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作物学报 ›› 2024, Vol. 50 ›› Issue (10): 2458-2467.doi: 10.3724/SP.J.1006.2024.44017

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

过表达野生大豆耐盐碱基因GsGSTU13提高了水稻苗期耐盐碱性

李宛鸿(), 胡冰霜, 孙晓丽, 才晓溪*(), 孙明哲*()   

  1. 黑龙江八一农垦大学农学院 / 作物逆境分子生物学实验室, 黑龙江大庆 163316
  • 收稿日期:2024-01-28 接受日期:2024-06-20 出版日期:2024-10-12 网络出版日期:2024-07-11
  • 通讯作者: *孙明哲, E-mail: sunmingzhe@byau.edu.cn;才晓溪, E-mail: 18746616279@163.com
  • 作者简介:E-mail: wh10161122@163.com; alisa961102@gmail.com
  • 基金资助:
    国家自然科学基金项目(U20A2025);国家自然科学基金项目(31971826);黑龙江省自然科学基金项目(YQ2023C035)

Overexpression of wild soybean salt-alkali tolerance gene GsGSTU13 increases salt-alkaline tolerance in rice seedlings

LI Wan-Hong(), HU Bing-Shuang, SUN Xiao-Li, CAI Xiao-Xi*(), SUN Ming-Zhe*()   

  1. College of Agriculture, Heilongjiang Bayi Agriculture University / Crop Stress Molecular Biology Laboratory, Daqing 163316, Heilongjiang, China
  • Received:2024-01-28 Accepted:2024-06-20 Published:2024-10-12 Published online:2024-07-11
  • Contact: *E-mail: sunmingzhe@byau.edu.cn;E-mail: 18746616279@163.com
  • Supported by:
    National Natural Science Foundation of China(U20A2025);National Natural Science Foundation of China(31971826);Natural Science Foundation of Heilongjiang Province(YQ2023C035)

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摘要：

谷胱甘肽S-转移酶(glutathione S-transferases, GSTs)是一类高度保守的功能酶, 在植物对非生物胁迫应答过程中发挥重要作用。本研究通过生物信息学分析发现, 野生大豆GsGSTU13与水稻GSTU17蛋白序列相似性最高, 均含有高度保守的结构域。为探究GsGSTU13基因对水稻耐盐碱性的影响, 通过遗传转化、PCR、RT-PCR和GST活性检测, 获得了2个GsGSTU13过表达转基因水稻纯合株系。苗期耐盐碱性分析显示, 200 mmol L-1 NaHCO3处理下, GsGSTU13转基因株系的活性氧积累显著低于野生型, 而存活率、相对含水量、超氧化物歧化酶、过氧化物酶、过氧化氢酶和GST活性均显著高于野生型。综上, 在水稻中过表达GsGSTU13基因增强了活性氧清除能力, 提高了转基因水稻耐盐碱性, 为耐盐碱水稻新种质创制提供了参考。

关键词: 水稻, 野生大豆, 耐盐碱性, 谷胱甘肽S-转移酶, GsGSTU13

Abstract:

Glutathione S-transferases (GSTs) are a class of highly conserved enzymes that play crucial roles in plant responses to environmental stresses. Bioinformatic analysis has revealed that Glycine soja GsGSTU13, which positively regulates salt-alkaline tolerance, shares the highest sequence identity with the OsGSTU17 protein. To investigate the potential contribution of GsGSTU13 to rice salt-alkaline tolerance, we transformed GsGSTU13 into rice and obtained two homozygous transgenic lines with significantly elevated GST activity. Phenotypic assays showed that after treatment with 200 mmol L-1 NaHCO3, the accumulation of reactive oxygen species was significantly lower in GsGSTU13 transgenic lines compared to wild-type. Additionally, the survival rates, relative water contents, and the activities of superoxide dismutases, peroxidases, catalases, and GSTs were significantly higher in GsGSTU13 transgenic lines than in the wild-type. In summary, overexpression of GsGSTU13 in rice enhanced salt-alkaline tolerance by promoting ROS scavenging, which could facilitate the breeding of new rice cultivars with improved tolerance to salt-alkaline stress.

Key words: rice, wild soybean, salt-alkaline tolerance, glutathione S-transferases, GsGSTU13

表1

本研究使用的引物"

引物名称
Primer name		 序列
Sequence (5°-3°)		 试验用途
Experimental purpose
pCAMBIA130035Su-GsGSTU13-F GGCTTAAUGGCTTCAAATCATGAAG 植物超量表达载体构建
Generation of plant expression construct
pCAMBIA130035Su-GsGSTU13-R GGTTTAAUCTACTTTTTAGCAGAAGCTTG
pCAMBIA35S-F ATAAGGAAGTTCATTTCATTTGGA 遗传转化抗性苗PCR检测
PCR identification of transgenic rice
GsGSTU13-PCR-R TCCTCCACTGCTTTCTCACG
GsGSTU13-RT-PCR-F TGCCTCGAGACCCTTATGAGA 遗传转化抗性苗RT-PCR检测
RT-PCR identification of transgenic rice
GsGSTU13-RT-PCR-R CCTCCTTGATCACAGGGTGATTG
OsGSTU17-qRT-F CTCCCAACTCCCAACTTCCC OsGSTU17基因表达Real-time PCR分析
Real-time PCR analysis of OsGSTU17 gene expression
OsGSTU17-qRT-R ACATGCCGAACACCTTCACT

图1

野生大豆GSTU13与水稻GSTU蛋白进化关系分析 A: GSTU13与水稻GSTU蛋白进化关系(红色五角星标记为GsGSTU13, 蓝色圆点表示有功能报道的OsGSTUs基因)。B: GsGSTU13与同源OsGSTU蛋白序列相似性热图。"

图2

GsGSTU13与水稻同源OsGSTU蛋白序列比对"

图3

pCAMBIA130035Su-GsGSTU13植物表达载体构建 A: pCAMBIA130035Su-GsGSTU13载体图谱示意图; B: GsGSTU13基因CDS区PCR产物(M: DNA marker 2K); C: pCAMBIA130035Su- GsGSTU13菌落PCR鉴定; M: DNA marker 2K; +: 阳性质粒对照(pGEM-T-GSTU13); -: 阴性H2O对照; 1~4: GsGSTU13单菌落。"

图4

转GsGSTU13基因水稻株系的分子鉴定 A: 转GsGSTU13基因水稻抗性苗PCR检测(M: DNA marker 2K Plus; +: 质粒阳性对照; -: 阴性H2O对照; WT: 野生型对照; #1~#9: 转GsGSTU13基因水稻抗性苗); B: GsGSTU13转基因水稻株系RT-PCR检测(WT: 野生型对照; #1和#2: GsGSTU13转基因水稻株系; OsElf1-α作为内参基因); C: GsGSTU13转基因水稻株系GST活性检测; 差异显著性分析采用t测验方法, *: P < 0.05, n = 20。D: GsGSTU13转基因水稻株系中OsGSTU17基因表达分析; ns: 无显著性差异, n = 3。"

图5

GsGSTU13转基因水稻苗期耐盐碱性分析 A: NaHCO3处理前后各株系生长状态; B: 水稻幼苗存活率; C: 水稻幼苗相对含水量; 差异显著性分析采用t测验方法, *: P < 0.05, **: P < 0.01, n = 20。"

图6

GsGSTU13转基因水稻盐碱胁迫下ROS积累检测 A: NBT染色; B: DAB染色。图中标尺为1 cm。"

图7

盐碱胁迫处理下GsGSTU13转基因水稻抗氧化物酶检测 A: 谷胱甘肽S-转移酶(GST)活性; B: 超氧化物歧化酶(SOD)活性; C: 过氧化物酶(POD)活性; D: 过氧化氢酶(CAT)活性。差异显著性分析采用Student’s t测验方法, *: P < 0.05, **: P < 0.01, n = 20。"

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