不同启动子驱动下<i>GL</i>6基因表达对水稻叶表皮毛发育的影响

朱永生; 肖开转; 王福祥; 连玲; 何炜; 许惠滨; 魏毅东; 陈丽萍; 蒋家焕; 谢华安; 张建福

doi:10.19303/j.issn.1008-0384.2019.02.001

不同启动子驱动下GL6基因表达对水稻叶表皮毛发育的影响

doi: 10.19303/j.issn.1008-0384.2019.02.001

朱永生^{1, 2, 3,},
肖开转^{1, 2, 3},
王福祥^{1, 2, 3},
连玲^{1, 2, 3},
何炜^{1, 2, 3},
许惠滨^{1, 2, 3},
魏毅东^{1, 2, 3},
陈丽萍^{1, 2, 3},
蒋家焕^{1, 2, 3},
谢华安^{1, 2, 3},
张建福^{1, 2, 3, ,}

1.
福建省农业科学院水稻研究所, 福建福州 350019
2.
闽台作物有害生物生态防控国家重点实验室, 福建福州 350003
3.
农业部华南杂交水稻种质创新与分子育种重点实验室/福州(国家)水稻改良分中心/福建省作物种质创新与分子育种省部共建国家重点实验室培育基地/杂交水稻国家重点实验室华南研究基地/水稻国家工程实验室/福建省作物分子育种工程实验室/福建省水稻分子育种重点实验室, 福建福州 350003

基金项目:

国家重点研发计划项目 2017YFD0100100

福建省自然科学基金项目 2016J01135

福建省自然科学基金项目 2018J01039

福建省科技计划项目——省属公益类科研院所基本科研专项 2018R1021-5

福建省财政专项——福建省农业科学院科技创新团队项目 STIT2017-1-1

福建省农业科学院科技服务团队项目 kjfw02

详细信息

作者简介:
朱永生(1982-), 男, 博士研究生, 助理研究员, 主要从事水稻遗传育种研究(E-mail:zysfaas@qq.com)

通讯作者:
张建福(1971-), 博士, 研究员, 主要从事水稻分子生物学与分子育种研究(E-mail:jianfzhang@163.com)

中图分类号: S511
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- 被引次数: 0
出版历程
- 收稿日期: 2018-12-20
- 修回日期: 2019-01-05
- 刊出日期: 2019-02-28

Correlation Between GL6 Expression Driven by Varied Promoters and Development of Epidermal Hairs on Leaves of Rice Plant

ZHU Yong-sheng^{1, 2, 3
,},
XIAO Kai-zhuan^{1, 2, 3},
WANG Fu-xiang^{1, 2, 3},
LIAN Ling^{1, 2, 3},
HE Wei^{1, 2, 3},
XU Hui-bin^{1, 2, 3},
WEI Yi-dong^{1, 2, 3},
CHEN Li-ping^{1, 2, 3},
JIANG Jia-huan^{1, 2, 3},
XIE Hua-an^{1, 2, 3},
ZHANG Jian-fu^{1, 2, 3
, ,}

1.
Rice Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350019, China
2.
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China
3.
Key Laboratory of Germplasm Innovation and Molecular Breeding of Hybrid Rice for South China, Ministry of Agriculture, China/Fuzhou Branch, National Rice Improvement Center of China/Incubator of National Key Laboratory of Germplasm Innovation and Molecular Breeding Between Fujian and Ministry of Sciences & Technology/Base of South-China, National Key Laboratory of Hybrid Rice/National Rice Engineering Laboratory of China/Fujian Engineering Laboratory of Crop Molecular Breeding/Fujian Key Laboratory of Rice Molecular Breeding, Fuzhou, Fujian 350003, China

摘要

摘要: 目的研究水稻茸毛发育机制及相关基因的功能与调控模式，为深入研究相关基因功能及其在生产上的应用提供理论支撑。方法从不同水稻品种中克隆叶片表皮毛发育相关基因GL6的启动子序列，并将具有显著表皮毛特征的突变体品种75-1-127的GL6基因启动子与叶表无显著表皮毛特征的野生型品种相应基因的启动子序列进行比对，同时克隆突变体品种75-1-127中GL6基因的CDS序列，并分别构建以玉米泛素蛋白Ubiquitin和花椰菜花叶病毒CaMV35S为启动子驱动的过表达载体，以农杆菌介导的方法转化野生型粳稻品种Kitaake。结果不同品种中克隆的启动子序列区存在显著的序列差异，以玉米泛素蛋白Ubiquitin启动子驱动的过表达载体获得的转基因水稻出现了显著的表皮毛特征，以花椰菜花叶病毒CaMV35S为启动子驱动的过表达载体的转基因水稻则未出现典型的表皮毛特征。结论目标基因GL6的表达调控受启动子的影响，突变体品种75-1-127的叶表皮毛发育特征是因启动子区序列差异所致。
- 水稻 /
- 启动子 /
- 基因表达 /
- 表皮毛发育
Abstract: Objective Being one of the important agronomic traits that reflect the yield and physiological functions of a rice plant, the growth and photosynthesis of the fine hairs on the surface of rice leaves were studied. Method The sequences of GL6 gene promoters relating to leaf epidermal hair development were obtained and cloned from different rice varieties. That of Mutant 75-1-127 showing the characteristic epidermal hair development was compared with those of the wild type rice lacking it. The CDS sequence of the mutant gene was cloned. Then, the maize Ubiquitin and cauliflower mosaic virus CaMV35S were used as promoter-driven overexpression vectors to transform the wild-type Japonica rice, Kitaake, mediated by Agrobacterium. Result There were significant differences on the sequences in regions of the genes regulated by the promoters and cloned from different cultivars. The leaves of transgenic rice plants with GL6 driven by maize Ubiquitin promoter had significant hairy phenotype of the typical velvet appearance, but not those involved CaMV35S as a promoter. Conclusion It was clearly demonstrated that the promoters affected the expression of the target gene GL6 and that the epidermal hair development on leaves of Mutant 75-1-127 was derived by the promoter with a unique sequence.
- rice /
- promoter /
- gene expression /
- leaf epidermal hair development

HTML全文

图 1 候选基因CDS扩增产物的琼脂糖凝胶电泳分析

注：M=DL 5 000 marker; 1为GL6; 2为候选基因2;3为候选基因3。

Figure 1. Amplified products and agarose gel electrophoresis of candidate genes

Note: M: DL 5 000 marker; 1: GL6; 2: candidate gene 2; 3: candidate gene 3.

下载: 全尺寸图片幻灯片

图 2 GL6在75-1-127和MH63中启动子序列差异比对

Figure 2. Sequence alignments of GL6 promotors of Mutant 75-1-127 and MH63

下载: 全尺寸图片幻灯片

图 3 部分转基因苗PCR检测

注：M为DL 2 000 Marker；A为ddH₂O; B为pCubi1390flag-GL6载体的农杆菌菌株体；1~10泳道为部分转基因植株CDS序列阳性扩增。

Figure 3. Detection of transgenic plants

Note: M: DL 2 000 marker, A: ddH₂O, B: Agrobacterium tumefaciens with pCubi1390flag-GL6, 1-10: CDS sequences of transgenic seedling by PCR amplification.

下载: 全尺寸图片幻灯片

图 4 不同启动子驱动下的转基因植株叶片表皮毛表型

注：A和B: CaMV35S启动子驱动GL6转化Kitaake叶片上下表面扫描电镜观察；C和D：Ubiquitin启动子驱动GL6转化Kitaake叶片上下表面扫描电镜观察。

Figure 4. Phenotype of transgenic plants driven by CaMV35S promoter (A&B) or Ubiquitin promoter (C&D) as observed under SEM on adaxial and abaxial sides of leaf of Kitaake

Note:A&B: SEM observation on the adaxial side and the abaxial side of the leaf between KitaakeGL6trangneic driven by a CaMV35S promoter (A&B)and Kitaake-GL6trangneic driven by a Ubiquitin promoter(C&D).

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参考文献(24)

[1]	INOMURA K, BRAGG J, FOLLOWS M J. A quantitative analysis of the direct and indirect costs of nitrogen fixation:A model based on Azoto-bactervinelandii[J]. ISME J, 2017, 11:166. doi: 10.1038/ismej.2016.97
[2]	LAUTER D J, MUNNS D N. Water loss via the glandular trichomes of chickpea (Cicer arietinum L.)[J]. J Exp Bot, 1986, 37:640-649. doi: 10.1093/jxb/37.5.640
[3]	YAN A, PAN J, AN L, et al. The responses of trichome mutants to enhanced ultraviolet-B radiation in Arabidopsis thaliana[J]. J PhotochemPhotobiol B Biol, 2012, 113:29-35. doi: 10.1016/j.jphotobiol.2012.04.011
[4]	TATTINI M, GRAVANO E, PINELLI P, et al. Flavonoids accumulate in leaves and glandular trichomes of Phillyrealatifolia exposed to excess solar radiation[J]. New Phytol, 2000, 148:69-77. doi: 10.1046/j.1469-8137.2000.00743.x
[5]	尚宏芹, 刘建萍.干旱胁迫下不同茸毛性状辣椒植株抗旱性比较[J].核农学报, 2010, 24(4):835-839. http://d.old.wanfangdata.com.cn/Periodical/hnxb201004031 SHANG H Q, LIU J P. Comparison of drought resistance of pepper with different hairiness character under drought stress[J].Journal of Nuclear Agricultural Sciences, 2010, 24(4):835-839.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/hnxb201004031
[6]	CASTILLO-LÓPEZ J L, CANO-SANTANA Z, OYAMA K. Preferences and survival of Lophoceramicapyrrha, a shelter builder gregarious noctuid, in two host plants[J].Dugesiana, 2010, 17:229-236. https://www.cabdirect.org/cabdirect/abstract/20113106790
[7]	DALIN P, GREN J, BJRKMAN C, et al. Chapter 4:Leaf trichome formation and plant resistance to herbivory.Induced Plant Resistance to Herbivory[M]. Netherlands:Springer, 2008:89-105.
[8]	MCGINNIS A J, KASTING R. Dietary cellulose:Effect on food consumption and growth of a grasshopper[J]. Ca J Zool, 2011, 45:165-167. http://cn.bing.com/academic/profile?id=4ccd573840db1ff1b9cc035f3f792930&encoded=0&v=paper_preview&mkt=zh-cn
[9]	HANDLEY R, EKBOM B, AGREN J. Variation in trichome density and resistance against a specialist insect herbivore in natural populations of Arabidopsis thaliana[J]. EcolEntomol, 2005, 30:284-292. doi: 10.1111/j.0307-6946.2005.00699.x
[10]	AN L, ZHOU Z, SU S, et al. GLABROUS INFLORESCENCE STEMS (GIS) is required for trichome branching through gibberellic acid signaling in Arabidopsis[J]. Plant Cell Physiol, 2012, 53:457. doi: 10.1093/pcp/pcr192
[11]	KHOSLA A, PAPER J M, BOEHLER A P, et al. HD-Zip proteins GL2 and HDG11 have redundant functions in Arabidopsis trichomes and GL2 activates a positive feedback loop via MYB23[J]. Plant Cell, 2014, 26:2184-2200. doi: 10.1105/tpc.113.120360
[12]	MOROHASHI K, ZHAO M, YANG M, et al. Participation of the ArabidopsisbHLH factor GL3 in trichome initiation regulatory events[J]. Plant Physiol, 2007, 145:736-746. doi: 10.1104/pp.107.104521
[13]	BERNHARDT C, LEE M M, GONZALEZ, et al.ThebHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root[J]. Development, 2003, 130:6431-6439. doi: 10.1242/dev.00880
[14]	CHOPRA D, WOLFF H, SPAN J, et al. Analysis of TTG1 function in Arabisalpina[J]. BMC Plant Biol, 2014, 14:16. doi: 10.1186/1471-2229-14-16
[15]	LARKIN J C, OPPENHEIMER D G, MARKS M D, et al. The GL1 gene and the trichome developmental pathway in Arabidopsis thaliana[J]. Results Probl Cell Differ, 1994, 20:259-275. doi: 10.1007/978-3-540-48037-2
[16]	SZYMANSKI D B, JILK R A, POLLOCK S M, et al. Control of GL2 expression in Arabidopsis leaves and trichomes[J]. Development, 1998, 125:1161-1171. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=6e5bae151798cddf42ed4ac8fb9edda9
[17]	SUN W Q, GAO D W, XIONG Y, et al.Hairy Leaf 6, an AP2/ERF Transcription Factor, Interacts with OsWOX3B and Regulates Trichome Formation in Rice[J].Molecular Plant, 2017, 10(11):1417-1433. doi: 10.1016/j.molp.2017.09.015
[18]	LI J J, YUAN Y D, LU Z F, et al. Glabrous Rice 1, encoding a homeodomainprotein, regulates trichome development in rice[J].Rice, 2012, 5(32):1-10. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4883694/
[19]	QIN B, TANG D, HUANG J, et al. Rice OsGL1-1 is involved in leaf cuticular wax and cuticle membrane[J]. Mol Plant, 2011, 4:985-995. doi: 10.1093/mp/ssr028
[20]	曾跃辉, 朱永生, 连玲, 等.水稻茸毛基因GL6的遗传学分析与精细定位[J].科学通报, 2013, 58(1):1027-1035. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=kxtb201311010 ZENG Y H, ZHU Y S, LIAN L, et al.Genetic analysis and fine mapping of the pubescence gene GL6 in rice (Oryzae sativa L.)[J].Chinese Science Bulletin, 2013, 58(1):1-8.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=kxtb201311010
[21]	ZHENG K, TIAN H, HU Q, et al. Ectopic expression of R3MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation[J]. Sci Rep, 2016, 6:19254. doi: 10.1038/srep19254
[22]	KASUGA M, LIU Q, MIURA S, et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor[J]. Nat biotechnol, 1999, 17(3):287-291 doi: 10.1038/7036
[23]	乔龙飞, 于延冲.LEAFY启动子中W-box对LEAFY表达及拟南芥开花的影响[J].植物生理学报, 2017, 53(4):713-720. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zwslxtx201704024 QIAO L F, YU Y C. Effect of W-box in LEAFY promoter on LEAFY expression and Arabidopsis flowering[J]. Plant Physiology Journal, 2017, 53(4):713-720.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zwslxtx201704024
[24]	陈兆骞, 陈熙, 张炜, 不同启动子在水稻悬浮细胞中诱导外源基因的表达, 江苏农业学报[J], 2014, 30(6):1208-1215. doi: 10.3969/j.issn.1000-4440.2014.06.004 CHEN Z Q, CHEN X, ZHANG W, et al. Promoter-induced expression of exogenous gene in rice suspension cell lines[J]. Jiangsu Journal of Agricultural Sciences, 2014, 30(6):1208-1215.(in Chinese) doi: 10.3969/j.issn.1000-4440.2014.06.004