7 showed the highest expression in the bark. The expressions of HbACO7 fluctuated greatly with the change of tapping months, showing the highest level in August. The prokaryotic expression vector HbACO7 was successfully constructed, and the expression of HbACO7 protein was induced in E. coli BL21. The purified HbACO7 recombinant protein successfully catalyzed ethylene synthesis in vitro, indicating its functionality as an active ACO enzyme. This study would lay a foundation for further research on the regulatory mechanism of ethylene biosynthesis in the bark of rubber tree.

Key words: Hevea brasiliensis, ethylene, biosynthesis, HbACO7, enzyme activity

CLC Number:

S794.1

WANG Yiwei, FENG Renjun, HUANG Yacheng, LIU Xiaodong, FANG Yongjun, LUO Hongli, TANG Chaorong. Expression and Function of an ACC Oxidase Gene (HbACO7) from the Bark of Hevea brasiliensis[J]. Chinese Journal of Tropical Crops, 2021, 42(9): 2435-2442.

Figures/Tables 10

References 30

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基因名称 Gene name	Scaffold序列号Scaffold number	CDS长度 CDS length/bp
HbACO1	scaffold0113_1714149	939
HbACO2	scaffold1057_27974	957
HbACO3	scaffold2408_724	957
HbACO4	scaffold0371_1076732	936
HbACO5	scaffold0611_59152	936
HbACO6	scaffold1057_68935	927
HbACO7	scaffold0894_322678	921
HbACO8	scaffold0075_154737	939

基因名称 Gene name	Scaffold序列号Scaffold number	CDS长度 CDS length/bp
HbACO1	scaffold0113_1714149	939
HbACO2	scaffold1057_27974	957
HbACO3	scaffold2408_724	957
HbACO4	scaffold0371_1076732	936
HbACO5	scaffold0611_59152	936
HbACO6	scaffold1057_68935	927
HbACO7	scaffold0894_322678	921
HbACO8	scaffold0075_154737	939

引物名称 Primer name	引物序列（5°-3°） Primer sequence (5°-3°)	用途 Purpose
HbACO1-F	ACTTCATTTCATACACATCAGAGACA	CDS扩增
HbACO1-R	CTTAGGACAATTATATCCCAAAGGTG
HbACO2-F	GCTGTCACCATGGCAAAGATCAAA
HbACO2-R	CAGAGAACAAACTGATGACAAAGA
HbACO3-F	ATGGAGTTTCCAGTAATTAACCTT
HbACO3-R	TTAAGCTGTTGCAATAGGACCCCA
HbACO4-F	TCTCTCTTAATTGTATTCCTTTCCTA
HbACO4-R	CTTTCAGGATTATTGAGGCAAATCTT
HbACO5-F	ATGGCAATTCCAGTGATTGAT
HbACO5-R	TCACACAGCCCTCACAGCTTG
HbACO6-F	ATGGAGTTCCCGGTCATTAAC
HbACO6-R	AGCTGTAGCAATGGGAACCAA
HbACO7-F	ACAATCTAAACTAGAAAGAAGAGAAGG
HbACO7-R	AAGGAAGAATTATTGGAGCTTGAGAAT
HbACO8-F	AGAAAATGGAGACCTTCCCTGTTA
HbACO8-R	CACCACTAATCAGTAAACTCATCA
M13-F	TGTAAAACGACGGCCAGT	菌落PCR
M13-R	CAGGAAACAGCTATGACC
pNC-ET- HbACO7-F	CCTGGTGCCGCGCGGCAGCATGGAGATACCAGTGATTGATTTTAGTG	原核表达
pNC-ET- HbACO7-R	GGTCTCAGCAGACCACAAGTTTAGACATAGAGACCGTTGTGCCC
T7	TAATACGACTCACTATAGGG	原核表达检测
T7 ter	GCTAGTTATTGCTCAGCGG	原核表达检测
UBC2a-F	CATTTATGCGGATGGAAGCA	qRT-PCR 分析 (内参基因)
UBC2a-R	CAGGGGAGTTTGGATTTGGA	qRT-PCR 分析 (内参基因)
HbACO7- qP-F	TGAGAACAGGAGCAAAATGATGG	qRT-PCR 分析 (HbACO7)
HbACO7- qP-R	TTCACCAACTGCTTTACTTTCTCC	qRT-PCR 分析 (HbACO7)

引物名称 Primer name	引物序列（5°-3°） Primer sequence (5°-3°)	用途 Purpose
HbACO1-F	ACTTCATTTCATACACATCAGAGACA	CDS扩增
HbACO1-R	CTTAGGACAATTATATCCCAAAGGTG
HbACO2-F	GCTGTCACCATGGCAAAGATCAAA
HbACO2-R	CAGAGAACAAACTGATGACAAAGA
HbACO3-F	ATGGAGTTTCCAGTAATTAACCTT
HbACO3-R	TTAAGCTGTTGCAATAGGACCCCA
HbACO4-F	TCTCTCTTAATTGTATTCCTTTCCTA
HbACO4-R	CTTTCAGGATTATTGAGGCAAATCTT
HbACO5-F	ATGGCAATTCCAGTGATTGAT
HbACO5-R	TCACACAGCCCTCACAGCTTG
HbACO6-F	ATGGAGTTCCCGGTCATTAAC
HbACO6-R	AGCTGTAGCAATGGGAACCAA
HbACO7-F	ACAATCTAAACTAGAAAGAAGAGAAGG
HbACO7-R	AAGGAAGAATTATTGGAGCTTGAGAAT
HbACO8-F	AGAAAATGGAGACCTTCCCTGTTA
HbACO8-R	CACCACTAATCAGTAAACTCATCA
M13-F	TGTAAAACGACGGCCAGT	菌落PCR
M13-R	CAGGAAACAGCTATGACC
pNC-ET- HbACO7-F	CCTGGTGCCGCGCGGCAGCATGGAGATACCAGTGATTGATTTTAGTG	原核表达
pNC-ET- HbACO7-R	GGTCTCAGCAGACCACAAGTTTAGACATAGAGACCGTTGTGCCC
T7	TAATACGACTCACTATAGGG	原核表达检测
T7 ter	GCTAGTTATTGCTCAGCGG	原核表达检测
UBC2a-F	CATTTATGCGGATGGAAGCA	qRT-PCR 分析 (内参基因)
UBC2a-R	CAGGGGAGTTTGGATTTGGA	qRT-PCR 分析 (内参基因)
HbACO7- qP-F	TGAGAACAGGAGCAAAATGATGG	qRT-PCR 分析 (HbACO7)
HbACO7- qP-R	TTCACCAACTGCTTTACTTTCTCC	qRT-PCR 分析 (HbACO7)

基因名称 Gene name	序列来源 Sequence source	CDS度 CDS length /bp	序列比对 Sequence alignment
基因名称 Gene name	序列来源 Sequence source	CDS度 CDS length /bp	单个碱基差别 Nucleobase difference	氨基酸差别 Amino acid difference
HbACO1	本文克隆	939	5	5
HbACO1	基因组预测	939	5	5
HbACO2	本文克隆	957	无	无
HbACO2	基因组预测	957	无	无
HbACO3	本文克隆	956	1	无
HbACO3	基因组预测	956	1	无
HbACO4	本文克隆	937	2	1
HbACO4	基因组预测	937	2	1
HbACO5	本文克隆	936	2	1
HbACO5	基因组预测	936	2	1
HbACO6	本文克隆	927	1	无
HbACO6	基因组预测	927	1	无
HbACO7	本文克隆	921	无	无
HbACO7	基因组预测	921	无	无
HbACO8	本文克隆	939	1	无
HbACO8	基因组预测	939	1	无