Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (2): 201-214.doi: 10.3864/j.issn.0578-1752.2019.02.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning Expression Analysis and Transformation of MsGAI Gene from Medicago sativa L

ZHANG Han,WANG XueMin,LIU XiQiang,MA Lin,WEN HongYu,WANG Zan()   

  1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193
  • Received:2018-07-18 Accepted:2018-09-07 Online:2019-01-16 Published:2019-01-21
  • Contact: Zan WANG E-mail:wangzan@caas.cn

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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

Table 1

Sequence of primers used in the experiment"

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

Fig. 1

Structure of 35S:MsGAI-gus over-expression vector"

Fig. 2

PCR amplified product of MsGAI M: DL5000 marker; 1: PCR amplified product"

Fig. 3

Amino acid sequence alignment of MsGAI with other homologous proteins"

Fig. 4

Prediction of second structure of MsGAI Blue: α-helix; Red: Extended chain; Green: β-turn; Orange: Random coil"

Table 2

Subcellular localization prediction"

名称
Name		 长度
Length(bp)		 叶绿体
cTP		 线粒体
mTP		 信号肽
SP		 Loc RC
MsGAI 605 0.634 0.097 0.036 c 5

Fig. 5

Phylogenetic tree of MsGAI and other GAI proteins"

Table 3

Promoter prediction of PlantCARE"

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

Fig. 6

Expression pattern of MsGAI in different tissues and different treatments"

Fig. 7

Enzyme digestion identification of 35S:MsGAI-gus M: DL 5000 DNA marker; 1: pBI121 vector; 2: Enzyme digestion fragments of 35S:MsGAI-gus"

Fig. 8

Transgenic Medicago sativa L. plants obtained after Agrobacterium tumefaciens-mediated A: Exophyte; B: Callus; C: Mature embryo; D: Positive plants; E: Gus staining; F: Transgenic plant"

Fig. 9

PCR detection results of partial transgenic medicago sativa L. A: Detection results used pBI121 vector primers; B: Detection results used MsGAI gene primers; C: Detection results used combination of pBI121 vector primers and MsGAI gene primers"

Fig. 10

Expression pattern of MsGAI in transgenic medicago sativa L. and different treatments"

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