异源表达盐单胞属(Halomonas)周质磷酸盐结合蛋白基因PBP降低转基因烟草砷积累研究

doi:10.11924/j.issn.1000-6850.casb2023-0114

中国农学通报 ›› 2024, Vol. 40 ›› Issue (12): 70-79.doi: 10.11924/j.issn.1000-6850.casb2023-0114

所属专题：烟草种植与生产

异源表达盐单胞属(Halomonas)周质磷酸盐结合蛋白基因PBP降低转基因烟草砷积累研究

黄仁权¹^,²(), 袁婷³, 宋拉拉⁴, 秦利军¹()

¹ 贵州大学山地植物资源与种质创新教育部重点实验/农业生物工程研究院/生命科学学院，贵阳 550025
² 海南大学热带农林学院，海口 570228
³ 贵州农业职业学院，贵阳 550014
⁴ 贵州省农业科学院辣椒研究所，贵阳 550006

收稿日期:2023-02-23 修回日期:2023-05-15 出版日期:2024-04-25 发布日期:2024-04-22
通讯作者:
秦利军，男，1982年出生，贵州遵义人，副教授，博士，主要从事植物逆境胁迫研究。通信地址：550025 贵州省贵阳市花溪区贵州大学南校区，Tel：0851-85360812，E-mail：leequine_chin@126.com。
作者简介:
黄仁权，男，1991年出生，贵州从江人，在读博士，主要从事植物次生代谢研究。通信地址：550025 贵州省贵阳市花溪区贵州大学南校区，Tel：0851-85360811，E-mail：1747920941@qq.com。
基金资助:
贵州省科技计划项目“异源表达盐单胞菌(Halomonas salina)磷酸盐ABC转运蛋白基因HsPbp降低烟草As积累研究”(黔科合基础[2020]1Y063)

Reduction of As Accumulation by Heterologous Expression of Halomonas Peripheral Phosphate-binding Protein Gene PBP in Transgenic Tobaccos

HUANG Renquan¹^,²(), YUAN Ting³, SONG Lala⁴, QIN Lijun¹()

¹ Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education/ Institute of Agro-Bioengineering and College of Life Sciences, Guizhou University, Guiyang 550025
² College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228
³ Guizhou Vocational College of Agriculture, Guiyang 550014
⁴ Institute of Pepper Science, Guizhou Academy of Agricultural Sciences, Guiyang 550006

Received:2023-02-23 Revised:2023-05-15 Published:2024-04-25 Online:2024-04-22

摘要/Abstract

摘要：

为探讨PBP基因在异源烟草中的生物学功能，筛选出低砷(As)积累亲本烟草种质。以转PBP基因烟草T₂代种子和野生型烟草(Nicotiana tabacum K326)种子为材料，利用Real-time PCR分析外源PBP的时空表达模式，测定As胁迫下转基因烟株抗氧化酶(AOEs)活性变化及分析转基因烟草株系中As积累差异。AOEs活性测定表明，As胁迫第5天时CK植株中SOD和POD酶活性达到极值，分别为80.25 U/g和152.02 U/g，显著高于转基因植株；在胁迫后期(3~5 d) CK植株中H₂O₂和MDA含量也显著高于转基因植株。另外，在第7天和14天时转基因植株中As含量比CK植株均显著降低。离子转运蛋白基因表达分析表明，As胁迫不仅诱导转基因植株PBP基因表达显著上调，而且也引起转基因和CK植株中HAK1和PHT4等通道蛋白基因的显著上调，且CK植株中HAK1和PHT4的平均表达水平为转基因植株的1.5倍和3.75倍。外源PBP基因导入可有效降低转基因烟草对As的积累。

关键词: 盐单胞属, 周质磷酸盐结合蛋白, PBP基因, 转基因烟草, 低As积累

Abstract:

In order to explore the biological function of PBP gene in transgenic tobaccos and select the low arsenic (As) accumulation parent tobacco germplasm, taking T₂ generation seeds of transgenic tobacco with PBP gene and wild-type tobacco (Nicotiana tabacum K326) seeds as materials, Real-time PCR was used to study the spatial and temporal expression profile of exogenous PBP in transgenic tobaccos, and the activities of antioxidant enzymes (AOEs) in transgenic tobacco lines under As stress were also determined. AOEs activity showed that SOD and POD activities reached the maximum value of 80.25 U/g and 152.02 U/g in CK plants at 5 d after As stress, which were significantly higher than those in transgenic plants. Also, H₂O₂ and MDA contents in CK plants were significantly higher than those in transgenic plants at the later stage of As stress (3-5 d). Additionally, at 7 d and 14 d after stress, the content of As in transgenic tobacco plants was significantly reduced. Based on the expression profiling of ion transporter genes, the results showed that As stress not only induced significant up-regulation of PBP in transgenic plants, but also caused significant up-regulation of channel protein genes such as HAK1 and PHT4 in transgenic and CK plants, and the average expression levels of HAK1 and PHT4 in CK were 1.5 and 3.75 times of that in transgenic plants. The introduction of exogenous PBP gene can effectively reduce the accumulation of As in transgenic tobaccos.

Key words: Halomonas, periplasmic phosphate-binding protein, PBP gene, transgenic tobacco, low-As accumulation

黄仁权, 袁婷, 宋拉拉, 秦利军. 异源表达盐单胞属(Halomonas)周质磷酸盐结合蛋白基因PBP降低转基因烟草砷积累研究[J]. 中国农学通报, 2024, 40(12): 70-79.

HUANG Renquan, YUAN Ting, SONG Lala, QIN Lijun. Reduction of As Accumulation by Heterologous Expression of Halomonas Peripheral Phosphate-binding Protein Gene PBP in Transgenic Tobaccos[J]. Chinese Agricultural Science Bulletin, 2024, 40(12): 70-79.

图/表 8

参考文献 32

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基因	GenBank登录号	引物序列(5’→3’)
Actin	AB158612	GATCTTGCTGGTCGTGATCT ACTTCCGGACATCTGAACCT
PBP	CP016490.1	AATTCAAGGAGCTGGATCTGG CTCATAGCTCTAGACATTGGTCC

基因	GenBank登录号	引物序列(5’→3’)
Actin	AB158612	GATCTTGCTGGTCGTGATCT ACTTCCGGACATCTGAACCT
PBP	CP016490.1	AATTCAAGGAGCTGGATCTGG CTCATAGCTCTAGACATTGGTCC

基因	GenBank登录号	引物序列(5’→3’)
Actin	AB158612	GATCTTGCTGGTCGTGATCT ACTTCCGGACATCTGAACCT
PBP	CP016490.1	AATTCAAGGAGCTGGATCTGG CTCATAGCTCTAGACATTGGTCC
HAK1	DQ841950	ATCCACACCGAGCTTGTTTCAGGA TGGGTCCAATTCTTCCCACCAAGA
HMA	HF675181.1	TTCAAAAGCAGCAACGTCCG GCATCGCGGTTTTGATACCC
PHT4	AB042956.1	CATGGGAATTCGTTCGTCGC TCTTTATGCCGATACCCGCC
ATQ1	KF938567.1	CAAGTGGCATCGGTTTGCAA CAATCTTGAGGTCTCCCGGG

基因	GenBank登录号	引物序列(5’→3’)
Actin	AB158612	GATCTTGCTGGTCGTGATCT ACTTCCGGACATCTGAACCT
PBP	CP016490.1	AATTCAAGGAGCTGGATCTGG CTCATAGCTCTAGACATTGGTCC
HAK1	DQ841950	ATCCACACCGAGCTTGTTTCAGGA TGGGTCCAATTCTTCCCACCAAGA
HMA	HF675181.1	TTCAAAAGCAGCAACGTCCG GCATCGCGGTTTTGATACCC
PHT4	AB042956.1	CATGGGAATTCGTTCGTCGC TCTTTATGCCGATACCCGCC
ATQ1	KF938567.1	CAAGTGGCATCGGTTTGCAA CAATCTTGAGGTCTCCCGGG

株系	胁迫处理前		胁迫处理7 d		胁迫处理14 d
株系	As含量/(mg/kg)	P含量/(mg/kg)	As含量/(mg/kg)	P含量/(mg/kg)	As含量/(mg/kg)	P含量/(mg/kg)
CK	1.102±0.15a	0.158±0.01c	4.562±0.86a	0.152±0.01b	9.508±1.02a	0.165±0.01c
L3	1.011±0.12a	0.116±0.02d	3.215±0.77b	0.127±0.03c	5.071±0.87b	0.139±0.01d
L4	0.975±0.05a	0.187±0.01a	2.226±0.82c	0.204±0.01a	4.534±0.94c	0.239±0.03a
L5	1.004±0.14a	0.154±0.03c	3.031±0.88b	0.162±0.01b	4.823±0.87c	0.180±0.01b
L10	0.885±0.02a	0.175±0.02b	1.534±0.04d	0.192±0.02a	1.647±0.06f	0.228±0.02a
L13	0.972±0.01a	0.173±0.01b	1.305±0.13e	0.206±0.02a	2.095±0.23e	0.195±0.01a
L15	1.031±0.16a	0.152±0.02c	2.217±0.86c	0.158±0.02b	4.223±0.59c	0.174±0.02b
L21	0.875±0.04a	0.122±0.01d	1.223±0.08e	0.132±0.01c	3.524±0.44d	0.154±0.01d
L25	0.952±0.01a	0.141±0.01c	1.676±0.17d	0.167±0.03b	1.821±0.78e	0.182±0.02b

异源表达盐单胞属(Halomonas)周质磷酸盐结合蛋白基因PBP降低转基因烟草砷积累研究

Reduction of As Accumulation by Heterologous Expression of Halomonas Peripheral Phosphate-binding Protein Gene PBP in Transgenic Tobaccos

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