Cloning of Thioredoxin Peroxidase Gene in Sugar Beet M14 Line and Its Function Responsive to Oxidative Stress in Prokaryotic Expression System

doi:10.11924/j.issn.1000-6850.casb2020-0147

Abstract

Abstract:

The purpose is to study the oxidative-resistance function of BvM14-Tpx gene. In this study, the monosomic addition line M14 with the alien No. 9 chromosome from the wild white flower sugar beet species was used as the experimental material. The cDNA full length of thioredoxin peroxidase gene (BvM14-Tpx) in sugar beet M14 line was obtained by RACE technique, and the bioinformatics analysis was carried out. The tissue-specific expression of BvM14-Tpx gene was analyzed by Real-time PCR and semi-quantitative RT-PCR. The BvM14-Tpx gene in response to oxidative stress was studied in prokaryotic expression system. The results of bioinformatics analysis showed that the total cDNA length of BvM14-Tpx gene was 1044 bp including the largest ORF 489 bp, which encoded 162 amino acids and contained the conserved domain of peroxidase Ⅱ (Prx Ⅱ). BvM14-Tpx protein had close genetic relationship with the Tpx protein from Pisum sativum L. and Medicago truncatula L.. The results of tissue specific expression analysis showed that the expression level of BvM14-Tpx gene from high to low was root, stem, leaf and flower. The research on BvM14-Tpx gene in response to oxidative stress in prokaryotic expression system showed that BvM14-Tpx gene could improve the adaptability of Escherichia coli to oxidative stress and reduce the inhibition of H₂O₂ on bacterial growth. This research is of certain significance for mining the high quality genes of sugar beet M14 line, improving the resistance of sugar beet to abiotic stress and carrying out genetic improvement of sugar beet.

Key words: sugar beet M14 line, thioredoxin peroxidase gene, bioinformatics, prokaryotic expression, oxidative stress

CLC Number:

S566.3

Zhang Tingyue, Zhao Chenxi, Chen Sixue, Li Hongli. Cloning of Thioredoxin Peroxidase Gene in Sugar Beet M14 Line and Its Function Responsive to Oxidative Stress in Prokaryotic Expression System[J]. Chinese Agricultural Science Bulletin, 2020, 36(32): 30-38.

Figures/Tables 14

References 28

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引物功能	引物名称	序列
3′ RACE	M4	5'-GTTTTCCCAGTCACGAaC-3'
3′ RACE	S	5'-CGCATCGTCTACACCCAC-3'
5′ RACE	AS	5'-TGAGCCATTTCGCCTACA-3'
BvM14-Tpx基因cDNA全长的扩增	S1	5'-AATCAGGTGGTGAATTTTCTGTCTC-3'
BvM14-Tpx基因cDNA全长的扩增	AS1	5'-ATTTCGCCTACAATGATTTGCTTTT-3'
内参基因18S rRNA的上游及下游引物	18S-F	5'-CCCCAATGGATCCTCGTTAa-3'
内参基因18S rRNA的上游及下游引物	18S-R	5'-aTGACGGAGAATTAGGGTTCG-3'
BvM14-Tpx基因组织表达分析	S2	5'-AATCGCATCGTCTACACCCACTTTT-3'
BvM14-Tpx基因组织表达分析	AS2	5'-TTTCGCCTACAATGATTTGCTTTTA-3'
原核表达载体构建扩增 BvM14-Tpx基因特异引物	NdeIs	5'- GCGCATATGTCATCACTCATTTTCTCTCTCA -3'
原核表达载体构建扩增 BvM14-Tpx基因特异引物	XhoIas	5'- GCGCTCGAGCAAGGCTTTGAGGATTTC -3'

引物功能	引物名称	序列
3′ RACE	M4	5'-GTTTTCCCAGTCACGAaC-3'
3′ RACE	S	5'-CGCATCGTCTACACCCAC-3'
5′ RACE	AS	5'-TGAGCCATTTCGCCTACA-3'
BvM14-Tpx基因cDNA全长的扩增	S1	5'-AATCAGGTGGTGAATTTTCTGTCTC-3'
BvM14-Tpx基因cDNA全长的扩增	AS1	5'-ATTTCGCCTACAATGATTTGCTTTT-3'
内参基因18S rRNA的上游及下游引物	18S-F	5'-CCCCAATGGATCCTCGTTAa-3'
内参基因18S rRNA的上游及下游引物	18S-R	5'-aTGACGGAGAATTAGGGTTCG-3'
BvM14-Tpx基因组织表达分析	S2	5'-AATCGCATCGTCTACACCCACTTTT-3'
BvM14-Tpx基因组织表达分析	AS2	5'-TTTCGCCTACAATGATTTGCTTTTA-3'
原核表达载体构建扩增 BvM14-Tpx基因特异引物	NdeIs	5'- GCGCATATGTCATCACTCATTTTCTCTCTCA -3'
原核表达载体构建扩增 BvM14-Tpx基因特异引物	XhoIas	5'- GCGCTCGAGCAAGGCTTTGAGGATTTC -3'

序号	开放阅读框分析内容	分析结果	在图5上的标注
1	全长	1044 bp	无
2	最长ORF	489 bp	无
3	编码氨基酸	162个	无
4	理论分子量	17.5 kDa	无
5	等电点	5.49	无
6	起始密码子		黑色方框
7	终止密码子		星号
8	β折叠		橙色双向箭头
9	α螺旋		蓝色横双向箭头
10	糖基化位点		黄色方框
11	蛋白激酶C磷酸化位点		蓝色方框
12	酪蛋白激酶磷酸化位点		紫色方框
13	酰基化位点		粉色方框
14	Tpx过氧化的半胱氨酸		绿色方框
15	Tpx游离的半胱氨酸		红色方框

序号	开放阅读框分析内容	分析结果	在图5上的标注
1	全长	1044 bp	无
2	最长ORF	489 bp	无
3	编码氨基酸	162个	无
4	理论分子量	17.5 kDa	无
5	等电点	5.49	无
6	起始密码子		黑色方框
7	终止密码子		星号
8	β折叠		橙色双向箭头
9	α螺旋		蓝色横双向箭头
10	糖基化位点		黄色方框
11	蛋白激酶C磷酸化位点		蓝色方框
12	酪蛋白激酶磷酸化位点		紫色方框
13	酰基化位点		粉色方框
14	Tpx过氧化的半胱氨酸		绿色方框
15	Tpx游离的半胱氨酸		红色方框