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作物学报 ›› 2019, Vol. 45 ›› Issue (8): 1166-1175.doi: 10.3724/SP.J.1006.2019.84166

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

AtDREB1A基因过量表达对马铃薯生长及抗非生物胁迫基因表达的影响

贾小霞1,2,齐恩芳1,2,刘石1,2,文国宏1,2,*(),马胜1,2,李建武1,2,黄伟1,2   

  1. 1 甘肃省农业科学院马铃薯研究所/甘肃省马铃薯种质资源创新工程实验室, 甘肃兰州730070
    2 农业部西北旱作马铃薯科学观测实验站, 甘肃渭源748201
  • 收稿日期:2018-12-11 接受日期:2019-04-15 出版日期:2019-08-12 网络出版日期:2019-07-16
  • 通讯作者: 文国宏
  • 作者简介:E-mail: jiaxx0601@163.com
  • 基金资助:
    本研究由国家自然科学基金项目(31560412);本研究由国家自然科学基金项目(31060200);甘肃省农业科学院科技创新专项(2017GAAS38);国家现代农业产业技术体系建设专项资助(CARS-09-P06)

Effects of over-expression of AtDREB1A gene on potato growth and abiotic stress resistance gene expression

JIA Xiao-Xia1,2,QI En-Fang1,2,LIU Shi1,2,WEN Guo-Hong1,2,*(),MA Sheng1,2,LI Jian-Wu1,2,HUANG Wei1,2   

  1. 1 Potato Research Institute, Gansu Academy of Agricultural Sciences/Gansu Engineering Laboratory of Potato Germplasm Resources Innovation, Lanzhou 730070, Gansu, China
    2 The Ministry of Agriculture, Scientific Observation and Experiment Station of Dry Potato in the Northwest, Weiyuan 748201, Gansu, China
  • Received:2018-12-11 Accepted:2019-04-15 Published:2019-08-12 Published online:2019-07-16
  • Contact: Guo-Hong WEN
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31560412);This study was supported by the National Natural Science Foundation of China(31060200);Science and Technology Support Program of Gansu Academy of Agricultural Sciences(2017GAAS38);China Agriculture Research System(CARS-09-P06)

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摘要：

为明确AtDREB1A基因过量表达对马铃薯生长及基因表达的影响, 以马铃薯品种陇薯3号(L3)及其AtDREB1A转基因株系T2为材料, 采用盆栽试验, 在马铃薯盛花期将盆土含水量控制为田间最大持水量(FWC)的45%~50%, 观察转基因前后植株表型, 并研究叶片MDA含量、RWC、SOD和POD活性及其基因表达的差异。结果表明, 正常浇水条件下2个株系各指标差异不大。胁迫20 d后, 转基因植株T2的表型明显好于对照L3, 且RWC显著高于L3; 各株系叶片的MDA含量、SOD和POD活性均明显上升, 但转基因植株MDA含量上升幅度较对照小, 抗氧化保护酶SOD、POD活性升高幅度较对照大, 说明转基因植株细胞膜损伤和膜脂过氧化程度较轻, 植株的耐旱性明显提高。转录组测序及生物信息学分析发现, 转基因材料T2相对于L3的差异表达基因共430个, 其中上调表达基因287个, 下调表达基因143个。功能注释和显著性富集结果表明, 差异表达基因富集涉及 GO 功能分类体系中生物过程、细胞组分和分子功能3个大类别, 且大部分集中在细胞内和膜上, 主要涉及信号传导、氧化还原、生物调解、应激反应、发育过程、系统免疫过程、核酸和蛋白结合转录因子活性、转运活性及催化活性。其中抗非生物胁迫相关蛋白PPR、HSP、P450、MLO等家族的大量基因表达量发生较大变化, 说明这些基因在转AtDREB1A基因马铃薯抵御干旱过程中发挥着非常重要的作用。本研究为进一步解析AtDREB1A基因提高马铃薯抗旱性的调控网络奠定了基础。

关键词: 马铃薯, AtDREB1A基因, 非生物胁迫, 差异表达基因, 干旱

Abstract:

Longshu 3 (L3) and its AtDREB1A transgenic line T2 were planted in the pot experiment. The water content of the soil in pots was controlled to 45%-50% of the maximum water holding capacity (FWC) during the flowering stage of the potato. The phenotype, MDA content, RWC, activities of SOD and POD, and the gene expression level in leaves were analyzed, showing there were no significant differences between the two lines under normal watering conditions. After 20 d of stress treatment, the phenotype of transgenic plants T2 was significantly better than that of control L3, and RWC was significantly higher than that of L3. The MDA content, SOD and POD activities of leaves in each line increased significantly, while the MDA content of transgenic plants increased less than, and the activities of SOD and POD of transgenic plants increased more than those of the control, indicating that the cell membrane damage and membrane lipid peroxidation of the transgenic plants were lighter than those of the control, and the drought tolerance of transgenic plants was significantly improved. Transcriptome sequencing and bioinformatic analysis showed that compared with L3, there were 430 differentially expressed genes in T2, including 287 up-regulated genes and 143 down-regulated genes. Functional annotation and significance enrichment showed that all differentially expressed genes were involved in three broad categories of GO functional classification systems, that was biological processes, cell components and molecular functions. Most of these differentially expressed genes concentrated on the membrane in cells, and mainly related to signal transduction, redox, biological mediation, stress response, development process, system immune process, nucleic acid and protein binding transcription factor activity, transport activity and catalytic activity. The expression of a large number of abiotic stress-related genes was up-regulated, including PPR protein family, P450 protein family, heat shock protein family and MLO protein family, indicating that these genes play a very important role in drought-stress resistance of AtDREB1A transgenic potato. This study lays a foundation for further understanding AtDREB1A’s regulatory network to improve potato drought resistance.

Key words: potato, AtDREB1Agene, abiotic stress, differentially expressed genes, drought

表1

用于qRT-PCR表达验证的基因及其引物"

基因 ID
Gene ID		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
EF1α ATTGGAAACGGATATGCTCCA TCCTTACCTGAACGCCTGTCA
AtDREB1A GCCGATCAGCCTGTCTCAAT TCTGCCATATTAGCCAACAAACTC
PGSC0003DMG400016489 AAATGTGTCTTTCAAGCGAA TCCATCTTCCCACATTTACCGTA
PGSC0003DMG400011977 CTTTGCCTGAGAATATTGACT CTTTGGGATTACTACTAGCCTT
PGSC0003DMG400000534 CCGACGATACAAACATATACTGA TGAGTTCCTTCCAGCGATT
PGSC0003DMG402019042 CCAACTCAGAACTGGTACGGACA CGTAAACATCCGTTGGGACCAT
PGSC0003DMG400000242 AACAGCCAGAAGAAGTTGAT ATTCCCATCACAGGCATCTCA
PGSC0003DMG401018057 AGGATCCATTAAAGTTCAAGCCA AATCCTTGAAGAAGCCTAGCA

图1

干旱胁迫下转基因及其对照马铃薯表型分析"

图2

干旱胁迫下马铃薯叶片SOD和POD活性的变化 相同处理标以不同字母的柱值在P < 0.05水平上差异显著。"

图3

干旱胁迫下马铃薯叶片MDA含量和RWC的变化 相同处理标以不同字母的柱值在 P < 0.05 水平上差异显著。"

图4

马铃薯叶片中AtDREB1A基因表达的实时荧光定量分析 相同处理标以不同字母的柱值在 P < 0.05 水平上差异显著。"

图5

干旱胁迫后马铃薯叶片中差异基因表达量火山图 横坐标代表基因在不同样品中表达倍数变化; 纵坐标代表基因表达量变化的统计学显著程度。图中每个基因均由一个点表示, 黑色圆点表示无显著性差异的基因, 左侧绿色圆点表示有显著性差异的下调基因, 右侧红色圆点表示有显著性差异的上调基因。"

图6

差异表达基因GO注释聚类图 横坐标为GO各分类内容, 纵坐标左边为基因数目所占百分比, 右边为基因数目。"

图7

干旱胁迫下PPR蛋白家族基因表达模式聚类图 图中连线表示各个基因变化趋势的相近程度, 线越短越相近。红色表示上调表达水平, 绿色表示下调表达水平。"

图8

干旱胁迫下HSP蛋白家族基因表达模式聚类图 图中连线表示各个基因变化趋势的相近程度, 线越短越相近。红色表示上调表达水平, 绿色表示下调表达水平。"

图9

干旱胁迫下P450蛋白家族基因表达模式聚类图 图中连线表示各个基因变化趋势的相近程度, 线越短越相近。红色表示上调表达水平, 绿色表示下调表达水平。"

图10

干旱胁迫下MLO蛋白家族基因表达模式聚类图 图中连线表示各个基因变化趋势的相近程度, 线越短越相近。红色表示上调表达水平, 绿色表示下调表达水平。"

图11

干旱胁迫下L3 vs. T2差异表达基因的qRT-PCR验证"

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