紫花苜蓿MsGAI的克隆、表达及遗传转化

doi:10.3864/j.issn.0578-1752.2019.02.002

摘要/Abstract

摘要：

【目的】 DELLA蛋白属于GRAS家族,是赤霉素信号转导途径中重要的转录因子,负向调节GA转导途径。克隆获得紫花苜蓿GAI,分析其基因生物信息学特征并预测蛋白结构域。明确紫花苜蓿GAI组织表达特征及不同处理下的表达模式,构建该基因超表达载体并转入紫花苜蓿,以探究DELLA蛋白基因在紫花苜蓿赤霉素（GA）信号转导途径及胁迫条件下的作用机理。【方法】利用同源克隆的方法,从紫花苜蓿中克隆得到MsGAI。利用生物信息学方法分析该基因的序列特征,使用MEGA7.0对MsGAI蛋白序列及同源序列进行多序列比对,构建同源物种间的系统发育树。利用实时荧光定量PCR检测紫花苜蓿各组织GAI表达量以及在PEG、NaCl、GA、ABA和黑暗处理下,GAI的表达变化。同时对转基因GAI株系表达水平进行分析,选择表达量高、中、低株系（L5、L8、L11）分别进行PEG和NaCl处理,分析GAI的表达变化。以pBI121为基础载体,采用双酶切-连接的方法构建植物超表达载体35S:MsGAI-gus。将重组载体转入农杆菌GV3101菌株中,以紫花苜蓿叶片为外植体,采用农杆菌介导的愈伤组织转化法转化紫花苜蓿,经PCR检测和GUS组织化学染色,得到转基因阳性苗。【结果】该基因序列包含有一个1 818 bp的开放阅读框,编码605个氨基酸。生物信息学分析结果显示,MsGAI蛋白具有GRAS家族的典型结构域和保守区,其中包含N端保守结构域DELLA和TVHYNP,C端保守结构域SAW。多序列比对及系统进化树分析表明,该序列与其他物种的DELLA蛋白序列相似度均高达80%以上,将其命名为MsGAI。该基因与蒺藜苜蓿GAI亲缘关系最近,其次与鹰嘴豆、红三叶等双子叶豆科植物亲缘关系较近,与大麦等单子叶植物较远。实时荧光定量PCR分析表明,MsGAI在紫花苜蓿各组织中均有表达,根中的表达量最高。经PEG、NaCl、GA以及ABA处理后,均有明显响应;黑暗处理显著抑制MsGAI的表达。转基因株系经PEG、NaCl处理后,GAI表达量均上调。对构建完善的35S:MsGAI-gus植物超表达载体进行双酶切检测,琼脂糖凝胶电泳显示,条带大小与预期一致。对转基因植株进行GUS组织染色验证,结果表明,阳性植株呈现蓝色,对照组为白色。对超表达载体携带的MsGAI和GUS序列进行PCR检测均呈阳性。【结论】紫花苜蓿DELLA蛋白基因的克隆和超表达载体构建成功,MsGAI对逆境胁迫有响应。

关键词: 紫花苜蓿, DELLA蛋白, 赤霉素, 表达分析, 同源克隆, 遗传转化

Abstract:

【Objective】 DELLA, one of GRAS family proteins, is an important transcription factor, which negatively regulates the gibberellin (GA) signal pathway. In this study, the Medicago sativa L. DELLA gene MsGAI was cloned, and its secondary structure was also predicted through bioinformatics. To illustrate the molecular function of MsGAI in M. sativa L. gibberellin (GA) signal transduction pathway, we clarify the spatio-temporal expression pattern of MsGAI and its response to different treatments; and the transgenic alfalfa over-expressing MsGAI were obtained by Agrobacterium-mediated transformation to explore gene function as well.【Method】 MsGAI was cloned by homologous cloning and sequence characteristics of MsGAI were analyzed by online bioinformatics tools. The phylogenetic tree of MsGAI and its homologous genes from other species was constructed by MEGA 7.0. The real-time quantitative PCR (qRT-PCR) was used to detect the spatio-temporal expression pattern of GAI and its response to abiotic including PEG, NaCl, GA, ABA and dark. The plant overexpression vector 35S:MsGAI-gus was transferred into Agrobacterium GV3101 strain, and transgenic alfalfa were obtained by Agrobacterium-mediated callus transformation. The transgenic positive seedlings were verified by PCR and GUS histochemical staining. Three transgenic line (L5, L8, L11) were selected to analysis MsGAI expression under PEG, and NaCl treatment.【Result】 Sequence analysis showed that 605 amino acids constitute MsGAI and it contains the conserved DELLA and VHYNP sequences in N-terminal regulatory region, and SAW in C-terminal. Multiple sequence alignment and phylogenetic tree analysis showed that the sequence similarity with other DELLA protein was as high as 80%, and it has the closest relationship with M. truncatula GAI, followed by the dicotyledonous such as Cicer arietinum and Trifolium pratense, and is far away from monocotyledon such as Hordeum vulgare. Real-Time PCR analysis showed that MsGAI had the highest expression in roots and after PEG, NaCl, GA and ABA treatment, the response was obvious while dark treatment significantly inhibited the expression of MsGAI. The GUS tissue staining showed that the positive plants were blue and the control group was white and the expression of MsGAI was upregulated in positive plants under PEG and NaCl treatments.【Conclusion】 The cloning and overexpression vector of DELLA protein in alfalfa was successfully constructed and the MsGAI could respond to stress treatments.

Key words: Medicago sativa L., DELLA protein, GA, expression analysis, homologous cloning, transformation

张涵,王学敏,刘希强,马琳,温红雨,王赞. 紫花苜蓿MsGAI的克隆、表达及遗传转化[J]. 中国农业科学, 2019, 52(2): 201-214.

ZHANG Han,WANG XueMin,LIU XiQiang,MA Lin,WEN HongYu,WANG Zan. Cloning Expression Analysis and Transformation of MsGAI Gene from Medicago sativa L[J]. Scientia Agricultura Sinica, 2019, 52(2): 201-214.

图/表 13

表1

图1

图2

图3

图4

表2

图5

表3

PlantCARE启动子预测结果"

顺式作用元件 Cis-elements	序列 Sequence (5'-3')	功能 Function	数量 No.
3-AF1 binding site	AAGAGATATTT	光响应元件 Light responsive element	1
A-box	CCGTCC	顺式作用调控元件 Cis-acting regulatory element	2
ACE	AAAACGTTTA	光响应顺式元件 Cis-acting element involved in light responsiveness	1
ARE	TGGTTT	厌氧诱导必需元件 Cis-acting regulatory element essential for the anaerobic induction	2
ATCT-motif	AATCTAATCT	参与光响应的保守DNA模块的部分元件 Part of a conserved DNA module involved in light responsiveness	2
BOX-4	ATTAAT	参与光反应元件 Part of a conserved DNA module involved in light responsiveness	3
Box-W1	TTGACC	真菌诱导子反应元件 Fungal elicitor responsive element	1
CAAT-box	CAAAT	启动子和增强子区域中常见的顺式作用元件 Common cis-acting element in promoter and enhancer regions	47
CCAAT-box	CAACGG	结合位点MYBHv1 binding site MYBHv1	1
CCGTCC-box	CCGTCC	与分生组织特异性激活相关的顺式调控元件 Cis-acting regulatory element related to meristem specific activation	2
GAG-motif	AGAGAGT	部分光响应元件 Part of a light responsive element	1
GT1-motif	GGTTAA	光响应元件 Light responsive element	1
HSE	AAAAAATTTC	热应激响应顺式元件 Cis-acting element involved in heat stress responsiveness	1
LAMP-element	CCAAAACCA	部分光响应元件 Part of a light responsive element	1
LTR	CCGAAA	参与低温响应的元件 Cis-acting element involved in low-temperature responsiveness	1
MBS	TAACTG	参与干旱诱导MYB结合位点 MYB binding site involved in drought-induction	1
MRE	AACCTAA	参与光反应的MYB结合位点 MYB binding site involved in light responsiveness	1
Skn-1_motif	GTCAT	胚乳表达所需的顺式作用调控元件 Cis-acting regulatory element required for endosperm expression	2
TATA-box	ATTATA	在转录起始的-30附近的核心启动子元件 Core promoter element around -30 of transcription start	74
chs-CMA2a	GCAATTCC	部分光响应元件 Part of a light responsive element	1
circadian	CAANNNNATC	参与昼夜节律顺行调控元件 Cis-acting regulatory element involved in circadian control	3

表3

图6

图7

图8

图9

图10

参考文献 34

[1]	陈敏 . 紫花苜蓿赤霉素受体基因MsGID1b的克隆及功能分析. 北京: 中国农业科学院, 2017. doi: 10.7606/j.issn.1000-4025.2016.11.2159
	CHEN M . Cloning and functional analysis of gibberellin receptor gene MsGID1b from Medicago sativa L. Beijing: Chinese Academy of Agricultural Sciences, 2017. ( in Chinese) doi: 10.7606/j.issn.1000-4025.2016.11.2159
[2]	BLUMENTHAL J M, RUSSELLE M P . Subsoil nitrate uptake and symbiotic dinitrogen fixation by alfalfa. Agronomy Journal, 1996,88(6):909-915. doi: 10.2134/agronj1996.00021962003600060010x
[3]	RUSSELLE M P, LAMB J F, MONTGOMERY B R, ELSENHEIMER D W, MILLER B S, VANCE C P . Alfalfa rapidly remediates excess inorganic nitrogen at a fertilizer spill site. Journal of Environmental Quality, 2001,30(1):30-36. doi: 10.2134/jeq2001.30130x pmid: 11215664
[4]	HEDDEN P, PHILLIPS A L . Gibberellin metabolism: new insights revealed by the genes. Trends in Plant Science, 2000,5(12):523-530. doi: 10.1016/S1360-1385(00)01790-8 pmid: 11120474
[5]	SUN T . Gibberellin metabolism, perception and signaling pathways in Arabidopsis. The Arabidopsis Book, 2008: e0103. doi: 10.1199/tab.0103 pmid: 22303234
[6]	董玉琛, 郑殿升 . 中国小麦遗传资源. 北京:中国农业出版社, 2000: 101-107.
	DONG Y C, ZHENG D S. The Wheat Genetic Resources in China. Beijing:China Agricultural Press, 2000: 101-107. (in Chinese)
[7]	AUSTIN R B . Yield of wheat in the United Kingdom: Recent advances and prospects. Crop Science, 1998,39(6):1604-1610.
[8]	MONNA L, KITAZAWA N, YOSHINO R, SUZUKI J, MASUDA H, MAEHARA Y . Rice “Green Revolution Gene” encodes a mutant enzyme involved in gibberellin synthesis. DNA Research, 2002,9(1):11-17. doi: 10.1093/dnares/9.1.11 pmid: 11939564
[9]	SASAKI A, ASHIKARI M, UEGUCHI-TANAKA M, ITOH H, NISHIMURA A, SWAPAN D . Green revolution: A mutant gibberellin- synthesis gene in rice. Nature, 2002,416:701-702. doi: 10.1038/416701a
[10]	SPIELMEYER W, ELLIS M H, CHANDLER P M . Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene. Plant Biology, 2002,99(13):9043-9048.
[11]	PYSH L D, WYSOCKA-DILLER J W, CAMILLERI C, BOUCHEZ D, BENFEY P N . The GRAS gene family in Arabidopsis: Sequence characterization and basic expression analysis of the SCARECROW- LIKE genes. The Plant Journal, 1999,18(1):111-119. doi: 10.1046/j.1365-313X.1999.00431.x pmid: 10341448
[12]	PENG J, RICHARDS D E, HARTLEY N M, MURPHY G P, DEVOS K M, FLINTHAM J E . 'Green revolution' genes encode mutant gibberellin response modulators. Nature, 1999,400(6741):256 doi: 10.1038/22307
[13]	SILVERSTONE A L, JUNG H S, DILL A, KAWAIDE H, KAMIYA Y, SUN T P . Repressing a repressor: Gibberellin-induced rapid reduction of the RGA protein in Arabidopsis. The Plant Cell, 2001,13(7):1555-1566.
[14]	GUBLER F, CHANDLER P M, WHITE R G, LLEWELLYN D J, JACOBSEN J V . Gibberellin signaling in barley aleurone cells. Control of SLN1 and GAMYB expression. Plant Physiology, 2002,129(1):191-200. doi: 10.1104/pp.010918
[15]	ITOH H, UEGUCHI-TANAKA M, SATO Y, ASHIKARI M, MATSUOKA M . The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE1 in nuclei. The Plant Cell, 2002,14(1):57-70. doi: 10.1105/tpc.010319 pmid: 11826299
[16]	PENG J, CAROL P, RICHARDS D E, KING K E, COWLING R J, MURPHY G P . The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses. Genes & Development, 1997,11(23):3194-3205. doi: 10.1101/gad.11.23.3194 pmid: 9389651
[17]	TYLER L, THOMAS S G, HU J, DILL A, ALONSO J M, ECKER J R . DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis. Plant Physiology, 2004,135(2):1008-1019.
[18]	LEE S, CHENG H, KING K E, WANG W, HE Y, HUSSAIN A . Gibberellin regulates Arabidopsis seed germination via RGL2, a GAI/RGA-like gene whose expression is up-regulated following imbibition.Genes & Development, 2002,16(5):646-658. doi: 10.1101/gad.969002 pmid: 11877383
[19]	FU X, SUDHAKAR D, PENG J, RICHARDS D E, CHRISTOU P, HARBERD N P . Expression of Arabidopsis GAI in transgenic rice represses multiple gibberellin responses.The Plant Cell, 2001,13(8):1791-1802.
[20]	TALON M, KOORNNEEF M, ZEEVAART J A D . Accumulation of C19-gibberellins in the gibberellin-insensitive dwarf mutant gai of Arabidopsis thaliana(L.). Planta, 1990,182(4):501-505. doi: 10.1007/BF02341024
[21]	TALON M, KOORNNEEF M, ZEEVAART J A . Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants.Proceedings of the National Academy of Sciences of the USA, 1990,87(20):7983-7987. doi: 10.1073/pnas.87.20.7983 pmid: 2236013
[22]	WILSON R N, SOMERVILLE C R . Phenotypic suppression of the gibberellin-insensitive mutant gai of Arabidopsis. Plant Physiology, 1995,108(2):495-502. doi: 10.1104/pp.108.2.475 pmid: 12228487
[23]	FU X, HARBERD N P . Auxin promotes Arabidopsis root growth by modulating gibberellin response.Nature, 2003,421(6924):740. doi: 10.1038/nature01387 pmid: 12610625
[24]	DILL A, SUN T . Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana. Genetics, 2001,159(2):777-785.
[25]	FOSTER T, KIRK C, JONES W T . Characterization of the DELLA subfamily in apple (Malus x domestica Borkh.). Tree Genetics & Genomes, 2007,3(3):187-197.
[26]	LIVAK K J, SCHMITTGEN T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^{- ΔΔCT} method . Methods, 2001,25(4):402-408. doi: 10.1006/meth.2001.1262
[27]	李俊 . 东方山羊豆赤霉素受体和水通道蛋白基因功能研究. 北京: 中国农业科学院, 2014.
	LI J . Construction of plant expression Vector of GoGID gene and transformation of Medicago sativa L. Beijing: Chinese Academy of Agricultural Sciences, 2014. ( in Chinese)
[28]	黄先忠, 蒋才富, 廖立力, 傅向东 . 赤霉素作用机理的分子基础与调控模式研究进展. 植物学通报, 2006,23(5):499-510. doi: 10.3969/j.issn.1674-3466.2006.05.006
	HUANG X Z, JIANG C F, LIAO L L, FU X D . Progress on molecular foundation of GA biosynthesis pathway and signaling. Chinese Bulletin of Botany, 2006,23(5):499-510. (in Chinese) doi: 10.3969/j.issn.1674-3466.2006.05.006
[29]	FUKUDA A, TANAKA Y . Effects of ABA, auxin, and gibberellin on the expression of genes for vacuolar H⁺-inorganic pyrophosphatase, H⁺-ATPase subunit A, and Na⁺/H⁺ antiporter in barley . Plant Physiology and Biochemistry, 2006,44(5/6):351-358. doi: 10.1016/j.plaphy.2006.06.012
[30]	ZENTELLA R, ZHANG Z L, PARK M . Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis. The Plant Cell, 2007,19(10):3037-3057. doi: 10.1105/tpc.107.054999 pmid: 17933900
[31]	DU H, CHANG Y, HUANG F, XIONG L . GID1 modulates stomatal response and submergence tolerance involving abscisic acid and gibberellic acid signaling in rice. Journal of Integrative Plant Biology, 2015,57(11):954-968. doi: 10.1111/jipb.12313 pmid: 25418692
[32]	DJAKOVIC-PETROVIC T, DE WIT M, VOESENEK L . DELLA protein function in growth responses to canopy signals. The Plant Journal, 2007,51(1):117-126. doi: 10.1111/j.1365-313X.2007.03122.x pmid: 17488236
[33]	ZENTELLA R, ZHANG Z L, PARK M, THOMAS S G, ENDO A, MURASE K . Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis. The Plant Cell, 2007,19(10):3037-3057. doi: 10.1105/tpc.107.054999 pmid: 17933900
[34]	GÓMEZ-CADENAS A, ZENTELLA R, WALKER-SIMMONS M K, HO T H . Gibberellin/abscisic acid antagonism in barley aleurone cells: Site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules. The Plant Cell, 2001,13:667-679. doi: 10.2307/3871414

引物名称 Primers	序列 Sequence of primers (5′-3′)	用途 Application
MsGAI	F: AAACTTCAACCCATAAACTC	基因克隆 Gene cloning
MsGAI	R: ACTTAAGGGTACCCTGAG	基因克隆 Gene cloning
121-MsGAI	F: TGCTCTAGAATGAAGAGAGAACACCA	pBI121载体构建 Vector construction
121-MsGAI	R: CGCGGATCCTCACTTGGACTCATTTTG	pBI121载体构建 Vector construction
Ms_Actin	F: CAAAAGATGGCAGATGCTGAGGAT	内参基因 Internal control
Ms_Actin	R: CATGACACCAGTATGACGAGGTCG	内参基因 Internal control
QGAI	F: CCACCACCTTAACAGCAGCA	荧光定量 Real-time PCR
QGAI	R: GAGCACTACCCATAACCATCTC	荧光定量 Real-time PCR
M13	F: GTAAAACGACGGCCAGT	亚克隆引物 The primers for subcloning
M13	R: CAGGAAACAGCTATGAC	亚克隆引物 The primers for subcloning
35s	F: GGTGGCTCCTACAAATGCCA	pBI121载体构建 Vector construction
35s	R: GAAACGCAGCACGATACGC	pBI121载体构建 Vector construction
Promoter	F: GGTACACGCTAAGACGCTAC	启动子克隆 Promoter cloning
	R: TTGCTGCTGTTAAGGTGG	启动子克隆 Promoter cloning