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作物学报 ›› 2024, Vol. 50 ›› Issue (2): 394-402.doi: 10.3724/SP.J.1006.2023.34064

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

磷转运蛋白StPHO1.2提高马铃薯耐热性

李万1,*(), 李成2, 程敏3, 吴芳4   

  1. 1商洛学院生物医药与食品工程学院, 陕西商洛 726000
    2西安润壮稼农业科技有限公司, 陕西西安 710003
    3商洛学院科技处, 陕西商洛 726000
    4商洛学院国内合作与校友工作处, 陕西商洛 726000
  • 收稿日期:2023-03-24 接受日期:2023-06-29 出版日期:2024-02-12 网络出版日期:2023-07-20
  • 通讯作者: *李万, E-mail: 599092122@qq.com
  • 基金资助:
    陕西省自然科学基础研究计划资助项目(2022JQ-223);陕西省高校科协青年人才托举计划项目(20220213);商洛学院博士启动项目(21SKY112);陕西省科技创新团队项目(2022TD-56)

Phosphorus transporter StPHO1.2 improving heat tolerance in potato

LI Wan1,*(), LI Cheng2, CHENG Min3, WU Fang4   

  1. 1School of Biomedicine and Food Engineering, Shangluo University, Shangluo 726000, Shaanxi, China
    2Xi’an Runzhuangjia Agricultural Technology Co., Ltd, Xi’an 710003, Shaanxi, China
    3Science and Technology Division, Shangluo University, Shangluo 726000, Shaanxi, China
    4Domestic Cooperation and the Schoolfellow Bureau, Shangluo University, Shangluo 726000, Shaanxi, China
  • Received:2023-03-24 Accepted:2023-06-29 Published:2024-02-12 Published online:2023-07-20
  • Contact: *E-mail: 599092122@qq.com
  • Supported by:
    Natural Science Basic Research Program of Shaanxi(2022JQ-223);Young Talent fund of University Association for Science and Technology in Shaanxi, China(20220213);Research Fund for the Doctoral Program of Shangluo University(21SKY112);Science and Technology Innovation Team Project of Shaanxi(2022TD-56)

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摘要：

植物通过磷转运蛋白吸收和转运磷元素, 充足的磷元素能够提高作物产量、品质和抗逆性。高温是影响马铃薯生长发育的重要环境因子, 严重时会造成减产甚至绝收。本研究利用农杆菌介导, 在马铃薯(Solanum tuberosum L.)中过表达磷转运蛋白基因StPHO1.2, 比较转基因株系和野生型株系在高温(35℃)和常温(22℃±1℃)环境下的生长状况。结果表明, 过表达StPHO1.2能够提高马铃薯耐热性, 促进其生长, 且磷元素浓度越高, 抗性越强, 长势越好。本氏烟草中的亚细胞定位结果显示, StPHO1.2在细胞膜上表达, 因此, 选择膜系统文库质粒筛选StPHO1.2的互作蛋白。通过酵母双杂交试验和BiFC试验, 本研究证明磷转运蛋白StPHO1.2与钙离子转运相关蛋白(StCAX1)和光系统II蛋白亚基(StPsbR)均有相互作用。综上所述, 过表达StPHO1.2可能通过影响光合作用和信号转导, 从而提高马铃薯耐热性, 促进马铃薯生长。这些结果为深入理解磷转运蛋白的功能提供了理论依据和参考, 对马铃薯新品种的选育具有促进作用。

关键词: 马铃薯, 磷转运蛋白, 高温胁迫, 酵母双杂交, 双分子荧光互补

Abstract:

Plants absorb and transport phosphorus through phosphorus transporters, and sufficient phosphorus can improve crop yield, quality, and stress resistance. High temperature is an important environmental factor affecting the growth and development of potato. In this study, we overexpressed the phosphorus transporter StPHO1.2 in potato mediated by Agrobacterium tumefaciens, and then compared the growth of transgenic strains and wild-type strains at high temperature (35℃) and normal temperature (22℃±1℃). The results showed that overexpression of StPHO1.2 could improve the heat resistance and promote the growth of potato. And the higher the phosphorus concentration, the stronger the resistance, the better the growth. Subcellular localization in tobacco showed that StPHO1.2 was expressed on cell membrane. Therefore, the membrane system library plasmid was selected to screen the interacting proteins of StPHO1.2. This study demonstrated that the phosphorus transporter StPHO1.2 interacted with both the calcium ion transporter associated protein (StCAX1) and the photosynthetic system II protein subunit (StPsbR) by the yeast two-hybrid and BiFC. In conclusion, overexpression of StPHO1.2 may improve the heat tolerance and promote the growth of potato by affecting photosynthesis and signal transduction in potato. These results provide theoretical basis and reference for further understanding of the function of phosphorus transporters and promote the breeding of new potato varieties.

Key words: Solanum tuberosum L., phosphorus transporter, high temperature stress, yeast two-hybrid, BiFC

表1

引物、酶切位点、重组质粒名称和载体抗性"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 酶切位点
Endonuclease
site		 重组质粒名称
Recombinant
plasmid name		 载体抗性
Vector resistance
Ox2F GAACACGGGGGACTCTAGAGGATCCATGGTGAAGTTTTCTAAAGAACTTG BamH I
Sac I		 pBI121-StPHO1.2 Kan+
Ox2R AACGATCGGGGAAATTCGAGCTCCTAGCCATCAGAGTCTGTCTCG
RFP2F GTTCCAGATTACGCTGAGCTCATGGTGAAGTTTTCTAAAGAACTTG Sac I
Sac I		 p1300-RFP-StPHO1.2 Kan+
RFP2R CGATCGGGGAAATTCGAGCTCCTAGCCATCAGAGTCTGTCTCG
STE2F TTTTATGTAATGGCCATTACGGCCATGGTGAAGTTTTCTAAAGAACTT Sfi I
Sfi I		 pBT3-STE-StPHO1.2 Kan+
STE2R# TCCTGCAGATGGCCGAGGCGGCGCCATCAGAGTCTGTCTCGCG
SUC2F TATCTGCAATGGCCATTACGGCCATGGTGAAGTTTTCTAAAGAACTT Sfi I
Sfi I		 pBT3-SUC-StPHO1.2 Kan+
SUC2R# TCCTGCAGATGGCCGAGGCGGCGCCATCAGAGTCTGTCTCGCG
pBT3-N2F ATTCCTGCAGGGCCATTACGGCCATGGTGAAGTTTTCTAAAGAACTTGA Sfi I
Sfi I		 pBT3-N-StPHO1.2 Kan+
pBT3-N2R# ACTTACCATGGGGCCGAGGCGGCGCCATCAGAGTCTGTCTCGCG
pBT3-C2F TAATCTAGACGGCCATTACGGCCATGGTGAAGTTTTCTAAAGAACTTGA Sfi I
Sfi I		 pBT3-C-StPHO1.2 Kan+
pBT3-C2R# TGGAGGCCTTTGGCCGAGGCGGCGCCATCAGAGTCTGTCTCGCG
NY2F CGCCACAACATCGAGGGATCCATGGTGAAGTTTTCTAAAGAACTTG BamH I
Sma I		 NY-StPHO1.2 Kan+
NY2R GAATTCGAGCTCTATCCCGGGCTAGCCATCAGAGTCTGTCTCG

表2

载体筛选"

序号
Serial number		 质粒1
Plasmid 1		 质粒2
Plasmid 2		 目的
Purpose
1 pNubG-Fe65 pTSU2-APP 阳性对照 Positive control
2 NMY51 — 阴性对照 Negative control
3 pPR3N pBT3-STE-StPHO1.2 自激活检测 Self-activation detection
4 pPR3N pBT3-SUC-StPHO1.2 自激活检测 Self-activation detection
5 pPR3N pBT3-N-StPHO1.2 自激活检测 Self-activation detection
6 pPR3N pBT3-C-StPHO1.2 自激活检测 Self-activation detection
7 pOST1-NubI pBT3-STE-StPHO1.2 功能检测 Functional detection
8 pOST1-NubI pBT3-SUC-StPHO1.2 功能检测 Functional detection
9 pOST1-NubI pBT3-N-StPHO1.2 功能检测 Functional detection
10 pOST1-NubI pBT3-C-StPHO1.2 功能检测 Functional detection

图1

抗性基因PCR鉴定阳性转基因株系 M: DNA分子量标记; 1: StPHO1.2-Desiree; 2: pBI121空载质粒; 3: Desiree。"

图2

定量PCR鉴定阳性转基因株系"

图3

StPHO1.2-Desiree和Desiree的表型分析 A: 无磷(0 mmol L-1 Pi); B: 缺磷(0.1 mmol L-1 Pi); C: 富含磷(1.0 mmol L-1 Pi); 1: Desiree; 2: StPHO1.2-Desiree。“1”和“2”中, 左侧植株为高温下(35℃)生长, 右侧植株为常温下(22℃±1℃)生长。"

图4

StPHO1.2的亚细胞定位"

图5

酵母双杂质粒的筛选"

图6

酵母双杂交筛选StPHO1.2的互作蛋白 箭头表示代表性显色菌斑。"

图7

StPHO1.2与互作蛋白的“一对一”验证 1~5表示不同重复。"

图8

StPHO1.2与互作蛋白的BiFC验证"

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