欢迎访问作物学报，今天是
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).

RichHTML

9

PDF

1001

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
[1] Andrzej B.Metabolism of brassinosteroids in plants. Plant Physiol Biochem, 2007, 45: 95-107
[2] Sasse J.Physiological actions of brassinosteroids: an update.J Plant Growth Regul, 2003, 22: 276-288
[3] Nomura T, Bishop G J.Cytochrome P450s in plant steroid hormone synthesis and metabolism.Phytochem Rev, 2006, 5: 421-432
[4] Bancos S, Szatma A M, Castle J, Kozma-Bognár L, Shibata K, Tyokota T, Bishop G J, Nagy F, Szekeres M.Diurnal regulation of the brassinosteroid biosynthetic CPD gene in Arabidopsis. Plant Physiol, 2006, 141: 299-309
[5] Hammond S M, Bernstein E, Beach D, Hannon, G. J.RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature, 2000, 404: 293-296
[6] Zhang J, Wang Y, Zhu P, Wang X, Lv M, Feng H.SiRNA-mediated silence of protease-activated receptor-1 minimizes ischemic injury of cerebral cortex through HSP70 and MAP2.J neurol, 2012, 320: 6-11
[7] Warthmann N, Chen H, Ossowski S, Weigel D, Hervé P.Highly specific gene silencing by artificial mirnas in rice.Plant J, 2009, 18: 1121-1133
[8] Molnar A, Bassett A, Thuenemann E, Schwach F, Karkare S, Ossowski S, Weigel D, Baulcombe D.Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J, 2009, 58: 165-174
[9] Zhao L Q, Li H L, Li R, Li W, Hua J P, Guo Y D.Cloning of cotton delta-12 oleate desaturase gene FAD2-1 and construction of its ihpRNA and amiRNA interference vectors.Agric Sci Technol, 2012, 13: 2281-2283
[10] Alvarez J P, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Esheda Y.Endogenous and synthetic micrornas stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species.Plant Cell, 2006, 18: 1134-1151
[11] Niu Q W, Lin S S, Reyes J L, Chen K C, Wu H W, Yeh S D, Chua N H.Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance.Nat Biotechnol, 2006, 24: 1420-1428
[12] Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D.Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell, 2006, 18: 1121-1133
[13] 张献贺, 孔稳稳, 李勇, 李晶. 人工miRNA沉默基因的研究. 生物技术通报, 2014, (04): 50-56
Zhang X H, Kong W W, Li Y, Li J.Study of gene silencing by artificial miRNA.Biotechnol Bull, 2014, (04): 50-56 (in Chinese with English abstract)
[14] 张晓辉, 邹哲, 张余洋, 李汉霞, 叶志彪. 从反义RNA到人工miRNA的植物基因沉默技术革新. 自然科学进展, 2009, 19: 1029-1037
Zhang X H, Zou Z, Zhang Y Y, Li H X, Ye Z B.Technology innovation of plant gene silencing from antisense RNA to artificial miRNA.Prog Nat Sci, 2009, 19: 1029-1037 (in Chinese with English abstract)
[15] Ossowski S, Schwab R, Weigel D.Gene silencing in plants using artificial microRNAs and other small RNAs.Plant J, 2008, 53: 674-690
[16] Tzfira T, Tian G W, Lacroix B, Vyas S, Li J, Leitner-Dagan Y, Krichevsky A, Taylor T, Vainstein A, Citovsky V. pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants.Plant Mol Biol, 2005, 57: 503-516
[17] Choi K, Park C, Lee J, Oh M, Noh B, Lee I. Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development. Development, 2007, 134: 1931-1941
[18] Zhou X Y, Zhang N, Yang J W, Si H J.Functional analysis of potato gene: a rate-limiting enzyme in brassinosteroid biosynthesis under polyethylene glycol-induced osmotic stress.Crop Sci, 2016, 56: 2675-2687
[19] 司怀军, 谢丛华, 柳俊. 农杆菌介导的马铃薯试管薯遗传转化体系的优化及反义class I patatin基因的导入. 作物学报, 2003, 29: 801-805
Si H J, Xie C H, Liu J.An efficient protocol for Agrobacterium-mediated transformation with microtuber and the introduction of an antisense class I patatin gene into potato. Acta Agron Sin, 2003, 29: 801-805 (in Chinese with English abstract)
[20] Edwards K, Johnstone C, Thompson C.A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.Nucl Acids Res, 1991, 19: 1349
[21] Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-delta delta C(T)) method.Methods, 2001, 25: 402-408
[22] 武亮亮, 姚磊, 马瑞, 朱熙, 杨江伟, 张宁, 司怀军. 马铃薯HD-Zip I家族ATHB12基因的克隆及功能鉴定. 作物学报, 2016, 42: 1112-1121
Wu L L, Yao L, Ma R, Zhu X, Yang J W, Zhang N, Si H J.Cloning and functional identification of ATHB12 gene of HD-Zip I Family in potato (Solanum tuberosum L.). Acta Agron Sin, 2016, 42: 1112-1121 (in Chinese with English abstract)
[23] Bates L S, Waldren R P, Teare I D.Rapid determination of free proline for water-stress studies.Plant Soil, 1973, 39: 205-207
[24] 丁玉梅, 马龙海, 周晓罡, 姚春馨, 董禄风, 孙茂林. 干旱胁迫下马铃薯叶片脯氨酸、丙二醛含量变化及与耐旱性的相关性分析. 西南农业学报, 2013, 26: 106-110
Ding Y M, Ma L H, Zhou X G, Yao C X, Dong L F, Sun M L.Effects of drought stress on free proline and malonaldedyde contents in potato leaves and correlation analysis of drought-tolerant level among different varieties.Southwest China J Agric Sci, 2013, 26: 106-110 (in Chinese with English abstract)
[25] Sestili F, Janni M, Doherty A, Botticella E.D’Ovidio R, Masci S, Jones H D, Lafiandra D. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes. BMC Plant Biol, 2010, 10: 144
[26] 苏君艺, 陆璇璇, 朱维宁, 张林生. 小麦WZY2基因RNA干扰表达载体的构建及遗传转化. 西北农林科技大学学报(自然科学版), 2012, 40(11): 66-72
Su J Y, Lu X X, Zhu W N, Zhang L S.Construction of RNAi expression vector of WAY2 gene from wheat and its genetic transformation.J Northwest Univ (Nat Sci Edn), 2012, 40(11): 66-72 (in Chinese with English abstract)
[27] 王会平, 遇玲, 邹世慧, 陈璟, 郭余龙, 李名扬. 利用amiRNA技术沉默矮牵牛查尔酮合成酶基因. 园艺学报, 2012, 39: 2491-2498
Wang H P, Yu L, Zou S H, Chen J, Guo Y L, Li M Y.Silencing of chalcone synthase genes by artificial microRNA inPetunia. Acta Hortic Sin, 2012, 39: 2491-2498 (in Chinese with English abstract)
[28] 武海军. 胡杨油菜素内酯合成酶基因CPD (PeCPD)的克隆及功能分析. 兰州大学博士学位论文, 甘肃兰州, 2012
Wu H J.Cloning and Function Analysis of Populus euphratica Brassinosteroids Biosynthase Gene CPD (PeCPD). PhD Dissertation of Lanzhou University, Lanzhou,China, 2012 (in Chinese)
[29] 姜丽丽. 农杆菌介导的BcBCP1基因转化马铃薯抗旱性研究. 东北农业大学硕士学位论文, 黑龙江哈尔滨, 2008
Jiang L L.Agrobacterium-Mediated Transformation of Potato with Drought Resistance Gene BcBCP1. MS Thesis of Northeast Agricultural University, Harbin, Heilongjiang,China, 2008 (in Chinese)
[30] 惠非琼, 彭兵, 楼兵干, 林福呈, 聂长春, 刘剑. 印度梨形孢通过促进渗透调节物质的合成和诱导抗逆相关基因的表达提高烟草耐盐性. 农业生物技术学报, 2014, 22: 168-176
Hui F Q, Peng B, Lou B G, Lin F C, Nie C C, Liu J.Piriformospora indica improves salt tolerance in Nicotiana tobacum by promoting the synthesis of osmolyte and inducing the expression of stress resistance genes. J Agric Biotechnol, 2014, 22: 168-176 (in Chinese with English abstract)
[1] 王海波, 应静文, 何礼, 叶文宣, 涂卫, 蔡兴奎, 宋波涛, 柳俊. rDNA和端粒重复序列鉴定马铃薯和茄子体细胞杂种染色体丢失和融合[J]. 作物学报, 2022, 48(5): 1273-1278.
[2] 石艳艳, 马志花, 吴春花, 周永瑾, 李荣. 垄作沟覆地膜对旱地马铃薯光合特性及产量形成的影响[J]. 作物学报, 2022, 48(5): 1288-1297.
[3] 王霞, 尹晓雨, 于晓明, 刘晓丹. 干旱锻炼对B73自交后代当代干旱胁迫记忆基因表达及其启动子区DNA甲基化的影响[J]. 作物学报, 2022, 48(5): 1191-1198.
[4] 雷新慧, 万晨茜, 陶金才, 冷佳俊, 吴怡欣, 王家乐, 王鹏科, 杨清华, 冯佰利, 高金锋. 褪黑素与2,4-表油菜素内酯浸种对盐胁迫下荞麦发芽与幼苗生长的促进效应[J]. 作物学报, 2022, 48(5): 1210-1221.
[5] 冯亚, 朱熙, 罗红玉, 李世贵, 张宁, 司怀军. 马铃薯StMAPK4响应低温胁迫的功能解析[J]. 作物学报, 2022, 48(4): 896-907.
[6] 张霞, 于卓, 金兴红, 于肖夏, 李景伟, 李佳奇. 马铃薯SSR引物的开发、特征分析及在彩色马铃薯材料中的扩增研究[J]. 作物学报, 2022, 48(4): 920-929.
[7] 谭雪莲, 郭天文, 胡新元, 张平良, 曾骏, 刘晓伟. 黄土高原旱作区马铃薯连作根际土壤微生物群落变化特征[J]. 作物学报, 2022, 48(3): 682-694.
[8] 丁红, 徐扬, 张冠初, 秦斐斐, 戴良香, 张智猛. 不同生育期干旱与氮肥施用对花生氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 695-703.
[9] 余慧芳, 张卫娜, 康益晨, 范艳玲, 杨昕宇, 石铭福, 张茹艳, 张俊莲, 秦舒浩. 马铃薯CrRLK1Ls基因家族的鉴定及响应晚疫病菌信号的表达分析[J]. 作物学报, 2022, 48(1): 249-258.
[10] 荐红举, 尚丽娜, 金中辉, 丁艺, 李燕, 王季春, 胡柏耿, Vadim Khassanov, 吕典秋. 马铃薯PIF家族成员鉴定及其对高温胁迫的响应分析[J]. 作物学报, 2022, 48(1): 86-98.
[11] 许德蓉, 孙超, 毕真真, 秦天元, 王一好, 李成举, 范又方, 刘寅笃, 张俊莲, 白江平. 马铃薯StDRO1基因的多态性鉴定及其与根系性状的关联分析[J]. 作物学报, 2022, 48(1): 76-85.
[12] 张明聪, 何松榆, 秦彬, 王孟雪, 金喜军, 任春元, 吴耀坤, 张玉先. 外源褪黑素对干旱胁迫下春大豆品种绥农26形态、光合生理及产量的影响[J]. 作物学报, 2021, 47(9): 1791-1805.
[13] 李洁, 付惠, 姚晓华, 吴昆仑. 不同耐旱性青稞叶片差异蛋白分析[J]. 作物学报, 2021, 47(7): 1248-1258.
[14] 唐锐敏, 贾小云, 朱文娇, 印敬明, 杨清. 马铃薯热激转录因子HsfA3基因的克隆及其耐热性功能分析[J]. 作物学报, 2021, 47(4): 672-683.
[15] 李鹏程, 毕真真, 孙超, 秦天元, 梁文君, 王一好, 许德蓉, 刘玉汇, 张俊莲, 白江平. DNA甲基化参与调控马铃薯响应干旱胁迫的关键基因挖掘[J]. 作物学报, 2021, 47(4): 599-612.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2