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作物学报

作物学报 ›› 2018, Vol. 44 ›› Issue (04): 512-521.doi: 10.3724/SP.J.1006.2018.00512

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

amiRNA技术沉默C-3氧化酶编码基因StCPD对马铃薯抗旱性的影响

周香艳1(), 杨江伟1,2, 唐勋1,2, 文义凯1, 张宁1,*(), 司怀军1,2   

  1. 1甘肃农业大学生命科学技术学院, 甘肃兰州 730070
    2甘肃省作物遗传改良与种质创新重点实验室 / 甘肃省干旱生境作物学省部共建国家重点实验室培育基地, 甘肃兰州 730070
  • 收稿日期:2017-08-18 接受日期:2018-01-08 出版日期:2018-01-29 网络出版日期:2018-01-29
  • 通讯作者: 张宁
  • 作者简介:

    zhouxy@gsau.edu.cn

  • 基金资助:
    本研究由国家自然科学基金项目(31460370, 31660416)资助

Effect of Silencing C-3 Oxidase Encoded Gene StCPD on Potato Drought Resistance by amiRNA Technology

Xiang-Yan ZHOU1(), Jiang-Wei YANG1,2, Xun TANG1,2, Yi-Kai WEN1, Ning ZHANG1,*(), Huai-Jun SI1,2   

  1. 1 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
    2 Gansu Key Laboratory of Crop Genetic and Germplasm Enhancement / Gansu Provincial Key Laboratory of Aridland Crop Science, Lanzhou 730070, Gansu, China
  • Received:2017-08-18 Accepted:2018-01-08 Published:2018-01-29 Published online:2018-01-29
  • Contact: Ning ZHANG
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31460370, 31660416).

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摘要：

CPD (constitutive photomorphogenesis and dwarf)基因编码C-3氧化酶, 为油菜素内酯(brassinosteroid, BR)生物合成途径中的限速酶, 在植物响应逆境胁迫过程中具重要调控作用。本研究利用人工microRNA (artificial microRNA, amiRNA)技术, 构建马铃薯CPD基因(StCPD)的干扰表达载体pCPB121-amiRcpd, 通过根癌农杆菌介导法将其转入马铃薯栽培品种“紫花白”, 获得转基因植株(Ci1~Ci5), 其中Ci1和Ci3的StCPD基因干扰程度分别为78%和90%。基因组织表达特异性分析表明, StCPD在马铃薯试管苗叶片中表达量最高, 是茎和根中表达量的3.05倍和1.65倍。转基因植株株高、茎粗、根长、鲜重及薯的大小和鲜重等指标均较非转基因(NT)植株显著下降, 表明StCPD基因干扰表达后, 植株的长势明显受到抑制。模拟干旱胁迫处理下, 转基因植株叶片中丙二醛(malondialdehyde, MDA)含量显著高于NT植株, 而脯氨酸含量显著低于NT植株。转基因和NT马铃薯中, StCPD基因的表达量、MDA和脯氨酸含量均显著高于对照; 且随着胁迫处理时间延长, 基因表达量呈持续增强趋势, MDA和脯氨酸含量随之增加。结果表明, StCPD基因干扰表达能明显降低马铃薯对干旱胁迫的抵抗能力, 为进一步研究BR对马铃薯生长发育和对干旱胁迫的响应奠定了基础。

关键词: 马铃薯, 油菜素内酯, StCPD基因, RNA干扰, 干旱胁迫

Abstract:

The constitutive photomorphogenesis and dwarf (CPD) gene encodes C-3 oxidase as a key rate-limiting enzyme in the brassinosteroids (BRs) biosynthesis pathway, which plays a vital role in response to abiotic stress in plant. In this research, the potato CPD gene (StCPD) interference expression vector pCPB121-amiRcpd was constructed using the plant expression vector pRS-300 and amiRcpd skeleton precursor of Arabidopsis miR319a, and transferred into the potato genome mediated by Agrobactium tumefacienses L., so that transgenic plants (Ci1-Ci5) were obtained. The analysis of real time fluorescence quantitative polymerase chain reaction (qRT-PCR) showed that the interference degree of StCPD gene expression reached 78% and 90% in the transgenic lines Ci1 and Ci3 respectively. StCPD gene expressed in the roots, stems and leaves of the transgenic and non-transgenic (NT) plants, and expression level in the leaves was 3.05 and 1.65 fold higher than that in stems and roots. The malondialdehyde (MDA) content in transgenic plant leaves was significantly higher, whereas the proline content was significantly lower than those in NT under drought stress. The expression level of StCPD gene, MDA and proline contents in transgenic and NT plants under drought stress were significantly higher than those of control, and increased at different sampling times. These results indicated that StCPD gene interference expression could depress the effects of drought stress on potato. These results lay a foundation for further research on BRs regulation in potato development, which will help to reveal the molecular mechanisms of BRs regulation in potato.

Key words: potato, brassinosteroids, StCPD gene, RNA interference, drought stress

表1

重叠PCR引物"

amiRNA /amiRNA 引物序列 Primer sequences (5′-3′)
I GATATTATGTTAGCGATTCGCACTCTCTCTTTTGTATTCC
II GAGTGCGAATCGCTAACATAATATCAAAGAGAATCAATGA
III GAGTACGAATCGCTATCATAATTTCACAGGTCGTGATATG
IV GAAATTATGATAGCGATTCGTACTCTACATATATATTCCT

表2

产生amiRNA前体的标准反应体系"

PCR反应
PCR reaction		 正向引物
Forward primer		 反向引物
Reverse primer		 模板
Template		 产物长度
Length of PCR product (bp)
(a) A IV pRS300 272
(b) III II pRS300 171
(c) I B pRS300 298
(d) A B (a) + (b) + (c) 701

表3

马铃薯qRT-PCR分析相关基因引物"

引物Primer 引物序列Primer sequence (5'-3')
amiRcpd 正向引物 amiRcpd forward primer TACGAATCGCTATCATAAT
18S RNA TTAGAGGAAGGAGAAGTCGTAACAA
通用反向引物 Universal reverse primer miRNA cDNA synthesis试剂盒提供 miRNA cDNA synthesis kit provided

图1

合成amiRNA前体a、b和c片段的PCR扩增产物M: DL2000 marker; 1: 片段a; 2: 片段b; 3: 片段c。"

图2

合成amiRNAs前体d片段检测 a: d片段菌落PCR扩增产物; b: 双酶切验证。M: DL2000 marker; 1: 片段d。"

图3

合成amiRNA前体d片段的测序结果上面蓝色部分代表原pRS300载体上miR319a的成熟序列和其反向序列; 下面红色部分代表经PCR扩增后替换的StCPD成熟序列和其反向序列。"

图4

以PCR法检测转基因植株 M: DL2000 marker (TaKaRa); 1: pBI121质粒阳性对照; 2: 非转基因植株阴性对照; 3~7: 转基因植株。"

图5

转基因植株StCPD基因表达的qRT-PCR检测 NT: 非转基因植株; Ci1~Ci5: 5个不同的转基因株系。"

图6

qRT-PCR分析StCPD 基因组织表达特异性 NT: 未转基因植株; Ci1, Ci3, Ci4: 转基因植株; NT: 非转基因植株; Ci1、Ci3和Ci4: 3个不同转基因株系; 显著性分析采用邓肯氏新复极差法, 同列不同小写字母表示在0.05水平差异显著性。误差线表示标准误(n = 9, 即3个生物学重复×3次技术重复)。"

表4

StCPD干扰表达转基因植株表型分析"

株系
Plant line		 株高
Plant height (mm)		 茎粗
Stem thickness (cm)		 根长
Root length (mm)		 植株鲜重
Fresh weight of plant (g)
NT 79.16±1.23 a 0.29±0.07 a 38.60±1.00 a 0.37±0.06 a
Ci1 57.84±1.91 b 0.12±0.04 b 18.52±1.40 b 0.30±0.02 b
Ci3 55.02±3.18 c 0.15±0.04 c 13.82±1.65 c 0.25±0.04 c
Ci4 52.56±4.00 d 0.11±0.05 b 19.21±0.86 b 0.20±0.02 d

图7

干旱胁迫对未转基因(NT, A)和转基因(B)马铃薯StCPD基因相对表达量的影响不同处理时间(d)的显著性差异采用邓肯氏新复极差法分析, 柱上不同小写字母表示在0.05水平差异显著。误差线表示标准误(n = 9, 即3个生物学重复×3次技术重复)。"

图8

转基因马铃薯和未转基因(NT)马铃薯丙二醛含量 MS培养基中培养4周后, 未转基因与转基因株系各自分为两组, 其中一组用新的MS培养基进行对照培养, 另一组用含有20% PEG-8000的MS培养基培养0~6 d。不同处理时间(d)的显著性差异采用邓肯氏新复极差法分析, 柱上不同小写字母表示在0.05水平差异显著。误差线表示标准误(n = 9, 即3个生物学重复×3次技术重复)。"

图9

转基因马铃薯和未转基因(NT)马铃薯脯氨酸含量 MS培养基中培养4周后, 未转基因与转基因株系各自分为两组, 其中一组用新的MS培养基进行对照培养, 另一组用含有20% PEG-8000的MS培养基培养0~6 d。不同处理时间(d)的显著性差异采用邓肯氏新复极差法分析, 柱上不同小写字母表示在0.05水平差异显著。误差线表示标准误(n = 9, 即3个生物学重复×3次技术重复)。"

图10

转基因植株试管苗在模拟干旱胁迫处理前后表型 Ci1和Ci3: 对照处理的转基因株系; Ci1’和Ci3’: 干旱胁迫下的转基因株系。图中的标尺为1 cm。"

表5

干旱胁迫对StCPD基因干扰表达植株表型影响"

株系
Plant line		 株高
Plant height
(mm)		 茎粗
Stem thickness (cm)		 根长
Roots length
(mm)		 植株鲜重
Fresh weight of plant (g)		 薯鲜重
Fresh weight of tuber (g)		 薯直径
Diameter of tuber (cm)
NT1’ 76.34±3.22 a 0.23±0.03 a 20.53±0.56 b 0.35±0.03 b 0.42±0.04 b 0.67±0.07 b
NT2’ 73.41±2.87 b 0.22±0.01 a 23.29±1.21 a 0.33±0.01 bc 0.44±0.06 b 0.77±0.08 a
NT3’ 77.32±11.56 a 0.25±0.05 a 22.31±0.37 a 0.41±0.02 a 0.57±0.02 a 0.80±0.05 a
Ci1’ 35.16±0.25 d 0.10±0.01 b 18.82±1.34 bc 0.28±0.02 c 0.25±0.07 d 0.51±0.04 c
Ci3’ 36.77±0.55 c 0.11±0.02 b 19.17±0.88 b 0.22±0.03 d 0.23±0.02 de 0.38±0.01 e
Ci4’ 37.15±0.37 c 0.15±0.01 b 16.65±1.94 c 0.24±0.02 d 0.31±0.03 c 0.43±0.02 d
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